1.1. Chemical and physical data

1.1.2. Structural and molecular formulae and relative molecular mass

1.2. Production

Hydroxyurea has been prepared by the reaction of calcium cyanate with hydroxylamine nitrate in absolute ethanol and by the reaction of potassium cyanate and hydroxylamine hydrochloride in aqueous solution. Hydroxyurea has also been prepared by converting a quaternary ammonium anion exchange resin from the chloride form to the cyanate form with sodium cyanate and reacting the resin in the cyanate form with hydroxylamine hydrochloride (Graham, 1955).

Information available in 1999 indicated that hydroxyurea was manufactured and/or formulated in 25 countries (CIS Information Services, 1998; Royal Pharmaceutical Society of Great Britain; 1999; Swiss Pharmaceutical Society, 1999).

1.3. Use

Hydroxyurea is a chemically simple antimetabolite, which is cytostatic by inhibiting ribonucleotide reductase, an enzyme important in creating deoxynucleosides for DNA replication in growing cells (Gao et al., 1998). Hydroxyurea was initially synthesized over 120 years ago, but its potential biological significance was not recognized until 1928. In the late 1950s, the drug was evaluated in a large number of experimental murine tumour systems and shown to be active against a broad spectrum of tumours. Phase I trials with hydroxyurea began in 1960 and by the late 1960s it was in clinical use (Donehower, 1992).

Hydroxyurea has been used, or investigated for use, in the treatment of a number of diseases.

1.4. Occurrence

Hydroxyurea is not known to occur as a natural product. No data on occupational exposure were available to the Working Group.

1.5. Regulations and guidelines

Hydroxyurea is listed in the British, French and US pharmacopoeias (Royal Pharmaceutical Society of Great Britain, 1999; Swiss Pharmaceutical Society, 1999).

2.1. Case reports

Several case reports have been published on the occurrence of multiple squamous-cell and basal-cell carcinomas of the skin in patients who received prolonged treatment with hydroxyurea for chronic myeloid leukaemia (Disdier et al., 1991; Stasi et al., 1992; Best & Petitt, 1998; De Simone et al., 1998), essential thrombocythaemia or polycythaemia vera (Callot-Mellot et al., 1996; Best & Petitt, 1998). The skin carcinomas were typically seen in sun-exposed areas and had been preceded by other degenerative cutaneous manifestations.

Reiffers et al. (1985), van den Anker-Lugtenburg and Sizoo (1990) and Furgerson et al. (1996) described the occurrence of acute leukaemia in patients who had been treated with hydroxyurea for long periods for essential thrombocythaemia and had not received other treatments.

2.2. Cohort studies

These studies are summarized in Table 1.

Table 1

Cohort studies of acute leukaemia (AL) and myelodysplastic syndrome (MDS) in patients treated with hydroxyurea.

3.1. Intraperitoneal administration

Groups of 50 mice of each sex of the XVII/G strain were treated intraperitoneally with hydroxyurea [purity not specified] starting at two days of age and then at weekly intervals for one year. The doses per mouse were: 1 mg at two days of age, 3 mg at eight days, 5 mg at 15 days and 10 mg from 30 days to one year of age. One group of 50 mice was kept untreated as controls. The incidences of pulmonary tumours were 30/50 (60%) in control and 16/35 (46%) in treated mice. In a positive control group treated with urethane, 28/30 (93%) of mice had lung tumours (Muranyi-Kovacs & Rudali, 1972).

3.2. Administration with known carcinogens

Groups of 40 female Swiss mice, six to seven weeks of age, received dermal applications of 5 µg of 7,12-dimethylbenz[a]anthracene followed four weeks later by treatment with 1% croton oil for 14 weeks. Hydroxyurea at a dose of 500 mg/kg bw was injected intraperitoneally once at 24 h or twice at 24 and 48 h after the first painting with croton oil. Treatment with two doses of hydroxyurea significantly reduced the incidence of skin papillomas when compared with 7,12-dimethylbenz[a]anthracene and croton oil treatment alone (Chan et al., 1970).

Groups of 16 male and 16 female hairless (hr/hr) Oslo mice were given an intraperitoneal injection of 0 or 5 mg hydroxyurea in 0.5 mL distilled water 30 min before dermal application of 2 mg of N-methyl-N-nitrosourea (MNU). Hydroxyurea enhanced the production of skin tumours by MNU from about 50% to 80%, this effect being attributed to inhibition of DNA synthesis (Iversen, 1982a). No such effect was observed when hydroxyurea was administered simultaneously with or after dermal application of 1 mg of MNU (Iversen, 1982b).

Groups of 36–43 female Wistar rats, weighing about 200 g, were given intraperitoneal injections of hydroxyurea in 23 fractionated consecutive doses of 0.1 mg/kg bw each shortly before and during maximal urothelial cell proliferation (33–55 h after partial cystectomy) produced by MNU administered as a single intravesicular pulse dose of 5 mg/kg bw during the various cell cycle phases. Hydroxyurea inhibited MNU-induced urothelial tumour development, and the degree of this inhibition depended on the cell cycle phase during which MNU was instilled. The numbers of rats with urothelial bladder tumours were: 14/43 in the control group (G₀ phase) and 7/37, 4/43 (p < 0.02), 10/46, 10/38, 9/36 and 12/40 in groups receiving MNU during the late G₁, early and late S, G₂+M, and early and late postmitotic phases, respectively (Kunze et al.,1989).

4.1. Absorption, distribution, metabolism and excretion

4.2. Toxic effects

4.3. Reproductive and prenatal effects

4.4. Genetic and related effects

4.4.1. Humans

In studies of genetic alterations in leukaemic cells of patients treated with hydroxyurea, a statistically non-significant association was seen between treatment with hydroxyurea alone or in combination and the occurrence of leukaemia and myelodysplastic syndrome characterized by abnormalities of chromosome 17 in patients with essential thrombocythaemia [p = 0.11, Fisher’s exact test]. As discussed in section 2, Sterkers et al. (1998) monitored the occurrence of acute leukaemia and myelodysplastic syndrome in 251 patients with essential thrombocythemia who were treated with hydroxyurea. The findings in the leukaemic cells are summarized in Table 2. In seven cases of leukaemia treated with hydroxyurea, including three given the drug alone, there were rearrangements of chromosome 17, including unbalanced translocations, partial or complete deletions and isochromosome 17q, which resulted in 17p deletion in the leukaemic cells. P53 mutation was observed in six cases, including two treated with hydroxyurea alone. The authors suggested that the molecular characteristics of these leukaemias are consistent with 17p⁻ syndrome and that prolonged use of hydroxyurea in patients with essential thrombocythaemia may lead to acute myeloid leukaemia and myelodysplastic syndrome with loss of chromosome 17p material and P53 mutation. A review of the literature by these authors revealed similar 17p deletions in four of 11 patients treated for essential thrombocythaemia with hydroxyurea alone but in only one of 24 patients who did not receive this treatment. Tefferi (1998) cautioned, however, that the results of bone-marrow and cytogenetic investigations before treatment were not available for some of the patients. Monosomy 17 was also observed in complex karyotypes in two of three cases of leukaemia reported by Liozon et al. (1997) among 58 patients with essential thrombocythaemia treated with hydroxyurea; in the third case, which was chronic myelomonocytic leukaemia, the karyotype was normal.

Table 2

Karyotypic findings in the bone marrow of patients with essential thrombocythaemia treated with hydroxyurea.

Quesnel et al. (1993) identified the t(8;21) translocation in a case of leukaemia in which essential thrombocythaemia had been treated with hydroxyurea alone. The t(8;21) is associated with the French–American–British M2 (acute myeloblastic) subtype of denovo and treatment-related acute myeloid leukaemia. The complex karyotype, which also contained monosomy 17, was 46, XX[3]/47, XX, +8 [2]/43–44,XX,der(7)t(7;dup5), t(8;21)(q22;q22), −16,−17,t(18;?)(q;?)[10].

Ören et al. (1999) described a case of acute promyelocytic leukaemia with i(17q) after treatment of Philadelphia chromosome-positive chronic myeloid leukaemia with combination therapy including hydroxyurea, but i(17q) may occur in chronic myeloid leukaemia in blast crisis.

Diverse chromosomal aberrations have been seen in human bone-marrow cells after hydroxyurea treatment. Diez-Martin et al. (1991) reviewed studies of the chromosomes of 104 patients at various stages of polycythaemia vera. The bone-marrow cells of five of six patients treated with hydroxyurea alone had abnormalities, including an unbalanced t(1;7)(p11;p11), which can be associated with treatment-related myelodysplastic syndrome, but this abnormality may occur without prior treatment. Cytogenetic analyses in these five patients were performed only on bone-marrow samples obtained after treatment. One each of the other four abnormal marrows had t(8;13)(p21;q12), +9, del(6)(q13q21) and t(1;?)(q12;?). Furthermore, the authors observed several de-novo abnormalities in untreated patients which they related to the disease itself rather than to the therapy, including +9, +8 and 20q–, and suggested that the 13q– abnormality is related to disease progression.

Löfvenberg et al. (1990) examined 81 hydroxyurea-treated patients with Philadelphia chromosome-negative chronic myeloproliferative disorders, comprising 35 with polycythaemia vera, 32 with essential thrombocythaemia, 12 with myelofibrosis and two with myeloproliferative syndromes. Only three had received prior therapy with alkylating agents or radioactive phosphorus. Four of the 81 developed acute myeloid leukaemia or myelodysplastic syndrome. Five of 53 evaluable patients (9%) had clonal cytogenetic abnormalities involving chromosomes 1, 9, 20 and 21 before treatment, and 15% had these abnormalities at follow-up, during or after hydroxyurea treatment. Treatment was thus associated with a low frequency of cytogenetic abnormalities in a heterogeneous population, and the abnormalities observed before and after treatment were similar.

The series later reported on by Weinfeld et al. (1994) included 30 patients with polycythaemia vera, 10 with essential thrombocythaemia and 10 with myelofibrosis who were treated with hydroxyurea. Acute leukaemia developed in nine patients and myelodysplastic syndrome in one; seven of the leukaemia patients had been treated with hydroxyurea alone. The duration of therapy for patients who developed leukaemia or myelodysplastic syndrome was 5–111 months. Seven of 19 previously untreated patients with initially normal karyotypes treated with hydroxyurea alone developed clonal chromosomal abnormalities during therapy (37%).

Davidovitz et al. (1998) observed evolution of polycythaemia vera to myelofibrosis with a t(1;20)(q32;q13.3) in a patient who received chronic low-dose hydroxyurea. The t(1;20) affected the same region of chromosome 20 as the 20q– abnormality; it could not be determined whether the translocation was related to the treatment. Furgerson et al. (1996) described a patient in whom essential thrombocythaemia evolved to acute myeloid leukaemia after hydroxyurea treatment. The karyotype was normal at the time of diagnosis of essential thrombocythaemia but revealed del(5)(q23), del(7)(q31), inv(16)(p13;q22),+8 when acute myeloid leukaemia emerged.

4.4.2. Experimental systems

Early studies on the mutagenicity of hydroxyurea were summarized by Timson (1975). Reviews on the mutagenicity of anticancer drugs in general, including hydroxyurea, were provided by Ferguson (1995) and Jackson et al. (1996). Ferguson and Denny (1995) commented on some practical issues in testing antimetabolites, which may limit the usefulness (and meaning) of some types of in-vitro assays.

The results of tests for genotoxicity with hydroxyurea are summarized in Table 3.

Table 3

Genetic and related effects of hydroxyurea.

Hydroxyurea was inactive as either a frameshift or base-pair substitution mutagen in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100, and addition of an exogenous metabolic activation system did not affect these results. Hydroxyurea induced SOS repair in Escherichia coli K12 cells. In various Saccharomyces cerevisiae strains, hydroxyurea induced mitotic crossing over, mitotic gene conversion, intrachromosomal recombination and aneuploidy, but not ‘petite’ mutations. It also increased the frequency of ultraviolet-induced mitotic gene conversion and induced recombination in dividing but not G1 or G2 arrested cells of the RS112 strain of yeast. In meiotic yeast cells, hydroxyurea increased the frequency of meiotic recombination.

Hydroxyurea is a clastogen in mammalian cells in vitro and in vivo.

Hydroxyurea caused chromosomal aberrations in cultured Chinese hamster cells, in mouse cells and in various human cell lines. Karon and Benedict (1972) found that hydroxyurea induced chromosomal aberrations when given during S phase but not when given during G2 phase. It did not induce micronuclei in human peripheral blood lymphocytes but increased the frequency of sister chromatid exchange and of gene amplification in L5178Y mouse lymphoma cells. Hydroxyurea induced sister chromatid exchange in various Chinese hamster cell lines in vitro. It caused DNA strand breaks in Ehrlich ascites tumour cells and in human T lymphoma cells in vitro.

It did not induce mutations at the HPRT locus in a cultured human T-lymphocyte cell line at doses of 50–250 µmol/L, which had substantial effects on DNA synthesis, but induced mutants at the Tk locus in L5178Y cells.

Hydroxyurea caused cell transformation in mass cultures of embryonic cells from BN/a, mice, but not in cultures derived from two other strains of mice—DBA/2 and Swiss, nor in BALB/c 3T3 cells. Although hydroxyurea alone did not induce morphological transformation in Syrian hamster embryo cells, the cell cycle arrest caused by the drug led to enhancement of cell transformation by bromodeoxyuridine (Tsutsui et al., 1979). Hydroxyurea did not enhance metabolic cooperation between V79 cells (Toraason et al., 1992).

Hydroxyurea treatment led to hypermethylation of DNA in hamster fibrosarcoma cells (Nyce et al., 1986). In rats, this resulted in nitric oxide production (Jiang et al., 1997) and induced a cytokine response (Navarra et al., 1995, 1997). These effects may indicate an enhanced effect on chromosomal damage in certain situations in vivo. Hydroxyurea also induced DNA hypermethylation in normal human embryonic lung fibroblasts (WI-38) and their simian virus 40-transformed counterparts (SVWI-38) (De Haan & Parker, 1988).

Hydroxyurea induced micronuclei in the bone marrow of non-tumour-bearing male NMRI mice but did not induce micronucleated cells in female C57BL/6 × C3H/He hybrid mice, although it produced sperm abnormalities in male mice of this strain.

Although hydroxyurea is a mutagen in somatic cells, there is no evidence that it mutates germ cells. It did not cause dominant lethal mutation or specific locus mutation in mice. It did not induce chromosomal damage in spermatogonial cells of male Swiss mice, although it enhanced damage induced by X-rays.

Minford et al. (1984) showed that hydroxyurea at 0.1 mmol/L enhanced both DNA breakage and cytotoxicity caused by the intercalating DNA topoisomerase II inhibitor, amsacrine. Lambert et al. (1983) found similar results in relation to adriamycin. Palitti et al. (1984a) showed that treatment with hydroxyurea after mitomycin C enhanced the frequencies of chromosomal aberrations and sister chromatid exchange induced by mitomycin C alone in both Chinese hamster cells and human lymphocytes. Hydroxyurea had a synergistic effect on ultraviolet-induced sister chromatid exchange (Ishii & Bender, 1980) and enhanced X-radiation-induced damage in spermatogonial cells of Swiss mice (van Buul & Bootsma, 1994).

4.5. Mechanistic considerations

Hydroxyurea does not bind or bond to DNA but acts by inhibiting ribonucleotide reductase, which converts ribonucleoside diphosphates to deoxyribonucleotide diphosphates, the precursers for de-novo DNA synthesis. Hydroxyurea depletes intracellular deoxyribonucleotide pools and is known and used as an inhibitor of DNA synthesis (Timson, 1975).

The differences in the results of various studies may depend on the exact cell culture conditions, especially in regard to the amounts of deoxyribonucleotides available. Hansen et al. (1995) found that they could partially attenuate the embryotoxic effects of hydroxyurea by providing additional deoxyribonucleotides.

In a number of experiments, hydroxyurea appeared to enhance the susceptibility of cells to mutagenesis by other agents (e.g. Palitti et al., 1983, 1984a,b; Ferguson, 1990; Jelmert et al., 1992). There are three possible reasons for this:

1. It halts the progression of cells in the late G1 phase of the cycle, allowing synchronization of the culture (e.g. Tsutsui et al., 1979). Thus, if cells are sensitive to a certain agent in a particular phase of the cell cycle, hydroxyurea may reveal this effect.
2. Although hydroxyurea inhibits normal DNA synthesis, it does not appear to inhibit unscheduled DNA synthesis at the same doses after treatment with various genotoxic agents, including X-rays (e.g. Painter & Cleaver, 1967). This justifies its inclusion in protocols of unscheduled DNA synthesis.
3. Prempree and Merz (1969) suggested that hydroxyurea could inhibit the repair of chromosomal breaks without itself inducing breaks.

5.1. Exposure data

Hydroxyurea is a chemically simple antimetabolite that inhibits the enzyme ribonucleotide reductase. It has been in clinical use since the 1960s and is widely used for the treatment of severe sickle-cell disease, chronic myeloid leukaemia, myeloproliferative disorders such as polycythaemia vera and essential thrombocythaemia and, increasingly, in combination with didanosine in HIV infection. Hydroxyurea is sometimes used for the treatment of psoriasis and various solid tumours.

5.2. Human carcinogenicity data

The risk for leukaemia associated with administration of hydroxyurea in the treatment of chronic myeloproliferative disorders has been evaluated in a number of small cohort studies. Overall, 5–6% of patients developed either acute leukaemia or myelodysplastic syndrome subsequent to the start of hydroxyurea treatment. Large variation in the length of active follow-up was not taken into account in the analyses. The risk for leukaemia in patients with chronic myeloproliferative disorders who were not treated with hydroxyurea or other agents (e.g. polycythaemia vera patients treated with phlebotomy alone) was also increased in comparison with that of the general population. The available data do not allow a conclusion about whether the occurrence of acute leukaemia and myelodysplastic syndrome in the hydroxyurea-treated patients represents progression of the myeloproliferative process or an effect of the treatment.

5.3. Animal carcinogenicity data

Hydroxyurea was tested in one experiment in mice by intraperitoneal administration beginning at two days of age. No increase in the incidence of tumours was reported. Hydroxyurea has also been tested in combination with other chemical carcinogens to assess the effect of inhibition of DNA synthesis on carcinogenesis. The experiments are inadequate to assess the carcinogenicity of hydroxyurea.

5.4. Other relevant data

Hydroxyurea is readily absorbed after oral administration. In one study, 35% of an administered dose was excreted unchanged in the urine of humans. Hydroxyurea is widely distributed in tissues. Its main toxic effect is neutropenia.

Hydroxyurea is teratogenic and causes postnatal behavioural deficits after prenatal exposure in all species of animals in which it has been tested. It has commonly been used as positive control substance in studies of developmental toxicity.

In one study of patients treated with hydroxyurea for essential thrombocythaemia who developed leukaemia, a statistically non-significant association was found with a 17p chromosomal deletion in leukaemic cells.

Hydroxyurea neither bonds chemically nor otherwise binds to DNA. Instead, it inhibits ribonucleotide reductase, which converts ribonucleoside diphosphates to deoxyribonucleotide diphosphate precursers for de-novo DNA synthesis. Hydroxyurea does not induce gene mutation in bacteria and does not cause mutation at the Hprt locus in mammalian cells. It causes chromosomal mutations and mutagenic effects at the Tk locus in mouse lymphoma cells. It is an effective recombinogen in yeast and induces sister chromatid exchange in mammalian cells. It also causes gene amplification in mammalian cells and may lead to transformation of some but not all cell lines. Although it has been reported to be ineffective in causing germ-cell mutation, it has not been extensively tested for that end-point.

5.5. Evaluation

There is inadequate evidence in humans for the carcinogenicity of hydroxyurea.

There is inadequate evidence in experimental animals for the carcinogenicity of hydroxyurea.

Overall evaluation

Hydroxyurea is not classifiable as to its carcinogenicity to humans (Group 3).

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1.1. Chemical and physical data

1.1.2. Structural and molecular formulae and relative molecular mass

1.2. Production

Phenolphthalein can be prepared from a mixture of phenol, phthalic anhydride and sulfuric acid which is heated to 120 °C for 10–12 h. The product is extracted with boiling water, and the residue is dissolved in dilute sodium hydroxide solution, filtered, and precipitated with acid (Gennaro, 1995).

It has been reported that 197 tonnes of phenolphthalein were produced by one US manufacturer in the early 1990s (National Toxicology Program, 1999). Information available in 1999 indicated that phenolphthalein was manufactured and/or formulated in 33 countries (CIS Information Services, 1998; Royal Pharmaceutical Society of Great Britain; 1999; Swiss Pharmaceutical Society, 1999).

1.3. Use

Phenolphthalein is a stimulant laxative which has been used for the treatment of constipation and for bowel evacuation before investigational procedures or surgery. The laxative effect of phenolphthalein was discovered in 1902, and it has been widely used since that time (Mvros et al., 1991). It usually has an effect within 4–8 h after oral administration, generally in tablets or capsules; it is also available as an emulsion with liquid paraffin. It is available without prescription in many countries. The usual oral laxative dose of phenolphthalein (white or yellow) is 30–200 mg daily taken at bedtime for adults and children aged ≥ 12 years (270 mg should not be exceeded); 30–60 mg daily for children aged 6–11 years; and 15–30 mg daily for children aged 2–5 years, given as a single or divided doses. A dose of 260 mg has been used in regimens for bowel evacuation (American Hospital Formulary Service, 1997; Royal Pharmaceutical Society of Great Britain, 1999).

The use of laxatives to relieve constipation and to maintain regularity in bowel habits is common in western cultures. Studies in Australia, the United Kingdom and the USA have found that about 20% of the general population reports regular use of laxatives (Kune, 1993). Two large surveys of the adult population in the USA found that about 10% of adults used some form of laxative at least once a month, that female users outnumbered male users and that the fraction of users increases with age (Everhart et al., 1989; Harari et al., 1989).

Few studies report the prevalence of use of phenolphthalein laxatives. One study of 424 cases of colon cancer and 414 controls in Washington State, USA, aged 30–62, found that 34% of the control subjects reported constipation requiring treatment (use of a laxative, enema or prunes), 2.7% reported ever having used phenolphthalein laxatives and 1.4% reported having used phenolphthalein laxatives at least 350 times in their lifetimes (Jacobs & White, 1998).

In three populations of 268–813 persons who had undergone endoscopy for colon polyps, two in North Carolina and one in California, USA, comprising approximately equal numbers of cases and controls, 0.8–4.4% of the control subjects had used phenolphthalein laxatives at least once per week. The two groups in North Carolina comprised subjects aged 30–89 years, 58% and 53% of whom were female; the group in California comprised subjects aged 50–74 years of whom 34% were female. The mean ages of the three groups were comparable (59–62 years). Among controls, the frequent users of phenolphthalein laxatives represented 5.2–30% of all frequent laxative users. In the two studies in North Carolina, 18% of case subjects and 25% of controls reported ever having used phenolphthalein laxatives, and 10% of cases and 7% of controls had used them at least once a month (Longnecker et al., 1997). In a study of colorectal cancer in Melbourne, Australia (Kune, 1993), 9.7% of the 723 subjects reported ever having used phenolphthalein laxatives.

Phenolphthalein in a 1% alcoholic solution is also used as a visual indicator in titrations of mineral and organic acids and most alkalis. Phenolphthalein-titrated solutions are colourless at pH < 8.5 and pink to deep-red at pH > 9 (Budavari, 1996).

1.4. Occurrence

Phenolphthalein is not known to occur as a natural product. No data on occupational exposure were available to the Working Group.

1.5. Regulations and guidelines

Phenolphthalein is listed in the Austrian, Belgian, British, Chinese, Czech Republic, Hungarian, Italian, Swiss and US pharmacopoeias (Royal Pharmaceutical Society of Great Britain, 1999; Swiss Pharmaceutical Society, 1999).

After the publication in 1996 of the results of studies in rodents indicating that phenolphthalein was carcinogenic and genotoxic in several test systems, with damage (loss) of the p53 tumour suppressor gene (Food & Drug Administration, 1999), many countries moved to restrict over-the-counter sales of phenolphthalein-containing laxatives. Both France and Italy have suspended use of phenolphthalein in prescription and over-the-counter pharmaceutical preparations, and the United Kingdom has changed the status of phenolphthalein from an over-the-counter to prescription agent in pharmaceutical preparations (WHO, 1997; Francesco International, 1998; WHO, 1998). Canada has suspended the sale of all products containing phenolphthalein (Canadian Pharmacists Association, 1999). The German Federal Institute for Drugs and Medical Devices recommended that holders of authorizations to market phenolphthalein-containing laxative products withdraw their products from the market because of the potential toxicological risks. The Japanese Pharmaceutical and Medical Safety Bureau of the Ministry of Health and Welfare issued a statement that laxative products containing phenolphthalein had been voluntarily withdrawn by the manufacturers (WHO, 1998). The Food and Drug Administration (1999) issued a final rule establishing that phenolphthalein is not generally recognized as safe and effective.

2.1. Colon cancer

Kune (1993) analysed data on laxative use reported by 685 subjects with colorectal adenocarcinoma diagnosed in 1980–81 in Melbourne, Australia, and 723 controls frequency matched with cases on age and sex. Laxative use throughout adult life was assessed by interview. The relative risk associated with use of commercially produced laxatives was 1.0 (95% confidence interval [CI], 0.86–1.4). Eighty-seven case subjects (13%) and 70 controls (9.7%) reported having used phenolphthalein-containing laxatives (relative risk, 1.4 [95% CI, 0.96–1.9]).

In a case–control study of the association between colon cancer, constipation and use of phenolphthalein–containing laxatives in Washington State, USA (Jacobs & White, 1998), of 659 potential cases identified, 102 died before being approached and 55 were found to be ineligible. Of the 502 remaining cases, data were obtained from 424. Potentially eligible controls were selected by stratified random sampling of subjects in households identified by random-digit dialling, to approximate the distribution by age, sex and county of residence of the case subjects. Of 549 controls thus identified, data were obtained from 414 subjects. Data on laxative use were obtained by telephone interview, and subjects were also asked to complete a mailed food frequency questionnaire. The reference period was up to two years before diagnosis. Regular use was defined as a total use of more than 90 days. The relative risk for colon cancer associated with up to 349 lifetime uses of phenolphthalein-containing laxatives compared with no regular use was 1.0 (95% CI, 0.3–3.7) after adjustment for fibre as percentage of calories. The relative risk for ≥ 350 lifetime uses was 3.9 (95% CI, 1.5–10). Frequent constipation during the 10 years before the reference date (two years before diagnosis) was associated with an increased risk for colon cancer (4.4; 95% CI, 2.1–8.9). When constipation and commercial laxative use were adjusted for mutually, the association with commercial laxative use was no longer apparent, whereas the association with constipation persisted (2.7; 95% CI, 1.4–5.3). The relative risk associated with use of phenolphthalein-containing laxatives adjusted for constipation was 0.42 (95% CI, 0.10–1.7) for < 350 lifetime uses and 1.4 (95% CI, 0.47–4.3) for > 350 uses. [The Working Group noted the difficulty of excluding possible confounding by indication.]

2.2. Colorectal adenomatous polyps

The association between phenolphthalein-containing laxatives and colorectal adenomatous polyps was investigated in a case–control study in Los Angeles (California, USA) in the period 1991–93 and in two case–control studies in North Carolina (USA) in 1988–90 and 1992–95 (Longnecker et al., 1997). In all three studies, cases and controls were selected from among people undergoing an endoscopic procedure (sigmoidoscopy in Los Angeles, colonoscopy in North Carolina); the cases were those found to have polyps. The main indication for this procedure was screening in the Los Angeles study and bleeding in the North Carolina studies. In the Los Angeles study, data on laxative use were collected by personal interview, and subjects were asked about use of specified agents in the year prior to sigmoidoscopy. The agents specified did not include phenolphthalein-containing laxatives but included ‘other laxative preparations’ as a category. If the subject reported use of laxatives in this category, the specific preparation was recorded. In North Carolina, subjects were asked over the telephone about the brand of laxative they used most often. For all three studies, the responses to questions about the preparation used were reviewed without knowledge of the subject’s case or control status, and laxatives were classified as containing phenolphthalein on the basis of brand. In view of these differences between the studies and differences in the eligibility criteria and matching, the three studies were analysed separately. The relative risk for colorectal polyps associated with use of phenolphthalein-containing laxatives at least once a week was 1.8 (95% CI, 0.5–6.2) in Los Angeles (488 cases, 488 controls), 1.0 (95% CI, 0.4–2.2) in North Carolina in 1988–90 (236 cases, 409 controls) and 1.1 (95% CI, 0.2–5.7) in North Carolina in 1992–95 (142 cases, 169 controls).

[The Working Group noted the low statistical power of these studies to detect associations, resulting from the low prevalence of use of phenolphthalein-containing laxatives.]

Oral administration

Mouse

Groups of 50 male and 50 female B6C3F₁ mice, six to seven weeks of age, were given diets containing phenolphthalein (purity, 99.9%) at a concentration of 3000, 6000 or 12 000 mg/kg for two years, equivalent to 0, 300, 600 or 1200 mg/kg bw in males and 0, 400, 800 or 1500 mg/kg bw in females. Only females treated with the highest dose had a significantly decreased rate of survival when compared with controls. The plasma concentrations of total phenolphthalein were similar at all doses. As shown in Table 2, the incidence of histiocytic sarcoma (principally in the liver but also at other sites) was significantly greater in males and females at the two higher doses than in controls. The incidence of malignant lymphoma (all types) was significantly increased in all groups of treated females, but not in males. The incidence of lymphoma of thymic origin was significantly increased in all groups of exposed females and in males at 6000 ppm. As shown in Table 2, the incidence of benign ovarian sex-cord stromal tumours was significantly increased in treated females; the mean historical incidence of all ovarian luteomas was 0.4% (Dunnick & Hailey, 1996; National Toxicology Program, 1996).

Table 2

Incidences of lesions in mice fed diets containing phenolphthalein.

Groups of 20 female p53^+/− heterozygous mice, 7–10 weeks of age, received diets containing phenolphthalein at a concentration of 0 (control), 200, 375, 750, 3000 or 12 000 mg/kg for 26 weeks, equivalent to average daily doses of phenolphthalein of 0, 43, 84, 174, 689 or 2375 mg/kg bw per day. The two lowest concentrations delivered doses of phenolphthalein that were approximately 0.5–1.5 times the recommended human dose based on a mg/m² body surface area comparison. The incidence of malignant lymphoma of the thymus was significantly increased in heterozygous p53-deficient female mice given the two higher doses. Atypical thymic hyperplasia, seen in 3/20 animals at 750 mg/kg, 3/20 at 3000 mg/kg and 5/20 at 12 000 ppm, was considered to represent proliferative change preceding lymphoma. The incidence of atypical hyperplasia or malignant lymphoma was increased in animals at 750 ppm. The incidence of malignant lymphomas was significantly increased at the two highest doses (0/19 in controls and 1/20, 0/20, 2/20, 17/20 (p < 0.01) and 14/20 (p < 0.01) at the five doses, respectively). Loss of the p53 wild-type allele was found in 2/2 thymic lymphomas from animals at 750 mg/kg, 13/13 at 200 mg/kg and 6/6 at 12 000 mg/kg (Dunnick et al., 1997).

In a study published as an abstract, p53^+/− knock-out mice [age not specified] were given phenolphthalein [purity not specified] for 26 weeks by gavage at a dose of 800 or 2400 mg/kg bw per day [number of treatments per week not specified] or in the diet at 2400 mg/kg bw per day [dietary concentration not specified]. [Details of the control groups were not reported.] The experiment was terminated at 26 weeks. The incidences of thymic lymphomas were 3/15, 4/15 and 12/15 in males and 5/15, 8/15 and 14/15 in females receiving 800 (by gavage), 2400 (by gavage) and 2400 (in the diet) mg/kg bw, respectively (Furst et al., 1999).

Rat

Groups of 50 male and 50 female Fischer 344 rats, seven weeks of age, were given diets containing phenolphthalein (purity, 99.9%) at a concentration of 0, 12 000, 25 000 or 50 000 mg/kg for two years, equivalent to 0, 500, 1000 or 2000 mg/kg bw for males and 0, 500, 1000 or 2500 mg/kg bw for females. As in the mice, the total plasma concentrations of phenolphthalein did not increase with increasing dose. The survival rate in all groups of treated animals was similar to that of controls. As shown in Table 3, the incidence of benign phaeochromocytoma of the adrenal medulla was significantly increased in all treated male groups, and most were bilateral. The incidence of malignant phaeochromocytoma was not increased by treatment at any dose. The incidence of benign phaeochromocytoma was also increased in female rats given the highest dose, but the incidences of bilateral tumours and malignant phaeochromocytoma were not increased in females. As seen in Table 4, the incidence of renal tubular adenoma (single and step sections combined) was also significantly increased in all treated male groups, and a few renal tubular carcinomas were also observed. In females, one renal tubular adenoma was observed at the highest dose (Dunnick & Hailey, 1996; National Toxicology Program, 1996). [The Working Group noted the high doses administered.]

Table 3.

Incidences of lesions in the adrenal medulla in Fischer 344 rats fed diets containing phenolphthalein.

Table 4

Incidences of renal tubular lesions in male Fischer 344 rats fed diets containing phenolphthalein.

4.1. Absorption, distribution, metabolism and excretion

4.1.2. Experimental systems

Phenolphthalein is absorbed in the intestine (Visek et al., 1956) and is almost completely converted to its glucuronide during extensive first-pass metabolism in the intestinal epithelium and liver (Parker et al., 1980) via uridine diphosphate glucuronosyltransferase (UDPGT) in rodents and dogs (Sund & Hillestad, 1982; National Toxicology Program, 1996). In guinea-pigs, small amounts of sulfate-conjugated metabolites have been detected in isolated mucosal sheets originating in the jejunum and colon (Sund & Lauterbach, 1986). Faecal excretion is the major route of elimination of phenolphthalein in rats, while in mice both urinary and faecal elimination are important. The metabolites identified in urine and faeces are phenolphthalein glucuronide, phenolphthalein sulfate and phenolphthalein hydroxide (Griffin et al., 1998; see Figure 1).

Figure 1

Metabolism of [¹⁴C]phenolphthalein in Fischer 344 rats and B6C3F₁ mice

Six hours after an intravenous injection of [³H]phenolphthalein to female Wistar rats, analysis of the systemic circulation showed that all of the radiolabel was associated with the glucuronide conjugate (Colburn et al., 1979). Enterohepatic recirculation is limited by the rate of hydrolysis of phenolphthalein glucuronide to aglycone by intestinal bacterial β-glucuronidase (Bergan et al., 1982; National Toxicology Program, 1996).

The extent of enterohepatic recirculation of phenolphthalein was examined in rats with cannulated bile ducts. Within 24 h, 95% of a dose of 25 mg/kg bw [³H]phenolphthalein administered intraperitoneally to female Wistar rats was recovered as glucuronide in the bile, with 0.2% in the urine. In rats without cannulated bile ducts, 86% of the same dose was recovered in the faeces, with little glucuronide, and 10% was recovered in the urine, primarily as the glucuronide (Millburn et al., 1967; Parker et al., 1980).

In male Sprague-Dawley CR-1 strain rats with cannulated femoral veins, femoral arteries and bile ducts given an intravenous dose of 3, 30 or 60 mg phenolphthalein, 99.5% of the dose was eliminated in the bile as the glucuronide. When the same rats were given 3, 30 or 100 mg phenolphthalein glucuronide by intravenous administration, no phenolphthalein was detected in the bile (Mehendale, 1990).

Studies in dogs and mice given [¹⁴C]phenolphthalein showed that the radiolabel is evenly distributed throughout the body. In newborn pups of bitches given 4.8 mg/kg bw orally 50 h before whelping, < 0.03% of the dose was found in the liver and gall-bladder and none in the blood, indicating extremely limited passage across the placenta (Visek et al., 1956).

Phenolphthalein is excreted in bile, urine, faeces and milk. In mice, 56% of an oral dose was recovered from the urine within 48 h and an additional 38% from the faeces. When an intravenous dose was given, 30% was recovered from the urine and 68% from the faeces (Visek et al., 1956). Some phenolphthalein is excreted into the bile, and the prolonged cathartic effect may be due to the ensuing enterohepatic recirculation (Hardman et al., 1996). Pre-treatment with hepatic microsomal enzyme inducers increased biliary excretion of metabolites in rats, but post-treatment with enzyme inhibitors decreased it (National Toxicology Program, 1996).

Within 72 h of oral administration of 4.8 mg/kg bw [¹⁴C]phenolphthalein to mongrel bitches, 51% of the radiolabel was excreted in the faeces and 36% in the urine. After an intravenous dose, 54% was found in the faeces and 37% in the urine. When the same animals received a cannula in the bile-duct and were given an oral dose, 31% of the radiolabel was found in faeces, 38% in urine and 22% in bile. After an intravenous dose, 11% was eliminated in faeces, 35% in urine and 43% in bile (Visek et al., 1956).

The profile of systemic blood concentration–time for phenolphthalein during 24 h after a single intravenous bolus injection was described by a classical compartmental pharmacokinetics model, with evidence of enterohepatic recirculation (Colburn et al., 1979).

In the two-year bioassays of the National Toxicology Program (1996), the concentrations of total phenolphthalein in plasma were 100–200 µg/mL.

Whole-body autoradiography of male BOM:NMRI mice showed high concentrations of radiolabel in the stomach, gall-bladder and small intestine 1 h after administration of an intragastric dose of 1 mL/100 g bw [¹⁴C]phenolphthalein (10 µCi/100g) [10 mL/kg bw or 3.2 mg/kg bw]. As evidenced by the presence of radiolabel in peripheral organs (including the kidney, liver and skin), the compound was absorbed. After 2 h, it had arrived in the large intestine, and 4 h after administration, maximum radiolabel was observed in the rectum. Two days after administration, no radiolabel was detected (Sund et al., 1986).

4.2. Toxic effects

4.3. Reproductive and prenatal effects

4.4. Genetic and related effects

4.4.2. Experimental systems

The results of these studies are summarized in Table 5.

Table 5

Genetic and related effects of phenolphthalein.

Phenolphthalein was not mutagenic in several assays in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence or absence of exogenous metabolic activation. It did not induce DNA damage in DNA repair-deficient strains of Bacillus subtilis.

Phenolphthalein did not induce sister chromatid exchange in Chinese hamster ovary cells in the presence or absence of exogenous metabolic activation, but it induced a dose-related response in chromosomal aberrations in these cells only in the presence of exogenous metabolic activation.

In experiments in which a number of end-points were studied in Syrian hamster embryo cells (a mixed population of cell types that retain some endogenous metabolizing enzymic activity, including oxidation and peroxidation), phenolphthalein induced chromosomal aberrations and Hprt mutations, but not ouabain mutations or aneuploidy. No evidence was found for adduct formation in DNA of these cells. The data for micronuclei failed to reach statistical significance (p = 0.057). Phenolphthalein caused cellular transformation in the same cell line, indicating that it is metabolized appropriately in this system.

Phenolphthalein increased the incidence of micronucleated erythrocytes in male and female B6C3F₁ mice and in male Swiss CD-1 mice. [The Working Group noted that the doses were significantly higher than those to which humans would be exposed.]

Tice et al. (1998) studied the effects of phenolphthalein at various concentrations in the diet of transgenic female mice heterozygous for the p53 gene, over a six-month period. They found significant increases in the frequency of micronucleated erythrocytes, most of which appeared to arise from whole chromosomes rather than chromosomal damage; these were observed at doses comparable to those to which humans are exposed. Inconclusive evidence was found for DNA damage in blood leukocytes, and there was no evidence for DNA damage, apoptosis or necrosis in liver parenchymal cells.

In phenolphthalein-induced thymic lymphomas in B6C3F₁ mice, p53 protein accumulated in most tumour cell nuclei, but detectable p53 protein was not seen in control thymuses in this model (Dunnick et al., 1997). Other studies have shown that accumulation of p53 protein results from p53 gene alterations (Hegi et al., 1993).

In p53^+/− heterozygous mice, phenolphthalein induced atypical hyperplasia and malignant lymphomas of thymic origin within six months in 0% of controls, 5% of animals at 200 mg/kg, 5% at 375 mg/kg, 25% at 750 mg/kg, 100% at 3000 mg/kg and 95% of animals at 12 000 mg/kg. Two of two thymic lymphomas examined from animals at 750 mg/kg, 13/13 from those at 3000 mg/kg and 6/6 from those at 12 000 mg/kg had lost the remaining p53 wild-type allele (Dunnick et al., 1997). No spontaneous thymic lymphomas were found in control mice in these studies, but in other studies in p53^+/− mice of spontaneous tumours (which may occur in mice after one year of age), only 55% showed loss of the remaining functional p53 allele (Harvey et al., 1993). The presence of functional p53 protein is essential for normal cell growth. When this protein is absent, as is the case in phenolphthalein-induced thymic lymphomas, regulation of cell cycle electrophoresis is lost and malignant progression may be enhanced.

4.5. Mechanistic considerations

Analogy with related biphenolic compounds suggests that phenolphthalein has oestrogenic activity; however, studies with MCF-7 human breast cancer cells in tissue culture (Ravdin et al., 1987) and in rat uterus in vivo (Nieto et al., 1990) suggested only a weak oestrogenic response. Tsutsui et al. (1997) used the nuclease P1 enhancement version of the ³²P-postlabelling assay to investigate whether (and what type of) DNA adducts were responsible for the morphological transformation induced by phenolphthalein. Although they found no adducts, they recognized the possible limitations of the techniques and suggested that small DNA adducts formed by free radicals could be involved in the effects. Sipe et al. (1997) showed free radical metabolism of phenolphthalein by peroxidases in vitro.

The observation of Witt et al. (1995) that chromosomal damage in Chinese hamster ovary cells occurred only when exogenous metabolic activation was added suggests that some as yet unidentified metabolite is responsible for these effects. Bishop et al. (1998) also interpreted differences in the micronucleus response between the two human lymphoblastoid cell lines, MCL-5 and AHH-1 TK^+/−, as being likely to reflect the importance of a metabolite in chromosome-damaging effects.

Tice et al. (1998) suggested that numerical chromosomal loss is responsible for the enhanced incidence of thymic tumours seen after treatment with phenolphthalein in p53 heterozygous mice. Dunnick et al. (1997) noted that these tumours uniformly showed loss of heterozygosity for the p53 allele rather than point mutations, suggesting either chromosome loss or deletions of large chromosomal segments.

The ability to detect micronuclei but not mutations at the TK locus in AHH cells may indicate that cells containing phenolphthalein-induced lesions are susceptible to apoptosis (Bishop et al., 1998).

5.1. Exposure data

Phenolphthalein has been widely used as a laxative for nearly a century. Generally available without prescription, it is now being withdrawn from the market in many countries because of recent toxicological concern. Phenolphthalein has also long been used in the laboratory as an indicator in acid–base titrations.

5.2. Human carcinogenicity data

In the few available studies, there was no consistent association between the occurrence of colon cancer or adenomatous colorectal polyps and use of phenolphthalein-containing laxatives. Cancers at other sites have not been studied.

5.3. Animal carcinogenicity data

Phenolphthalein was tested for carcinogenicity by oral administration in two experiments in mice and in one experiment in rats. In one experiment in mice, it induced histiocytic sarcomas and lymphomas in both males and females and benign ovarian tumours in females. In an experiment in mice lacking one allele of the p53 tumour suppressor gene, it increased the incidence of lymphomas. This result was confirmed in a separate study reported as an abstract. It induced benign renal tumours in male rats and benign phaeochromocytomas in males and females.

5.4. Other relevant data

Phenolphthalein is absorbed in the small bowel and is conjugated in the liver to form phenolphthalein glucuronide, which is eliminated in the bile. As it passes through the small intestine, it is partially deconjugated and reabsorbed.

Phenolphthalein and its glucuronide enhance oxygen radical production and cause oxidative damage in vitro. Phenolphthalein has also been shown to have low oestrogenic activity in some model systems. Phenolphthalein induced micronucleated erythrocytes in mice given multiple but not single treatments by gavage or in feed. Abnormal spermatozoa were induced in male mice but not male rats treated with phenolphthalein in the feed for 13 weeks. The malignant thymic lymphomas induced by phenolphthalein in female heterozygous p53-deficient mice showed loss of the normal p53 allele.

Phenolphthalein induced chromosomal aberrations, Hprt gene mutations and morphological transformation but not aneuploidy or ouabain-resistant mutations or sister chromatid exchange in cultured mammalian cells. It did not induce gene mutations in bacteria.

5.5. Evaluation

There is inadequate evidence in humans for the carcinogenicity of phenolphthalein.

There is sufficient evidence in experimental animals for the carcinogenicity of phenolphthalein.

Overall evaluation

Phenolphthalein is possibly carcinogenic to humans (Group 2B).

6. References

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1.1. Chemical and physical data

1.1.1. Nomenclature, structural and molecular formulae and relative molecular

Vitamin K (generic)

• Chem. Abstr. Serv. Reg. No.: 12001-79-5
• Chem. Abstr. Name: Vitamin K

Vitamin K₁ (generic)

• Chem. Abstr. Serv. Reg. No.: 11104-38-4
• Chem. Abstr. Name: Vitamin K₁

Phylloquinone

• Chem. Abstr. Serv. Reg. No.: 84-80-0
• Deleted CAS Reg. Nos.: 10485-69-5; 15973-57-6; 50926-17-5
• Chem. Abstr. Name: 2-Methyl-3-[(2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecenyl]-1,4-naphthalenedione
• IUPAC Systematic Name: [R-[R*,R*-(E)]]-2-Methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione
• Synonyms: Antihaemorrhagic vitamin; 2-methyl-3-phytyl-1,4-naphthoquinone; 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione; α-phylloquinone; trans-phylloquinone; phylloquinone K₁; phytomenadione; phytonadione; phytylmenadione; 3-phytylmenadione; phytylmenaquinone; vitamin K₁; vitamin K₁₍₂₀₎; 2′,3′-trans-vitamin K₁ [Note: The IUPAC recommends use of the name ‘phylloquinone’ and the abbreviation ‘K’ (rather than ‘K₁’). Both phylloquinone and vitamin K₁ are in common use. The United States Pharmacopeia uses the name ‘phytonadione’; The European Pharmacopoeia uses the name ‘phytomenadione’, which is a synonym occasionally found in the pharmaceutical and pharmacological literature.]

Menaquinone-4

• Chem. Abstr. Serv. Reg. No.: 863-61-6
• Deleted CAS Reg. Nos.: 15261-37-7; 20977-31-5; 39776-41-5
• Chem. Abstr. Name: 2-Methyl-3-[(2E,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl]-1,4-naphthalenedione
• IUPAC Systematic Name: 2-Methyl-3-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-1,4-naphthoquinone
• Synonyms: Menaquinone-K4; menatetrenone; (E,E,E)-2-methyl-3-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl]-1,4-naphthalenedione; MK4; vitamin K₂₍₂₀₎; vitamin MK4

Vitamin K₂ (generic)

• Chem. Abstr. Serv. Reg. No.: 11032-49-8
• Chem. Abstr. Name: Vitamin K₂

Menadione

• Chem. Abstr. Serv. Reg. No.: 58-27-5
• Chem. Abstr. Name: 1,4-Naphthalenedione, 2-methyl-
• IUPAC Systematic Name: 1,4-Naphthoquinone, 2-methyl-
• Synonyms: 1,4-Dihydro-1,4-dioxo-2-methylnaphthalene; 2-methyl-1,4-naphthalenedione; 2-methylnaphthoquinone; β-methyl-1,4-naphthoquinone; 2-methyl-1,4-naphthoquinone; 3-methyl-1,4-naphthoquinone; MK-0; vitamin K₀; vitamin K₂₍₀₎; vitamin K₃ [Note: ‘Menadione’ is the common name preferred by IUPAC for the chemical, previously called vitamin K₃]

Menadione sodium bisulfite

• Chem. Abstr. Serv. Reg. No.: 130-37-0
• Alternate CAS Reg. No.: 57414-02-5
• Deleted CAS Reg. Nos.: 8012-53-1; 8017-97-8; 8028-24-8; 8053-08-5
• Chem. Abstr. Name: 1,2,3,4-Tetrahydro-2-methyl-1,4-dioxo-2-naphthalenesulfonic acid, sodium salt
• IUPAC Systematic Name: 1,2,3,4-Tetrahydro-2-methyl-1,4-dioxo-2-naphthalenesulfonic acid, sodium salt
• Synonyms: 3,3-Dihydro-2-methyl-1,4-naphthoquinone-2-sulfonate sodium; menadione sodium hydrogen sulfite; menaphthone sodium bisulfite; menaphthone sodium bisulphite; 2-methyl-1,4-naphthalenedione, sodium bisulfite deriv.; 2-methyl-1,4-naphthoquinone sodium bisulfite; 2-methylnaphthoquinone sodium hydrogen sulfite; 2-methyl-1,4-naphthoquinone sodium hydrogen sulfite; MSBC; sodium menadione bisulfite; vitamin K injection; vitamin K₃ sodium bisulfite

Menadione sodium bisulfite trihydrate

• Chem. Abstr. Serv. Reg. No.: 6147-37-1
• Chem. Abstr. Name: 1,2,3,4-Tetrahydro-2-methyl-1,4-dioxo-2-naphthalenesulfonic acid, sodium salt, trihydrate

Menadiol

• Chem. Abstr. Serv. Reg. No.: 481-85-6
• Chem. Abstr. Name: 2-Methyl-1,4-naphthalenediol
• IUPAC Systematic Name: 2-Methyl-1,4-naphthalenediol
• Synonyms: Dihydrovitamin K₃; menaquinol; 2-methyl-1,4-dihydroxynaphthalene; 2-methylhydronaphthoquinone; 2-methylnaphthalene-1,4-diol; 2-methyl-1,4-naphthohydroquinone; 2-methyl-1,4-naphthoquinol; reduced menadione; reduced vitamin K₃; vitamin K₃H₂

Menadiol sodium phosphate

• Chem. Abstr. Serv. Reg. No.: 131-13-5
• Chem. Abstr. Name: 2-Methyl-1,4-naphthalenediol, bis(dihydrogen phosphate), tetrasodium salt
• IUPAC Systematic Name: 2-Methyl-1,4-naphthalenediol, diphosphate, tetrasodium salt
• Synonyms: Menadiol diphosphate tetrasodium salt; menadiol sodium diphosphate; menadiol tetrasodium diphosphate; menadione diphosphate tetrasodium salt; 2-methyl-1,4-naphthoquinol bis(disodium phosphate); tetrasodium 2-methyl-1,4-naphthalenediol bis(dihydrogen phosphate)

Menadiol sodium phosphate hexahydrate

• Chem. Abstr. Serv. Reg. No.: 6700-42-1
• Chem. Abstr. Name: 2-Methyl-1,4-naphthalenediol, bis(dihydrogen phosphate), tetrasodium salt, hexahydrate
• IUPAC Systematic Name: 2-Methyl-1,4-naphthalenediol, diphosphate, tetrasodium salt, hexahydrate
• Synonyms: Menadiol sodium diphosphate hexahydrate

Acetomenaphthone

• Chem. Abstr. Serv. Reg. No.: 573-20-6
• Chem. Abstr. Name: 2-Methyl-1,4-naphthalenediol, diacetate
• IUPAC Systematic Name: 2-Methyl-1,4-naphthalenediol, diacetate
• Synonyms: 1,4-Diacetoxy-2-methylnaphthalene; menadiol diacetate; 2-methyl-1,4-naphthohydroquinone diacetate; 2-methyl-1,4-naphthoquinol diacetate; 2-methyl-1,4-naphthylene diacetate; vitamin K diacetate; vitamin K₄

IUPAC recommends that 2-methyl-3-polyprenyl-1,4-naphthoquinone be referred to as menaquinone-n, previously vitamin K₂, n being the number of prenyl residues. Vitamin K₂₍₂₀₎ is so named because it contains 20 carbon atoms in the chain. In the biological literature, vitamin K₂ is frequently referred to as menaquinone and is further designated by the number of isoprene units in the side-chain. For example, vitamin K₂₍₂₀₎ is also called menaquinone-4 for the four isoprene units in the side-chain. The compound originally isolated from rotting fish meal and named vitamin K₂ was later identified as menaquinone-7 (2-methyl-3-farnesylgeranyl-geranyl-1,4-naphthoquinone). In the older literature, the designation vitamin K₂₍₃₅₎ is used for menaquinone-7, but this is no longer used. Menaquinones found in nature have side-chains of 4–13 isoprenoid residues and are usually in the all-trans configuration; however, menaquinones with the cis configuration and partially saturated side-chains also exist (Suttie, 1985, 1991; Weber & Rüttimann, 1996; Van Arnum, 1998).

1.2. Production

Although the predominant commercial form of phylloquinone is the synthetic race-mate, natural phylloquinone is accessible either by extraction from a natural source or from condensation of menadione with natural phytol. The stability of phylloquinone to heat made possible the use of commercially dehydrated alfalfa meal, for example, as a natural source (Hassan et al., 1988). The synthesis and spectral properties of all four stereoisomers of (E)-phylloquinone have been described and their biological potencies determined. When natural phylloquinone was used as a standard in bioassays, it was concluded that all four stereoisomers have essentially identical activity (Van Arnum, 1998).

The first syntheses and structural elucidation of phylloquinone were published in 1939 almost simultaneously by four groups. The starting materials were menadione or menadiol as the aromatic component and natural phytol or one of its derivatives. A breakthrough in commercial synthesis was achieved in the 1950s, when it was found that monoacylated menadiols (e.g. the monoacetate or the monobenzoate) could be used advantageously in the alkylation step and that natural phytol could be replaced by isophytol, which is easy to synthesize (Weber & Rüttimann, 1996).

In the Isler-Lindlar method, excess menadiol monobenzoate is condensed with isophytol in the presence of boron trifluoride etherate as a catalyst. The alkylation product is obtained as a 70:30 trans/cis mixture. The trans form can be enriched by recrystallization. The trans-enriched alkylation product (trans:cis 9:1) is saponified with potassium hydroxide and oxidized to phylloquinone with oxygen (Weber & Rüttimann, 1996).

The industrial synthesis of menaquinones parallels that of phylloquinone and involves as a key step alkylation of monosubstituted menadione with an appropriate (all-trans) polyisoprenyl derivative. Considerably more work has been done on fermentative approaches to menaquinones than for phylloquinone. Menaquinones of varying chain lengths, from C₅ to C₆₅, have been produced and isolated from bacteria. Menaquinone-4 is produced and used extensively in Japan (Van Arnum, 1998).

Menadione can be prepared by oxidizing 2-methylnaphthalene with chromic acid or hydrogen peroxide (Weber & Rüttimann, 1996). A process based on biotechnological techniques has been reported in Japan (Van Arnum, 1998).

Menadione sodium bisulfite can be prepared by reacting menadione with sodium bisulfite. The reaction may be visualized as consisting of the typical addition of sodium bisulfite to a ketone, forming the R(OH)(SO₃Na) compound, which then rearranges at the expense of one degree of unsaturation of the quinoid nucleus. The compound readily regenerates menadione on treatment with mild alkali and behaves as a typical ketone–sodium bisulfite addition compound (Gennaro, 1985; Van Arnum, 1998).

Menadiol sodium phosphate can be prepared by reducing menadione to the diol, followed by double esterification with hydriodic acid, metathesis of the resulting 1,4-diiodo compound with silver phosphate and neutralization of the bis(dihydrogen phosphate) ester with sodium hydroxide (Gennaro, 1995).

Information available in 1999 indicated that phylloquinone was manufactured and/or formulated in 41 countries, menadione in 26 countries, menadione sodium bisulfite in 21 countries, menadiol and menadiol sodium phosphate (as the hexahydrate) in two countries each and acetomenaphthone in seven countries (CIS Information Services, 1998; Royal Pharmaceutical Society of Great Britain; 1999; Swiss Pharmaceutical Society, 1999).

1.3. Use

1.3.2. Supplementation and therapy

Vitamin K is given as a supplement to prevent or cure vitamin K deficiency when the endogenous vitamin K supply from the diet is likely to become or has proven to be insufficient. Neonates are born with very limited vitamin K stores, but most infants do not show relevant hypoprothrombinaemia at birth (von Kries et al., 1987a, 1988; von Kries, 1991). Biochemical signs of vitamin K deficiency are common during the first week of life, however, unless sufficient amounts of vitamin K are ingested. The natural diet of newborns is human milk, which contains vitamin K at concentrations of 0.69–9.2 ng/mL (see Table 1). [The Working Group noted that some of the high values in the Table may reflect methodological problems with analysis and milk collection.] Bleeding, the classical clinical manifestation of vitamin K deficiency, is extremely rare on the first day of life, and the typical time of onset is during the first week, with bleeding from mucous membranes, the umbilicus, following circumcision, and rarely, into the central nervous system (von Kries et al., 1988; von Kries, 1991). This condition was originally called ‘classical haemorrhagic disease of the newborn’; the present nomenclature is ‘classical vitamin K deficiency bleeding’ (Sutor et al., 1999).

Table 1

Concentrations of phylloquinone in human and cow’s milk, infant formulae and various oils.

During the first three months of life, exclusively breast-fed infants remain at risk for vitamin K deficiency bleeding. In many of these infants, the bleeding episode, which is often intracranial haemorrhage, is the first perceived symptom of an underlying cholestatic disease. In 10–30% of the cases, however, no underlying disease can be found (von Kries et al., 1988).

After the first three months of life, vitamin K deficiency is almost completely confined to patients with cholestatic diseases (congenital or acquired obstruction of the bile duct), malabsorption syndromes or cystic fibrosis (Houwen et al., 1987; van den Anker & Sinaasappel, 1993; O’Brien et al., 1994; Kowdley et al., 1997; Nowak et al., 1997; see also section 1.3.4).

1.4. Occurrence

Phylloquinone is widely distributed in higher plants and in some blue–green algae. It is present in many foods, especially leafy green vegetables and some vegetable oils. Table 2 shows the concentrations in some common foods (Booth et al., 1995; Shearer et al., 1996; Booth & Suttie, 1998).

Table 2

Phylloquinone content of common foods.

The Total Diet Study of the US Food and Drug Administration is conducted periodically to monitor the safety and nutritional quality of the US food supply by assessing the levels of nutrients and contaminants in daily diets. It is based on the collection and analysis of 265 core foods. Intakes are estimated from the concentrations of individual nutrients and contaminants in the core foods and the mean consumption of the foods in each population group. The quantitative contributions of specific foods to the phylloquinone intake of the total population are presented in Table 3. Table 4 gives the estimated daily intake in 1990 for 14 categories of age and sex (Booth et al., 1996).

Table 3

Contribution of certain food groups to total adult intake (%) of phylloquinone in the USA, stratified by age and sex.

Table 4

Estimated and recommended mean dietary intakes of phylloquinone in the USA, stratified by age and sex.

Phylloquinone has been determined by several analytical methods in human milk, in cow’s milk, in many brands of infant formula and in the oils that have been added to infant formulas for many years. Some of the concentrations found in each of these sources are presented in Table 1.

Menaquinones are synthesized by bacteria. They have a more restricted distribution in the diet than phylloquinone, and nutritionally significant amounts probably occur only in animal liver and some fermented foods, including cheese. Menaquinones are also synthesized by specific inhabitants of the human gut microflora. The major intestinal forms are MK-10 and MK-11 produced by Bacteroides, MK-8 by Enterobacteria, MK-7 by Veillonella genus and MK-6 by Eubacterium lentum (Shearer et al., 1996). The total concentration of menaquinones in human distal colonic contents is about 20 µg/g dry weight, with MK-10 predominating (Conly & Stein, 1992; Shearer, 1995). It seems likely that menaquinones synthesized by the gut microflora make a significant contribution to human tissue stores and are used by the hepatic vitamin K-dependent carboxylase, but the extent of this contribution remains uncertain (Shearer, 1995; Suttie, 1995).

1.5. Regulations and guidelines

Phylloquinone is listed (as phytomenadione or phytonadione) in the British, Chinese, Czech Republic, European, French, German, International, Japanese, Swiss and US pharmacopoeias (Royal Pharmaceutical Society of Great Britain, 1999; Swiss Pharmaceutical Society, 1999).

The Food and Drug Administration (1999) requires that all infant formulae sold in the USA contain a minimum of 4 µg/100 kcal (0.2 mg/kg) vitamin K; and that any vitamin K added should be in the form of phylloquinone.

Menadione is listed in the Austrian, Belgian, British, Dutch, European, French, German, International, Italian, Portuguese, Swiss and US pharmacopoieas, and menadione sodium bisulfite is listed in the Belgian, International, Swiss and US pharmacopoeias (Royal Pharmaceutical Society of Great Britain, 1999; Swiss Pharmaceutical Society, 1999). Menadiol sodium phosphate is listed in the British, Czech Republic and US pharmacopoeias (Swiss Pharmaceutical Society, 1999).

2.1. Case–control studies

In most of the case–control studies, the reference group comprised infants who had not received vitamin K and/or those who had received it orally. This combination is justified because the plasma concentrations after intramuscular administration are more than 10 times higher than those after oral administration (McNinch et al., 1985).

In a second study (Golding et al., 1992), 195 children with cancer diagnosed in the period 1971–91 who had been born at two major maternity hospitals in Bristol, England, in the period 1965–87 were compared with 558 controls identified from the delivery books of these hospitals. The cases were ascertained from the oncology register of the regional paediatric oncology unit and from the National Registry of Children’s Tumours. The basic method of control selection was to select every 300th birth in each year in each hospital. In view of the observation that the immediate effects of identical oral and intramuscular doses of vitamin K are different, the investigators sought to distinguish the effects of administration by the two routes. When the route of vitamin K administration was not recorded in the neonatal notes, a route was imputed on the basis of year of birth, the type of delivery and whether or not the infant was admitted to special care; the imputed route was identified in the absence of knowledge of case or control status. On the basis of 180 cases (92% of those for which notes were available) and 544 controls (98% of those for which notes were available), the relative risk (adjusted for hospital and year of delivery) for childhood cancer associated with intramuscular vitamin K was 2.2 (95% CI, 1.1–4.4) when compared with no vitamin K and 1.2 (95% CI, 0.5–2.7) for oral vitamin K. In view of the absence of an association with oral vitamin K in these data, the authors conducted a subsequent analysis in which the reference group was defined to include infants who had not received vitamin K or who had received it orally. The relative risk for leukaemia associated with intramuscular vitamin K was 2.7 (95% CI, 1.3–5.2) and that for other types of childhood cancer was 1.7 (95% CI, 1.0–2.8). Thus, there was no clear difference in the association by type of childhood cancer. When the analysis was confined to records in which the route was clearly stated, the odds ratio for all childhood cancer was 2.0 (95% CI, 1.2–3.3). These results could not be accounted for by other factors associated with the administration of intramuscular vitamin K, such as type of delivery or admission to a special care unit. Data were collected on 319 variables for all controls and for 111 cases of cancer ascertained from the oncology register of the regional paediatric oncology unit; these data were not obtained for the remaining 84 cancer cases. Of these variables, the presence of rubella antibody, resuscitation by intermittent positive pressure and paediatric estimate of gestation were statistically significant at the 1% level, which is what would be expected by chance. Adjustment for these and other variables reported to be associated with childhood cancer or known to be indicators for administering intramuscular vitamin K had little effect on the odds ratio for childhood cancer associated with vitamin K. Nineteen of the cases were diagnosed in the first year of life, and the possibility was considered that these cancers might have been present before the child was born and could therefore not have been initiated by an injection of vitamin K; however, the association persisted after exclusion of these 19 cases from the analysis. When the analysis was restricted to subjects who would have been followed for at least 10 years, by considering only those born in the period 1971–80, the relative risk for all childhood cancer associated with intramuscular vitamin K was 1.9 (95% CI, 1.1–3.4), similar to that assessed for all subjects. [The Working Group noted, as acknowledged by the authors, a large number of instances in which the information on potentially confounding variables was not available, for example on smoking in pregnancy. Medical records are not necessarily reliable sources of information about pregnancy and childbirth (Hewson & Bennett, 1987; Oakley et al., 1990), and this, together with the fact that potential confounding was assessed only for a subset of cases, constitutes a limitation of the study. The relationship between the type of delivery and intramuscular administration of vitamin K differed markedly between the two maternity hospitals in Bristol in which the case and control subjects in the study had been born (Carstensen, 1992; Draper & Stiller, 1992). The association with childhood cancer is largely accounted for by data from one of the hospitals in which virtually all of the control infants who received intramuscular vitamin K had been born by an assisted delivery. This raises the issue as to whether bias arose in control selection in that hospital.]

A study in the USA was reported by Klebanoff et al. (1993) which was based on follow-up to the age of seven or eight years of 54 795 liveborn children of women enrolled between 1959 and 1966 in 12 centres contributing to the National Collaborative Perinatal Project. Neonates whose cancer was diagnosed or strongly suspected during the first day of life were excluded because vitamin K could not have been a factor in those cases. Vitamin K was administered in the delivery room or the nursery, and information about the administration was recorded with other events during and after delivery by observers who were not involved in the clinical care of the mother or the infant. Cancer was diagnosed in 48 of 54 795 liveborn children after the first day of life. For each case, five controls were selected and matched with the index case on length of follow-up. In spite of the prospective recording by the observers, the data on vitamin K administration were not recorded unambiguously for 43 infants; a review of hospital records without knowledge of case or control status resulted in data for 25 (58%) of these. The exposure status was unknown for four case children. The relative risk for all childhood cancer associated with vitamin K was 0.84 (95% CI, 0.41–1.7), and that for leukaemia was 0.47 (95% CI, 0.14–1.6; based on 15 cases). In the USA, only two brands, Aquamephyton and Konakion, have been approved for use (see Table 6). Konakion in the USA contains polysorbate-80 rather than Cremophor EL as an emulsifier and phenol as an antrimicrobial agent. In the study of Klebanoff et al. (1993), the relative risk for total childhood cancer associated with the two brands together was 0.6, whereas that for children who had received the phenol-containing preparation alone was 0.7. In this study, only one child had received vitamin K orally.

von Kries et al. (1996) carried out a case–control study of children born in 162 obstetric hospitals in Lower Saxony (Germany) during the period 1975–93 when only one vitamin K preparation, Konakion, the same as that used in the United Kingdom, was licensed for neonatal vitamin K prophylaxis. Of a total of 218 children with leukaemia identified as eligible, information on vitamin K prophylaxis was obtained for 136 (62%). For each leukaemia case, one control was selected from the municipality where the patient lived at the time of diagnosis (local control), and a second one (state control) from a municipality selected at random in Lower Saxony by means of a population-weighted sampling scheme. These controls were matched with cases by sex and date of birth. Case and control families were contacted initially by being sent a questionnaire. If a control family refused to collaborate in the study or did not return the questionnaire within three months, another control family was invited; control families that returned the questionnaire after more than three months were also included. Thus, a total of 305 local and 308 state controls were invited to participate. Information on vitamin K prophylaxis was obtained for 174 (57%) of the local controls and 160 (52%) of the state controls. As the study was performed as part of a population-based case–control study to explore possible causes of childhood leukaemia in Lower Saxony, a third control group for the leukaemia study was identified which comprised cases of brain tumours, nephroblastoma, neuroblastoma and rhabdomyosarcoma. No population-based controls were selected for these cases, but they were used as additional cases in the study of vitamin K. Of a total of 246 potentially eligible cases of this type, information on vitamin K prophylaxis was obtained for 136 (55%). Data on vitamin K prophylaxis were abstracted from the birth report with no knowledge of the case or control status of each child. Information on the dose and route of vitamin K prophylaxis was obtained from the birth record or in the delivery book for 72% of the 272 cases of leukaemia and other cancers and 64% of the 334 controls. When this information was not available, the index child was assumed to have had the same exposure to vitamin K as the child nearest to the index infant in the delivery book with the same route of delivery and same perinatal morbidity (nine cases and six controls). When this could not be established, staff who worked in the delivery unit at the time when the index child was born were asked what kind of vitamin K prophylaxis the index infant would have received, given the birth weight and route of delivery (63 cases and 109 controls). Finally, similar information was sought from medical staff who did not work in the delivery unit at the time the index child was born (four cases and four controls). In the comparison with local controls (n = 107), the risk for leukaemia (n = 107) associated with intramuscular or subcutaneous administration of vitamin K relative to that for oral or no vitamin K prophylaxis was 1.2 (95% CI, 0.68–2.25). In the comparison with state controls (n = 160; leukaemia cases = 136), the relative risk was 0.82 (95% CI, 0.50–1.4). When the control groups were pooled (n = 334), the relative risk was close to unity (136 leukaemia cases), and the relative risk for brain tumours, nephroblastoma, neuroblastoma and rhabdomyosarcoma combined (n = 136) associated with vitamin K prophylaxis was 1.2 (95% CI, 0.77–1.8). When the analyses were repeated for subjects for whom vitamin K prophylaxis had been documented in birth records or delivery books, the results were almost unchanged, except in the comparison of leukaemia cases with local controls, which gave a relative risk of 2.0 (95% CI, 0.69–6.0). When the analyses were repeated for parenteral prophylaxis versus no prophylaxis, most of the relative risks were slightly decreased. The risk of the subgroup of cases of leukaemia in children aged 1–6 years was analysed as this was considered to be a relatively homogeneous subgroup, most of the cases having common acute lymphoblastic leukaemia. [The Working Group noted that it is not clear whether the decision to make this subgroup analysis was specified in the original study protocol or was made post hoc.] The risk relative to both control groups combined was 1.2 (95% CI, 0.69–2.15), in the comparison with state controls it was 0.99 (95% CI, 0.52–1.9) and in the comparison with local controls it was 2.3 (95% CI, 0.94–5.5). There was no difference between cases and controls in the source of information on vitamin K prophylaxis. The increased relative risk in the comparison with local controls could not be explained by any of the potential confounders. It would be expected that the policy of administration of vitamin K would be more likely to be similar for cases and local controls than for cases and state controls. Therefore, the relative risk would be expected to be closer to unity in the comparison between cases and local controls than in the other comparison, whereas the opposite was observed. The non-significantly increased risk relative to local controls may be a chance result in subgroup analysis with multiple testing, as acknowledged by the authors.

In a case–control study of childhood leukaemia based on births in three hospitals in England (Cambridge, Oxford and Reading), no association with intramuscular vitamin K, either as determined from hospital records (91 cases, 171 controls) or as imputed from hospital policy (132 cases, 264 controls), was found. In addition, no association was found specifically for acute lymphoblastic leukaemia (Ansell et al. 1996). Subsequently, Roman et al. (1997) reported a more detailed analysis of data on leukaemia and non-Hodgkin lymphoma diagnosed before the age of 30 years in subjects whose obstetric records were stored in the same three hospitals. Ninety-two per cent (132/143) of the cases of leukaemia were diagnosed at age 14 or less; these cases and their controls were included in the report of Ansell et al. (1996). There was no association between leukaemia and intramuscular vitamin K administration either recorded in the notes (relative risk, 1.2; 95% CI, 0.7–2.1) or imputed from information about hospital policy (relative risk, 1.2; 95% CI, 0.5–2.4). In view of the finding of von Kries et al. (1996), acute lymphoblastic leukaemia diagnosed between the ages of 1–6 years was considered; the relative risk associated with recorded administration (based on hospital notes) was 0.6 (95% CI, 0.3–1.4), and that based on hospital policy was again 0.6 (95% CI, 0.2–1.7).

Parker et al. (1998) identified 1432 children born in northern England between 1960 and 1991 from the regional Children’s Malignant Disease Registry, in whom cancer was diagnosed in 1968–92 when they were aged between three months and 14 years while still resident in the region. The birth records of 701 of these children could not be traced, usually because the maternity unit had retained only its most recent records or because the unit had closed and the records could not be located. Thirty children who had been given vitamin K orally at birth and 16 cases in multiple births were excluded. The controls were selected by taking the fourth, eighth and 12th birth before and after the index birth from birth or admission registers for the hospital of birth of the index child. Towards the end of the study, the number of controls per case was reduced from six to three because of time constraints. When the birth notes for control children could not be located, or when the child selected was found to be on the Malignant Disease Register, the next possible control was selected. The fact of intramuscular administration of vitamin K or non-administration of vitamin K was recorded in the maternity unit records for 438 of 685 cases (case notes). [The Working Group noted that the corresponding proportion for controls was not specified.] There was no association between intramuscular vitamin K administration and either all cancers or all cancers other than acute lymphoblastic leukaemia. The relative risk for acute lymphoblastic leukaemia associated with vitamin K administration based on case notes was 1.4 (95% CI, 0.71–1.7; 132 cases). Two secondary analyses were conducted to consider cases typical of the peak incidence of leukaemia in early childhood. When the 51 children in the case note analysis who had T-cell leukaemia or for whom subtype characterization was not available were excluded, the relative risk for the 81 cases of non-T-cell lymphoblastic leukaemia was 1.8 (95% CI, 0.82–3.9). In an analysis of 94 children aged 1–6 years at diagnosis, the relative risk was 2.3 (95% CI, 0.98–5.2). In all of these analyses, adjusted relative risks were calculated separately for the specified potential confounding factors—sex, gestation, birth weight, opiates during labour, assisted delivery, signs of asphyxia at birth, admission to special care or neonatal blood transfusions. Except for adjustment for assisted delivery, admission to special care or opiate exposure in labour, none of these changed any of the relative risks by more than 10%. Adjustment for assisted delivery or admission to special care caused a larger rise in the relative risk. The relative risk for acute lymphoblastic leukaemia diagnosed at ages 1–6 was 2.4 (95% CI, 1.0–5.7) after adjustment for exposure to opiates and 3.6 (95% CI, 1.3–9.7) after adjustment for assisted delivery based on case note analysis. As in many of the other studies, information on hospital policy was obtained in order to impute exposure when this was unclear from medical records. This information was obtained by a research midwife and neonatal staff in each unit in the region and by a paediatrician from current and recently retired medical staff, and this independently obtained information was then cross-validated. When inconsistencies were identified, the case notes were sampled to determine what policy had actually been followed. This enabled a further 226 cases to be included at the analysis; 21 cases were excluded because the policy of the local unit could not be ascertained. The relative risks were similar to but somewhat lower than those in the analysis based exclusively on subjects for whom data on vitamin K exposure was obtained only from medical records. [The Working Group noted that it was unclear which hypotheses about subgroups had been pre-specified. Bias may have arisen from the fact that while a large proportion of cases had to be excluded there was a mechanism for adding controls when a control record was unobtainable. Availability of records might have associations with both perinatal health problems and subsequent development of childhood cancer.]

McKinney et al. (1998) carried out a case–control study on childhood cancer in Scotland using data abstracted from 76 hospital records. A total of 500 cases of cancer diagnosed in children aged 0–14 years during the period 1991–94 while resident in Scotland were identified. Controls matched on age, sex and health board of residence were randomly selected from among all eligible children registered for primary care within each health board. A total of 1338 eligible controls was identified. A total of 460 mothers of cases (92%) and 861 mothers of controls (64%) were interviewed, and medical notes were abstracted for 440 cases and 802 controls. The data set for statistical analysis was restricted to matched sets, and information was lost for 23 cases without matched controls and 25 controls without a matched case. Therefore, 417 cases and 777 controls were included in the matched case–control analysis. Vitamin K was recorded as given or definitely not given only when this was mentioned in the notes. Similarly, the route of administration was classified as intramuscular, oral or not recorded. None of the relative risks reported for leukaemias, acute lymphoblastic leukaemia, lymphomas, central nervous system tumours or other solid tumours, either crude or adjusted for social class and type of delivery, was statistically significantly different from unity. The adjusted relative risk for leukaemia associated with vitamin K given intramuscularly (recorded) in the neonatal period was 1.2 (95% CI, 0.77–2.0) and that for acute lymphoblastic leukaemia was 1.2 (95% CI, 0.70–2.0). In view of the findings of Parker et al. (1998, see above), the subset of acute lymphoblastic leukaemia diagnosed in children aged 1–6 years (90 cases, 174 controls) was also analysed, and the adjusted relative risk was found to be 1.2 (95% CI, 0.62–2.2). As nothing about vitamin K had been written in the medical records for a substantial proportion of children (37% of cases and 35% of controls), the authors also sought to impute exposure on the basis of hospital policies. Information on the vitamin K policies of hospitals in which over 500 infants were delivered annually was validated by abstraction of a sample of medical records and through consultations with hospital pharmacies and senior labour room midwives. For 100 (24%) cases and 191 (25%) controls, no hospital policy was available for any imputation. The relative risks for the specific diagnostic categories associated with intramuscular vitamin K administration in the neonatal period either as recorded in medical records or imputed from hospital policy were very similar to those calculated for subjects for whom only data from medical records were included. The adjusted relative risk for leukaemia was 1.3 (95% CI, 0.78–2.1), that for acute lymphoblastic leukaemia was 1.1 (95% CI, 0.65–1.9) and that for acute lymphoblastic leukaemia in children aged 1–6 years was 1.3 (95% CI, 0.70–2.5). Very few subjects were recorded as having or imputed to have been given vitamin K orally in the neonatal period (12 cases, 2.9%; and 33 controls, 4.3%).

Passmore et al. (1998a) identified cases of childhood cancer diagnosed at ages up to 14 years in persons who were resident in Great Britain and had been born in 16 hospitals with large maternity units in 1968 or later and diagnosed by the end of 1986 from the National Registry of Childhood Tumours (excluding retinoblastoma, Down syndrome or neurofibromatosis). The 16 hospitals were selected on the basis of a survey which showed that they had a selective policy for the use of vitamin K prophylaxis. Of 1092 cases initially identified as born in these hospitals, 523 were born in the years for which a policy was known and for whom the medical records were found. Four controls matched on sex, month of birth and hospital of birth were selected randomly from these registers. Medical records departments were asked to locate the records for each case and for one control. Initially, two out of each of the four potentially eligible controls were selected randomly for location by the medical records department. If the records department was unable to locate the notes of either of these, details were supplied of the other two. Controls with illegible records, twins, stillbirths and neonatal deaths were excluded. In addition, infants with severe neural tube defects or a birth weight of less than 1000 g were excluded, as they were unlikely to have survived to the age at which the case patient developed cancer. For these, an alternative control was selected by using the next suitable birth in the hospital birth register. [The numbers of control replacements were not specified.] A second group of cases from the same period was chosen from records of the National Registry of Childhood Tumours in order to identify cases of cancer among children included in a survey of more than 100 000 births in South Glamorgan, Wales. For each case, two controls matched for sex, month of birth and hospital were selected, applying the same set of exclusions. Medical records were sought for all cases and controls, and information on vitamin K administration taken from these records was supplemented by data from the birth survey, which was available for most but not all of the period of study. This added three further hospitals to the study, all of which had selective policies of vitamin K administration, and 74 cases. In the combined data (16 maternity units in England and Wales and the three hospitals included in the survey in South Glamorgan), the relative risk for childhood cancer of all types associated with intramuscular vitamin K administration was 1.4 (95% CI, 1.0–2.1). In the data for the 16 maternity units in England and Wales, the relative risk was 1.2 (95% CI, 0.77–1.9), while in the data from South Glamorgan, the relative risk was 2.1 (95% CI, 1.1–4.1). For the combined data and for the data from South Glamorgan, mode of delivery (forceps, vacuum extraction, breech or caesarean) was a statistically significant confounding variable, and adjustment for this reduced the relative risks to 1.1 for the combined data and 1.3 for the South Glamorgan data. In the combined data, the relative risk for leukaemia was 1.5 (95% CI, 0.82–2.85), that for acute lymphoblastic leukaemia was 1.7 (95% CI, 0.89–3.3) and that for acute lymphoblastic leukaemia diagnosed at ages 1–5 years was 1.0 (95% CI, 0.48–2.2). Again, adjustment for mode of delivery reduced the relative risks. [The Working Group noted that the substantially lower relative risk for the 1–5 year-old group than for all ages combined implies that the effect for children of other ages is higher than that for this group, in contrast to the observations of von Kries et al. (1996) and Parker et al. (1998).] The relative risk for nonleukaemia cancers was 1.4 (95% CI, 0.88–2.2) in the combined data and 2.4 (95% CI, 1.1–5.4) in the data from South Glamorgan. In the South Glamorgan data, none of the potential confounders that were adjusted for reduced the magnitude of the relative risk. [The Working Group noted that in the absence of an effect in the data from the 16 maternity units in England and Wales, the South Glamorgan finding may reflect an unidentified bias or be a chance finding.]

[The Working Group noted that in the subgroup analyses of acute lymphoblastic leukaemia diagnosed at 1–6 years carried out by Parker et al. (1998) and 1–5 years by Passmore et al. (1998a), adjustment for mode of delivery had contrasting effects. In the study of Passmore et al. it attenuated the relative risk associated with vitamin K, while in the study of Parker et al. the relative risk was increased.]

2.2. Ecological studies

These studies are summarized in Table 7.

Table 7

Ecological studies on childhood cancer and vitamin K administered intramuscularly during the perinatal period as Konakiana.

Ekelund et al. (1993) investigated the association between childhood cancer and intramuscular administration of vitamin K in a study in Sweden based on linkage of the medical birth registry to the national cancer registry. The study was restricted to fullterm infants (gestation, 37–42 weeks) who had survived and who were born in 1973–89 after a delivery without use of forceps or vacuum extraction. The infants were followed up to 1 January 1992. Cancers diagnosed within 30 days of birth were regarded as congenital and were excluded from the analysis. Routines for administration of vitamin K were obtained from all 95 maternity hospitals and validated for a subset of 102 children with cancer and 100 control children randomly selected from among those who, according to the information on routine exposure, received intramuscular vitamin K, and 94 children with cancer and 100 control children from among those who should have received oral vitamin K. The doses of vitamin K given in Sweden were similar to those given in the United Kingdom, and the same preparation was used (phylloquinone, Konakion, see Table 6). When the method of administration of vitamin K was recorded, it agreed with the stated routine method of administration in 92% of the 235 cases for which individual information could be found. The relative risk for all childhood cancer associated with a hospital policy of intramuscular administration of vitamin K as compared with oral administration was 1.0 (95% CI, 0.88–1.2, after stratification for year of birth). The relative risk for leukaemia was 0.90 (95% CI, 0.70–1.2).

Olsen et al. (1994) compared the cumulative risk of childhood cancer among children aged 1–15 years who were born during the period 1945–54 (n = 835 430), in which no vitamin K was administered, those aged 1–15 years born during the period 1960–69 (n = 797 472), in which pregnant women received oral vitamin K, and those aged 1–13 years born during the period 1975–84 (n = 586 378), in which virtually all newborns received vitamin K intramuscularly. There was a small increase in risk for all tumour types combined, due mainly to lymphoma in boys and neuroblastoma in boys and girls. There was no trend for childhood leukaemia. The preparation was the same as that used in the United Kingdom (Draper & McNinch, 1994).

In addition to the case–control study in northern England described above, Parker et al. (1998) compared the incidence of acute lymphoblastic leukaemia diagnosed in children aged up to 14 years who were born in hospital units in which all infants received vitamin K, with those born in units where less than a third received this prophylaxis. As described above, information on hospital policy was obtained separately and independently by two people and then cross-validated. In units with a policy of selective prophylaxis, less than 30% of infants received intramuscular vitamin K at birth, while in units offering universal prophylaxis, sampling of case notes showed that more than 95% of babies received vitamin K. The risk for acute lymphoblastic leukaemia in children born in hospitals with a policy of universal prophylaxis relative to those born in hospitals with a policy of selective prophylaxis was 0.95 (95% CI, 0.78–1.2). The relative risk of the subgroup diagnosed at 1–6 years was 1.05 (95% CI, 0.82–1.35). [The Working Group noted that the cases included in this analysis overlapped with those included in the case–control study, so that the results are not independent].

Passmore et al. (1998b) carried out a similar comparison of cancers of all types other than retinoblastoma or associated with Down syndrome or neurofibromatosis diagnosed in children aged 1–14 years who were born in 94 hospital units in Great Britain. Information on hospital policy for neonatal vitamin K was obtained during the case–control studies of Passmore et. al. (1998a) and Ansell et al. (1996), described above, for 30 hospitals in Scotland from members of the Scottish Neonatal Network and from paediatricians for 41 of a further 80 hospitals in England and Wales in which more than 25 children who subsequently developed cancer had been born in the period 1968–85. The observed numbers of cases in hospitals with universal and selective policies were compared with the numbers expected on the basis of national rates. Separate analyses were carried out for births in hospitals that followed one policy throughout the period of study and births in hospitals in which the policy changed during the period of study. A large number of observed:expected ratios were calculated. The ratio for all cancers was 0.97, that for leukaemia at 1–14 years was 1.03, and that for acute lymphoblastic leukaemia at 1–5 years was 1.01 for hospitals with a consistent, non-selective policy. The ratio tended to be smaller in hospitals with a selective policy than in those offering universal prophylaxis. The only statistically significant (p < 0.05, two-tailed test) departure from unity indicated a lower risk for cancer other than leukaemia among children born in hospitals offering universal prophylaxis that those born in hospitals consistently offering selective prophylaxis in Scotland. [The Working Group noted that the cases included in this analysis overlapped with those in the case–control studies of Parker et al. (1998) and Ansell et al. (1996), so that the results are not independent.]

4.1. Absorption, distribution, metabolism and excretion

4.1.1. Humans

(a) Intestinal absorption and plasma transport in adults

The major dietary form of vitamin K is phylloquinone (Shearer et al., 1996). It is absorbed chemically unchanged from the proximal intestine after solubilization into mixed micelles composed of bile salts and the products of pancreatic lipolysis. In healthy adults, the efficiency of absorption of phylloquinone in its free form is about 80% (Shearer et al., 1974), but the efficiency of absorption from green leafy vegetables such as spinach is < 10% (Gijsbers et al., 1996).

Within the intestinal mucosa, phylloquinone is incorporated into chylomicrons, is secreted into the lymph and enters the blood via the lacteals (Shearer et al., 1970, 1974). After a phylloquinone-containing meal, the plasma concentration peaks between 3 and 6 h (Shearer et al., 1970; Lamon-Fava et al., 1998). Once in the circulation, phylloquinone is rapidly cleared at a rate consistent with its continuing association with chylomicrons and the chylomicron remnants that are produced by lipoprotein lipase hydrolysis at the surface of capillary endothelial cells. During the postprandial phase and after an overnight fast, more than half of the circulating phylloquinone is associated with triglyceride-rich lipoproteins, and the remainder is carried by low-density and high-density lipoproteins (Kohlmeier et al., 1996; Lamon-Fava et al., 1998). Although phylloquinone is the major circulating form of vitamin K, menaquinone-7 is present in plasma at lower concentrations and has a similar lipoprotein distribution to phylloquinone. While phylloquinone in blood is derived exclusively from the diet, it is not known what proportion of circulating menaquinones such as menaquinone-7 derives from the diet or the intestinal flora (Shearer et al., 1996).

(b) Plasma pharmacokinetics of phylloquinone in adults

The plasma clearance of an intravenous dose of 1 mg [³H]phylloquinone during the first 6 h resolved approximately into two exponential functions, the first with a half-time of 20–24 min and the second with a half-time of 121–150 min (Shearer et al., 1972). The curves for clearance up to 12 h after an intravenous injection of a 10-mg dose of phylloquinone (Konakion MM) were similar to those after 1 mg and were consistent with a two-compartment (sometimes three-compartment) model in which the log-linear terminal phase over 3–12 h had a half-time of about 3 h (Soedirman et al., 1996). A gradual slowing of the clearance rate was seen after the first 6 h (Shearer et al., 1972, 1974), as was also found in a study of the clearance of pharmacological doses of 10–60 mg by Øie et al. (1988), who reported that the log-linear terminal elimination phase was not reached before 8–12 h and that the average half-time was 14 h (range, 8–22 h). This slowing of the clearance rate may be explained by the complexity of the plasma transport of phylloquinone, in which the proportion of phylloquinone associated with low-density and high-density lipoproteins increases progressively (Lamon-Fava et al., 1998).

The plasma disposition of oral doses of 5–60 mg phylloquinone (Konakion or AquaMephyton) is similar to that found after a more physiological dose (≤ 1 mg), with peak plasma concentrations at 4–6 h followed by a rapid clearance phase (Shearer et al., 1974; Park et al., 1984; Øie et al., 1988; Hagstrom et al., 1995). After an oral dose of 10 or 50 mg Konakion, the plasma concentration declined from the peak absorptive level at a similar log-linear rate as that seen after intravenous administration, with a terminal half-time of about 2 h for measurements up to 9–12 h (Park et al., 1984). The absorption of oral preparations of phylloquinone shows inter- and intra-individual variation and, for doses of Konakion ranging from 10 to 60 mg, the bioavailability was 10–63% (Park et al., 1984) and 3.5–60% (Øie et al., 1988).

The pharmacokinetics of phylloquinone after an intramuscular dose is completely different, showing sustained, slow release from the muscle site over many hours and marked inter-individual variation (Hagstrom et al., 1995; Soedirman et al., 1996). The pharmacokinetics may also be influenced by the solubilizing agent. The systemic availability of intramuscularly injected Konakion MM, which is a mixed-micellar solution of phylloquinone in natural solubilizers, the bile acid glycocholic acid and the phospholipid lecithin (Schubiger et al., 1997; see Table 6), was irregular and < 65% in 20% of subjects (Soedirman et al., 1996). After intramuscular injection of phylloquinone (AquaMephyton R), most of the substance was carried by low-density and high-density lipoproteins instead of by triglyceride-rich (very-low-density) lipoproteins as found after oral administration (Hagstrom et al., 1995).

(c) Plasma pharmacokinetics of phylloquinone in neonates

The pharmacokinetics of phylloquinone during the early clearance phase up to 6 h in neonates (of low birth weight) after intravenous injection was very similar to that of adults (Shearer et al., 1972), declining bi-exponentially with median half-times of 23 and 109 min (Sann et al., 1985).

An early study of the plasma disposition of 1 mg Konakion given orally or intramuscularly at birth showed wide inter-individual differences during the first 24 h, especially after oral administration (McNinch et al., 1985). The peak plasma concentration after an oral dose occurred after 4 h; the median concentration was 73 ng/mL, which fell to 23 ng/mL after 24 h. The plasma concentration after administration of 1 mg of Konakion intramuscularly exceeded those after oral administration at all times, and after 24 h the median was 444 ng/mL. Physiologically, these concentrations compare with adult endogenous levels of about 0.5 ng/mL (Shearer, 1992).

In a comparison of the plasma concentrations of Konakion and Konakion MM in exclusively breast-fed infants at 24 h and 4 and 24 days after a single oral dose of 2 mg at birth (Schubiger et al., 1997), the mixed-micellar Konakion MM preparation resulted in higher median concentrations at all times, suggesting greater bioavailability. The largest difference was seen after four days, with median concentrations of 41 ng/mL Konakion MM and 12 ng/mL Konakion. By 24 days, the concentrations in both groups were mainly within the adult physiological range (0.3–0.4 ng/mL). An earlier study by the same group (Schubiger et al., 1993) had shown that a single oral dose of 3 mg Konakion MM resulted in higher plasma concentrations than a single dose of 1.5 mg of the same preparation given intramuscularly after four days. In this study, however, the plasma concentrations after 24 days were significantly higher after intramuscular injection, consistent with the hypothesis of the depot effect of intramuscular phylloquinone (Loughnan & McDougall, 1996; see also section 4.1.1(f)).

Stoeckel et al. (1996) pointed out that the terminal elimination plasma half-time of phylloquinone in neonates is probably longer than that in adults. They calculated from published studies that a realistic estimate of the terminal plasma half-time in neonates was 26–193 h (median, 76 h), as compared with 8–22 h (median, 14 h) in adults after intravenous administration (Øie et al., 1988). This longer terminal half-time may reflect the poorly developed organ systems of neonates and a reduced capacity to metabolize and excrete vitamin K (Stoeckel et al., 1996).

(d) Plasma pharmacokinetics of menaquinone-4

Oral preparations of menaquinone-4 are used in Japan for the prophylaxis of vitamin K deficiency bleeding. The plasma profile of an oral dose of this preparation in five-day-old infants appeared to be similar to that of phylloquinone; after a 4-mg dose, a peak concentration of about 100 ng/mL was achieved after 3–4 h, before declining to about 30 ng/mL by 12 h (Shinzawa et al., 1989). The half-time of menaquinone-4 was not calculated.

(e) Adult tissue reserves and distribution of vitamin K

Dietary vitamin K is delivered to the liver and possibly other tissues, including bone marrow, in the form of chylomicron remnants (Kohlmeier et al., 1996). The liver has often been assumed to be a major depot for vitamin K because it is the site of synthesis of the vitamin K-dependent coagulation proteins. Measurements of phylloquinone in livers obtained at autopsy from 32 adults in the United Kingdom revealed hepatic concentrations ranging from 1.1 to 21 ng/g wet tissue [2.4–47 pmol/g], with a median concentration of 5.5 ng/g [12 pmol/g]. The corresponding total liver stores of phylloquinone were 1.7–38 µg [3.8–85 pmol/g], with a median total store of 7.8 µg [17 pmol/g] (Shearer et al., 1988). Similar hepatic concentrations of phylloquinone were found in a smaller number of analyses of post-mortem samples from adults in Japan (10 ng/g) (Uchida & Komeno, 1988) and in The Netherlands (11 ng/g) (Thijssen & Drittij-Reijnders, 1996). The limited ability of the liver to store vitamin K is illustrated by the observation that the phylloquinone reserves are about 40 000-fold lower than those of vitamin A despite a daily dietary intake of vitamin K (∼100 µg) which is only about 10-fold lower than that of vitamin A (∼1000 µg). The distribution of the various forms of vitamin K in the liver is quite different from that in plasma in that the major transport form, phylloquinone, represents the minority of total hepatic stores (about 10%); the remainder comprises bacterial menaquinones, mainly menaquinones-6–13 (Shearer et al., 1988; Shearer, 1992; Shearer et al., 1996). The pattern of individual menaquinones in the liver varies considerably between individuals (Shearer et al., 1988; Uchida & Komeno, 1988; Thijssen & Drittij-Reijnders, 1996), perhaps reflecting their origin from the intestinal microflora (Shearer et al., 1996). This proposal is supported by the finding that two menaquinones, -10 and -11, which are major forms in most liver samples (Uchida & Komeno, 1988; Thijssen & Drittij-Reijnders, 1996), are known to be synthesized by Bacteroides species which are predominant members of the human intestinal flora (Conly & Stein, 1992); yet menaquinone-10 and menaquinone-11 do not make appreciable contributions to normal diets (Shearer et al., 1996).

Phylloquinone is also present in other human tissues. The concentration in the heart (∼5 ng/g) [∼10 pmol/g] is comparable to those in the liver, and even higher concentrations (∼13 ng/g) [∼25 pmol/g] are found in the pancreas, but lower concentrations (< 1 ng/g) [< 2 pmol/g] were detected in brain, kidney and lung. These tissues do not appear to contain appreciable concentrations of menaquinones except for the short-chain menaquinone-4. Particularly high concentrations of menaquinone-4 relative to phylloquinone are present in the kidney, brain and pancreas. Although these and other tissues contain the enzymes of the vitamin K epoxide cycle (see Figure 1) and carry out vitamin K-dependent carboxylation of protein precursors, this would not appear to account for the tissue-specific accumulation of menaquinone-4 and may suggest a hitherto unrecognized physiological role for menaquinone-4 in certain tissues (Shearer, 1992; Thijssen & Drittij-Reijnders, 1996). Indeed, menaquinone-4 may arise by tissue synthesis from phylloquinone itself (Davidson et al., 1998).

Figure 1

Cyclic metabolism of vitamin K for conversion of glutamate (Glu) residues to γ-carboxy glutamate (Gla) residues in vitamin K-dependent proteins

Osteocalcin is a major vitamin K-dependent bone protein synthesized by osteoblasts and therefore requires a source of vitamin K for γ-glutamyl carboxylation. Both trabecular and cortical bone contain ample reserves of vitamin K, with phylloquinone predominating and smaller amounts of shorter-chain menaquinones (Hodges et al., 1993; Shearer, 1997). With the absence of the typical hepatic forms menaquinones-10–13, the vitamin K content of bone resembles that of other extrahepatic tissues.

(f) Tissue stores and blood concentrations in neonates and infants

Information on liver stores (the site of synthesis of vitamin K-dependent clotting proteins) in infants and their response to vitamin K prophylaxis is limited (Shearer et al., 1988; Guillaumont et al., 1993). The endogenous stores of vitamin K in the liver of the newborn differ both quantitatively and qualitatively from those of adults because the concentrations and total reserves of phylloquinone are lower than those of adults (Shearer et al., 1988) and because bacterial menaquinones are undetectable (Shearer et al., 1988; Guillaumont et al., 1993). The endogenous hepatic concentrations of phylloquinone ranged from 0.3 to 6.0 ng/g (median, 1.4 ng/g) in preterm infants and from 0.1 to 8.8 ng/g (median, 1.0 ng/g) in term infants. The median hepatic concentration of 1 ng/g in term infants is equivalent to a total liver pool of about 0.1 µg phylloquinone, whereas the concentration is 5.5 ng/g and the pool 7.8 µg in adult liver. In infants who had received 0.5 or 1 mg phylloquinone at birth by intramuscular injection, these liver reserves were raised by some two to three orders of magnitude within 24 h. Hepatic phylloquinone concentrations may remain elevated for several weeks after injection: in two infants known to have received 1 mg phylloquinone by the intramuscular route and who survived 13 and 28 days, the total hepatic stores were 24 and 15 µg, respectively (Shearer et al., 1988). Guillaumont et al. (1993) measured hepatic concentrations in post-mortem liver samples obtained within the first 48 h of death from infants who had received 2 mg phylloquinone intravenously or orally (in some cases combined with extra intravenous or oral doses of 1, 5 or 10 mg). In three newborns who survived < 24 h, the hepatic concentrations of phylloquinone ranged from 63 to 94 µg/g (total liver stores, 2800–7300 µg), which were four orders of magnitude higher than the endogenous concentrations of 0.002–0.008 µg/g (total liver stores, 0.1–0.9 µg). Between 24 and 48 h, the hepatic concentrations in 10 infants had fallen to a median of 8.4 µg/g (total liver stores, 550 µg), and in one infant who survived for five days it was 2.9 µg/g (110 µg). The quite rapid fall in hepatic stores presumably reflects the relatively rapid metabolism and excretion of vitamin K via the urine and bile (Shearer et al., 1974). The lower hepatic concentration after intramuscular injection (Shearer et al., 1988) compared with intravenous injection (Guillaumont et al., 1993) is consistent with the idea that phylloquinone injected intramuscularly is released relatively slowly from the injection site (Loughnan & McDougall, 1996).

The reduced hepatic reserves of vitamin K in the human neonate are best explained by the existence of a barrier to placental uptake or transfer. This suggestion was originally made on the basis of the large concentration gradient of physiological concentrations of phylloquinone between maternal and cord blood plasma and the inefficient maternal–fetal transfer of pharmacological doses administered as an intravenous injection to the mother just before delivery (Shearer et al., 1982). The poor placental transport of phylloquinone has been confirmed by others (Mandelbrot et al., 1988; Yang et al., 1989). There is now general agreement that the cord plasma concentration of phylloquinone is < 50 pg/mL [110 pmol/L] and that the average maternal–fetal concentration gradient is within the range 20:1 to 40:1 (Shearer, 1992).

Few longitudinal studies have been conducted of plasma concentrations in infants who were not given vitamin K prophylaxis. In one such study, cord plasma concentrations were compared for breast-fed and formula-fed infants and in blood on days 3, 7 and 28 after birth (Pietersma-de Bruyn et al., 1990). In entirely breast-fed infants, the blood concentration rose from undetectable (< 20 pg/mL) at birth to mean values of 0.76, 0.49 and 0.49 ng/mL [1.7, 1.1 and 1.1 pmol/mL] on days 3, 7 and 28, respectively. In infants fed a milk formula containing 68 ng/mL phylloquinone, the plasma concentration rose steadily, with mean values of 1.4, 3.1 and 4.4 ng/mL [3.2, 6.8, and 9.9 pmol/mL] on days 3, 7 and 28, respectively. In another group of infants, Pietersma-de Bruyn et al. (1990) found that phylloquinone was undetectable in cord blood and in venous blood taken at 30 min but became measurable in venous blood after 12 h in 30% of infants (range, 0.04–0.40 ng/mL) and after 24 h in 60% of infants (range, 0.04–0.63 ng/mL).

A more detailed longitudinal comparison of plasma concentrations in breast-fed and formula-fed infants at 6, 12 and 26 weeks was made by Greer et al. (1991). This study is of special interest because the intakes of phylloquinone were also estimated at each time by measuring the vitamin K content of the milk and the volume of milk ingested (by weighing the infant). Such an assessment of the intake of phylloquinone depends on both the analytical accuracy of the measurements in breast milk and validation of the milk collection and sampling technique; both have proved problematical. The study of Greer et al. (1991) seems to have met the requisite criteria, and, although the concentrations were at the lower end of published values, they were in the same range as those in a carefully designed longitudinal study of the phylloquinone content of breast milk over the first five weeks of lactation (1–2 ng/mL) (von Kries et al., 1987b). The results, summarized in Table 8, illustrate the extreme differences in intakes between breast-fed and formula-fed infants, which are also reflected in the plasma concentrations. The plasma concentrations in the formula-fed infants agree with those found by Pietersmade Bruyn et al. (1990) after 28 days (4.5 ng/mL), and suggest that they plateau at around one month. The concentrations in entirely breast-fed infants aged one month and beyond tend, as in this study, to be at the lower end of the normal range in adults (∼0.15–1.0 ng/mL; mean, ∼0.5 ng/mL), even when the infants have received prophylaxis during the first week of life (Cornelissen et al., 1992; Schubiger et al., 1993, 1997). In contrast, the plasma concentrations in formula-fed infants are about 10-fold higher than the average values in adults (Pietersma-de Bruyn et al., 1990; Greer et al., 1991).

Table 8

Dietary intakes and plasma concentrations of phylloquinone in breast-fed and formula-fed infants aged 0–6 months in the USA.

(g) Hepatic catabolism

The liver plays an exclusive role in the metabolic transformations leading to the elimination of vitamin K from the body. After intravenous doses of 45 µg to 1 mg [³H]phylloquinone, about 20% of the radiolabel was excreted in the urine within three days, and 35–50% was excreted as metabolites in the faeces via the bile (Shearer et al., 1974). Rapid depletion of hepatic reserves of phylloquinone was also seen in surgical patients placed on a low-phylloquinone diet (Usui et al., 1990). These results suggest that the body stores of vitamin K are replenished constantly.

The route of hepatic catabolism leading to urinary excretion of vitamin K proceeds by oxidative degradation of the phytyl side-chain, probably involving the same enzymes used for ω-methyl and β-oxidation of fatty acids, steroids and prostaglandins. Two major metabolites or aglycones have been identified, which are carboxylic acids with five- and seven-carbon atom side-chains and are excreted in the urine as glucuronide conjugates (McBurney et al., 1980). The biliary metabolites have not been clearly identified but are initially excreted as water-soluble conjugates and become lipid-soluble during their passage through the gut, probably through deconjugation by the gut flora. There is no evidence that the body stores of vitamin K are conserved by enterohepatic circulation. Vitamin K itself is too lipophilic to be excreted in the bile, and the side-chain-shortened carboxylic acid metabolites are not biologically active.

(h) Vitamin K-epoxide cycle

In all tissues and cells found to carry out vitamin K-dependent carboxylation, the reaction has been shown to be intimately linked to a metabolic sequence known as the vitamin K-epoxide cycle. This cycle and the associated enzyme activities are shown in Figure 1. Its function seems to be to serve as a salvage pathway to conserve tissue reserves of vitamin K. In the course of γ-glutamyl carboxylation, vitamin K quinol is transformed into vitamin K epoxide, and the epoxide product is recycled in two steps; firstly by vitamin K epoxide reductase activity to produce vitamin K quinone and secondly by quinone reductase activity to produce the co-enzyme vitamin K quinol. Both these activities are thiol-dependent and are probably effected by the same enzyme (Suttie, 1987).

An important property of the dithiol-dependent epoxide and quinone reductase is their sensitivity to certain antagonists, especially those based on 4-hydroxycoumarin (e.g. warfarin) or indandione structures, which have long been used as oral anticoagulants. It is now clear that their anticoagulant action is based on their ability to inhibit epoxide reductase activity and block the recycling of the vitamin. The dithioldependent quinone reductase is also sensitive to warfarin, but the activity of a second quinone reductase catalysed by an NAD(P)H-dependent enzyme is less sensitive to warfarin inhibition and provides an alternative pathway for the reduction of vitamin K quinone to quinol in the presence of warfarin and other oral anticoagulant drugs (Shearer, 1992).

(i) Menadione and related water-soluble derivatives

No studies appear to have been conducted on the absorption, distribution, metabolism or excretion of menadione and related compounds in humans. Water-soluble salts of menadione (vitamin K₃) were introduced for vitamin K prophylaxis in newborns in the early 1940s and, until their use was almost entirely superseded by phylloquinone in the early 1960s, there were no suitable techniques for measuring menadione, its salts or their metabolites other than by radioisotopic techniques.

4.2. Toxic effects

4.3. Reproductive and prenatal effects

4.4. Genetic and related effects

4.4.2. Experimental systems

Limited data are available on the genetic and related effects of phylloquinone and menaquinones (Table 9). Phylloquinone did not induce mutation in Salmonella typhimurium. It enhanced the frequency of sister chromatid exchange in cultured human maternal lymphocytes at concentrations that are relevant in vivo, and a similar increase in sister chromatid exchange frequency was observed in cultured lymphocytes from human placental blood. In fetal sheep that received a catheter in the femoral vein 10–15 days before term, phylloquinone significantly increased the frequency of sister chromatid exchange in peripheral blood lymphocytes sampled 24 h later.

Table 9

Genetic and related effects of phylloquinone and menadione.

Menaquinone-4 but not phylloquinone inhibited osteoclastic bone resorption by inducing osteoclast apoptosis (Kameda et al., 1996). Menaquinone-4 and its derivatives also induced apoptosis in various human leukaemic cell lines (Yaguchi et al., 1997).

In preincubation protocols with Ames Salmonella tester strains, menadione did not induce reverse mutation in strains TA100, TA102, TA1535, TA1537, TA1538 or TA2638 in the presence or absence of an exogenous metabolic activation system. It was mutagenic in TA98 with metabolic activation and in TA2637 with or without activation. Menadione also induced mutation in strain TA104, but only with metabolic activation by purified NADPH–cytochrome P450 reductase; in another study it was mutagenic in this strain without activation. Menadione did not induce reverse mutation in Escherichia coli WP2/pKM101 or WP2uvrA/pkM101 in the absence of metabolic activation.

In tests with derivatives of E. coli WP2s (uvrA trpE) that are defective in 7,8-dihydro-8-oxoguanine DNA glycosylase activity (mutM) or MutY glycosylase activity on an A:7,8-dihydro-8-oxoguanine mispair (mutY) or give an adaptive response to oxidative stress by superoxide (soxRS), to compare the mutability of various reactive oxygen-generating compounds, menadione was not mutagenic; however, it was mutagenic in two strains of E. coli WP2 that contain deficiencies in the oxyR function. Menadione induced forward mutation to l-arabinose resistance (Ara^R) in E. coli K-12 strains with diminished concentrations of superoxide dismutase and induced a SOS response in PQ37.

This agent induced concentration-dependent single-strand and double-strand DNA breaks in a human breast cancer MCF-7 cell line, in cultured rat hepatocytes, in human fibroblasts, in human chronic myeloid leukaemic K562 cells and in a single-cell gel electrophoresis assay to measure DNA strand breaks in human lymphocytes at doses as low as 1 µmol/L. At concentrations of 15–100 µmol/L, menadione induced extensive DNA fragmentation in human chronic myeloid leukaemic K562 cells which could be measured in alkaline elution assays. At these doses, no oxidative stress appeared to occur in these cells.

Cantoni et al. (1991) reported that hydrogen peroxide produced during the metabolism of menadione does not contribute to the cytotoxic action of the quinone. In isolated rat hepatocytes, menadione induced DNA fragmentation consistent with apoptosis. These effects occurred in the absence of 8-oxo-2′-deoxyguanosine production, and the authors concluded that oxidative modification of DNA bases was unlikely to be involved (Fischer-Nielsen et al., 1995). Menadione induced protein-linked DNA breaks in the presence of purified human DNA topoisomerase II but not DNA topoisomerase I (Frydman et al., 1997), and it seems likely that DNA topoisomerase II poisoning is involved in DNA breakage by menadione at the lower concentrations, at which oxygen stress does not occur.

Menadione induced morphological transformation of BALB/c 3T3 cells, but only when tested in the presence of the tumour promotor 12-O-tetradecanoylphorbol 13-acetate.

Andrew et al. (1999) found that menadione enhanced the spontaneous mutation frequency and induced a novel mutation spectrum of lacI genes recovered from a rat embryonic fibroblast line transfected with a λ-phage shuttle vector, in both the traditional plaque assay and a positive selection assay.

4.5. Mechanistic considerations

5.1. Exposure data

The term ‘vitamin K’ refers to a group of 2-methyl-1,4-naphthoquinone derivatives which can fulfil an essential co-factor function in humans in the biosynthesis of a number of calcium-binding proteins, some of which are essential for haemostasis. In nature, vitamin K occurs as phylloquinone in plants and as menaquinones produced by bacteria. The major dietary sources of vitamin K are green leafy vegetables and certain vegetable oils. Clinically, vitamin K is used primarily to prevent or cure deficiency-related bleeding in newborns and patients with malabsorption syndromes and to reverse the anticoagulative effects of vitamin K antagonists.

5.2. Human carcinogenicity data

An association between childhood leukaemia and vitamin K prophylaxis given by the intramuscular route was found in two reports but was not confirmed in a number of studies in various countries. A major limitation of most of the studies is that the fact of intramuscular administration of vitamin K was difficult to establish retrospectively for a substantial proportion of subjects, although the results of the analyses based on individual records and on imputed hospital policies for vitamin K administration are similar. In the studies in which a suggestion of an association was observed, selection bias may have accounted for the result. The possibility cannot be entirely excluded of a small increase in the risk for acute lymphoblastic leukaemia occurring at ages around those of the peak incidence in childhood in children given intramuscular administration of vitamin K.

The few studies that investigated oral administration of vitamin K found no increase in the relative risk for leukaemia.

5.3. Animal carcinogenicity data

No adequate study on the carcinogenicity of vitamin K substances was available to the Working Group.

5.4. Other relevant data

Phylloquinone and menaquinones are absorbed from food into the lymphatic system and carried by triglyceride-rich lipoproteins in the blood. Menaquinones synthesized by the gut microflora may also be absorbed. Phylloquinone is rapidly cleared from the circulation by the liver, metabolized to metabolites with shortened side-chains and excreted in the bile and urine. In animals, menadione is absorbed predominantly by the portal route, does not accumulate in specific organs and is extensively excreted unchanged in the urine. A fraction of menadione is converted in tissues to menaquinone-4.

Phylloquinone rarely has toxic effects, and the few serious immunological complications observed have been attributed to the vehicle of solubilization. Menadione may cause haemolytic anaemia and induce cellular damage by arylating protein-bound and soluble thiols or by inducing oxidative stress.

No adverse effects have been reported in mothers or infants after administration of vitamin K during pregnancy, whereas vitamin K deficiency is teratogenic. The safety of vitamin K in pregnancy has not been adequately studied experimentally.

Neither phylloquinone nor menaquinones have been adequately studied for mutagenicity. Menadione acts as a bacterial mutagen in several specific strains of Salmonella typhimurium and Escherichia coli. In mammalian cells, menadione leads to DNA breakage, and there are isolated reports of chromosomal aberrations and sister chromatid exchange.

5.5. Evaluation

There is inadequate evidence in humans for the carcinogenicity of vitamin K substances.

There is inadequate evidence in experimental animals for the carcinogenicity of vitamin K substances.

Overall evaluation

Vitamin K substances are not classifiable as to their carcinogenicity to humans (Group 3).

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