3.1.1. Mouse

A group of 13 male and 12 female IF mice, weighing 25 g, were given 5 mg of 2-naphthylamine (BDH; purified by distillation and crystallization) in arachis oil via stomach tube twice weekly at a dose of 400 mg/kg bw per week for life (90 weeks). A group of six males and five females served as arachis-oil controls. Liver cholangiomas were observed in 5/13 (38%) male and 5/12 female (41%) mice. No tumours were observed in control animals (Bonser et at., 1952).

Groups of nine male and 14 female CBA mice, weighing 30 to 35 g (age not specified) were given five mg 2-naphthylamine (BHD; purified by distillation and crystallization) in arachis oil by gavage, twice weekly for a weekly dose of 240 mg/kg bw for life (90 weeks). A group of seven male and seven female mice served as arachis-oil controls. Hepatomas were observed in 6/9 males (67%) and 7/14 females (50%). No tumours were observed in the control animals. The authors reported the incidence of hepatomas in the control breeding mice of the laboratory to be 8% (Bonser et al., 1952).

Groups of 14–15 male and 12–15 female CBA mice (weight and age not given) were fed four different synthetic diets containing 2-naphthylamine (concentration not specified) for a dose of 160 mg/kg bw per week for 90 weeks. No information on use of controls was provided. Hepatomas occurred at similar incidences in all four diet groups, with a total of 24/57 (42%) hepatomas observed in males and 25/54 (46%) in females. The authors reported that malignant hepatomas were found in 16 mice (sex not specified) (Bonser et al., 1952). [The Working Group noted the lack of controls.]

Groups of 20 female BALB/c mice, eight weeks of age, were fed a diet containing 2000 ppm 2-naphthylamine [purity not specified] for 40 weeks, followed by basal diet for 15 weeks. At 55 weeks, hyperplasia of the bladder epithelium was seen in 6 of 16 (37%) surviving mice. In the livers of these mice, hyperplastic nodules were seen in 14 animals (87%), adenomas in 10 (62%) and hepatomas in three (19%) (Yoshida et al., 1979).

A group of 16 female and 16 male A/J mice, 6–8 weeks of age, received 2-naphthylamine in tricaprylin by gavage three times per week for eight weeks, resulting in a total dose per animal of 600 mg/kg bw. At 24 weeks, 8 of 14 (57%) male and 4 of 13 (31%) female survivors had lung tumours. This was not significantly different from the lung tumour rate in the vehicle controls. Tumour multiplicity was significantly increased in male mice (0.93 ± 1.00 vs 0.27 ±0.59; P< 0.05) (Stoner et al., 1986).

3.1.2. Rat

A group of 18 male, 29 female and three unknown albino rats were fed 0.01% 2-naphthylamine [purity unspecified] in the diet for life; this resulted in four bladder papillomas among 50 rats that survived for up to 102 weeks. No carcinomas were seen in the treated group. There were no tumours observed in the 43 controls fed an untreated diet (Bonser et al., 1952).

Oral administration by gavage of 0.5–150 mg 2-naphthylamine [purity unspecified] per week to rats for 52 weeks, followed by an observation period until up to 80 weeks did not result in a significant increase in tumours compared with controls (Hadidian et al., 1968).

A group of 25 female Wistar rats, 6–8 weeks of age, received 2-naphthylamine [purity unspecified] suspended in arachis oil by stomach tube at a dose rate of 300 mg/kg bw per week for one year, and were then kept until symptoms of bladder disease were seen. A group of 55 males and females served as controls. The first tumour appeared at 57 weeks. Histology indicated that five of 17 (29%) animals showed the presence of bladder tumours. Three animals were reported to be “still alive” (Hicks & Chowaniec, 1977).

A group of 20 female Wistar rats, 10 weeks of age, received 2-naphthylamine (purity not specified) at a dose of 300 mg/kg bw in arachis oil by gastric intubation once a week for 57 weeks. Animals were killed when moribund, or after 100 weeks. A group of 20 animals served as arachis-oil controls. Nineteen animals survived 57 weeks or longer. The bladders of 18 rats were examined histologically: eight of 18 (44%) had urothelial hyperplasia, and four of these eight had also carcinomas (Hicks et al., 1982).

3.1.3. Hamster

A group of 30 male and 30 female Syrian golden hamsters, nine weeks of age, received 1% or 0.1% (w/w) 2-naphthylamine (purity unspecified) in the diet for life. The estimated intake was 600 mg 2-naphthylamine/week. Bladder carcinomas were observed in 10/23 (43%) effective males and in 8/16 (50%) effective females treated with the 1% concentration, with the first tumours appearing after 45 weeks. One male and one female hamster also developed hepatomas. Dietary concentrations of 0.1% did not produce carcinomas in 60 animals after 97 weeks. Repeated administration of the chemical by gavage (10 mg, twice weekly for 40 weeks) did not produce any tumours. In two control groups of 60 and 40 animals, fed the diet only, no bladder carcinomas were observed (Saffiotti et al., 1967).

3.1.4. Rabbit

Oral doses (200 mg, twice weekly) of 2-naphthylamine (purity unspecified) given to six rabbits (age and sex unspecified) for more than five years induced a Iransitional-cell bladder papilloma at 4.75 years in one animal, and epithelial hyperplasia with downgrowth in the bladder in another after 5.25 years (Bonser et al., 1952).

3.1.5. Dog

A group of 16 medium-sized to large female mongrel dogs, weighing 8–20 kg, received daily subcutaneous injections of commercial 2-naphthylamine (4 mg/day for dogs ≤ 12 kg: 5 mg/day for larger dogs) for 14 weeks. The dose was doubled during the next 12 weeks and tripled during the subsequent 63 weeks. During the last 54 weeks of this period, the dogs also received a daily dose of 2-naphthylamine (by capsule) in the food. The number of dogs on this dual regimen was gradually increased, as was the oral dose (starting with 100 mg/day). By the time the subcutaneous injections were terminated, all dogs received a daily oral dose of 300 mg of 2-naphthylamine. During the first five months of this experimental period, eight dogs received sodium bicarbonate (100 mg/kg bw) to alkalinize the urine. Four control dogs received no treatment. Over a total treatment period of 20–26 months, 13 of the 16 (81%) dogs had developed papillomas and carcinomas in the bladder (Hueper et al., 1938). [This evidence was based partly on autopsy and partly on cystoscopy and biopsy.]

A group of four medium-sized mongrel Airedale dogs (one female and three males) received 2-naphthylamine (BDH; purified by distillation and crystallization) at 150 mg per day in milk for three weeks. The dose was then reduced to 100 mg/day (for an unspecified period). The dose was gradually increased to 700 mg per day after the end of the first year. One male dog died after one year of treatment. Multiple papillomas in the bladder were seen in the three other dogs after 3.7–5 years of treatment. In one animal, one anaplastic carcinoma was found. There were no metastases, and the renal pelves, ureters and urethra were tumour-free, as were all other organs (Bonser, 1943).

A group of four female mongrel dogs received 200 mg 2-naphthylamine (purity unspecified) orally in a gelatin capsule on six days per week for six months. The daily dose was then increased to 600 mg and continued for up to two years. The maximum cumulative dose of 2-naphthylamine given to a dog was 310 g. One dog died after 14.5 months of treatment without neoplastic changes in the bladder. Another dog received 2-naphthylamine for one year and was killed nine months later with no neoplastic changes in the bladder epithelium. Multiple transitional-cell tumours, some of which were histologically malignant, were seen in the bladder of the remaining two dogs. One of these died after two years of treatment; the other was killed one year later. The latter dog had developed an invasive bladder carcinoma (Bonser et al., 1956a).

A group of four female mongrel dogs, each weighing 12–14 kg, were fed 400 mg 2-naphthylamine [purity unspecified] daily for two years. Bladder tumours were noted and confirmed by microscopic examination of biopsy sections in all four animals within 9–18 months after the start of the experiment. The dogs received intravenous and intracavitary therapy with 5-fluorouracil, whereupon transient tumour regression was observed. All dogs were then kept on standard diet and followed for 23 to 55 additional months until death or sacrifice. Metastases of the bladder carcinomas were seen in the lungs of two dogs and in the kidney in one (Harrison et al., 1969).

A group of 34 beagle dogs, 8–10 months of age, received 2-naphthylamine [purity unspecified] mixed with lactose, orally in gelatin capsules on 6 days per week in doses of 6.25, 12.5, 25, and 50 mg/kg bw (dose groups A, B, C, D) per day for 2–26 months. Two male and two female control dogs received capsules with lactose only. Within 30 months, bladder tumours were induced across all dose groups in 24/34 (70%) dogs: invasive transitional-cell carcinomas were seen in two dogs each in dose groups A, B, and D, and in five dogs in dose group C; invasive squamous-cell carcinomas were observed in one, two, three and two dogs in dose groups A, B, C and D, respectively; papillary carcinomas were noted in one, three and four dogs in dose groups B, C and D, respectively. Overall, carcinomas were present in 9/11 (81%) dogs that had received a total of 100–200 g 2-naphthylamine and in 6/23 (26%) dogs receiving a total amount of < 100 g. No tumours were seen in the controls. The total amount of 2-naphthylamine required for tumour induction was less for dose group A after 24–30 months than for dose groups C and D after 9–18 months (Conzelman & Moulton 1972).

In a study designed to investigate the morphology of 2-naphthylamine-induced bladder tumours, eight female dogs, weighing 18–30 kg, were treated with 2-naphthylamine (5–30 mg/kg bw, 4–6 days per week, for 7.5 months and 30 mg/kg bw, 4–6 days per week, for 8.5 months). After 55 months since the beginning of the experiment, bladder carcinomas developed in seven (87%) animals (Romanenko & Martynenko, 1972).

A group of 15 female dogs, weighing 16–22 kg, received an oral capsule containing 500–600 mg 2-naphthylamine [purity not specified] daily for 20–26 months. All dogs had haematuria, and all developed bladder carcinoma (Rigotti et al., 1977).

Eight female pure-bred beagle dogs were given a daily dose of 2-naphthylamine (5 mg/kg bw; source and purity not specified; route of administration not given) for 30 days. Four dogs then started on a daily supplement of 6 g D,L-tryptophan admixed into 300 g dog chow during the rest of the experiment. The other four dogs were kept on standard diet. Four additional dogs received only the dietary supplement but no 2-naphthylamine. After three years, all 12 dogs were killed and the bladders were examined grossly and by histology. The bladders of dogs that received only 2-naphthylamine showed no abnormal effects. The dogs that were given tryptophan with 2-naphthylamine showed gross and histological changes, such as papillary tumours and epithelial hyperplasia. The bladder mucosa of dogs given tryptophan alone showed similar hyperplastic changes (Radomski et al., 1977).

Five male purebred beagle dogs received capsules with 2-naphthylamine in corn oil orally in doses of 25 mg/kg bw, daily on five days per week for 26 weeks. In a second experiment, groups of four dogs (two females, two males) were similarly treated for one, six or 36 weeks, then promptly killed. In a third experiment, eight dogs (four females, four males) were treated with the same regimen for 26 weeks, then four dogs were killed and the others kept without further treatment for three years. Loss of bladder luminal membrane, hyperplasia of the bladder epithelium, and lymphocyte infiltration of the submucosa were seen after one and six weeks of treatment in some dogs, and to a more severe degree after 36 weeks of treatment in all dogs. Two of the four dogs that were kept for three years after a 26-week treatment period had papillary carcinomas of the bladder (Rdomski et al., 1978).

A group of two female and three male beagle dogs, approximately nine months of age, received 400 mg pure 2-naphthylamine daily on five days per week for 34 months. The chemicals were given as tablets enclosed in a gelatin capsule. Control dogs (four males and four females) received capsules with lactose. All treated dogs developed transitional cell carcinomas of the bladder within 34 months. No neoplasia of the bladder was found in the controls (Purchase et al., 1981).

3.1.6. Monkey

Twenty-four young male and female rhesus monkeys (Macaca mulatta; weighing 2–3 kg) received 2-naphthylamine mixed with lactose, orally in gelatin capsules on six days per week in doses of 6.25, 12.5, 25, 50, 100, 200, and 400 mg/kg bw per day for 33–60 months. Some animals received a fixed dose during the entire experiment; others received different doses over the course of five years. Papillary carcinoma, transitional-cell carcinoma or carcinoma in situ were observed in the bladder of nine treated monkeys, the earliest of these tumours appearing after 33 months. The majority of tumours occurred in animals given high doses of 2-naphthylamine. No other types of tumour were observed, and no tumours occurred in three control monkeys (Conzelman et al. 1969).

3.2.1. Mouse

Swiss albino mice, approximately 12 weeks of age, received 0.1 ml of a freshly made 3% solution of 2-naphthylamine (BDH; purified by distillation and crystallization) in arachis oil by subcutaneous injection, twice weekly for 50 weeks. None of 13 mice surviving 33 weeks of treatment had subcutaneous sarcomas; two of four mice (50%; all females) surviving > 77 weeks had hepatomas. In a similar experiment with 2-naphthylamine (RCH; purified by gradient sublimation), local subcutaneous sarcomas were seen in two of 12 mice (17%; at 37 and 41 weeks, respectively) and one hepatoma at 52 weeks. When a 3% solution of 2-naphthylamine (BDH; purified by distillation and crystallization) was allowed to stand for four weeks and injected subcutaneously into Swiss albino mice (0.1 ml per mouse; twice weekly for 50 weeks), 10 of 16 (62%) mice that survived ≥ 20 weeks had subcutaneous injection-site sarcomas, and four of five (80%) mice that survived ≥ 80 weeks had hepatomas. No sarcomas or hepatomas developed in 17 male and female controls treated with arachis oil only (Bonser et al., 1956a).

CBA mice, 12 weeks of age, received 0.1 mL of a freshly made 3% solution of 2-naphthylamine-HCl (BDH; purified by distillation and crystallization) by subcutaneous injection, twice weekly for six months and then once weekly for a further four months. No sarcomas were seen in 11 mice surviving ≥ 56 weeks of treatment, and hepatomas were observed in four of 11 (36%) mice dying after 82 weeks. In a similar experiment with 2-naphthylamine-HCl purified by gradient sublimation, no subcutaneous sarcomas were seen in ten mice surviving ≥ 58 weeks of treatment, and hepatomas were observed in four of 11 (36%) mice dying after 82 weeks. One (7%) hepatoma developed in 15 male and female control animals (Bonser et al., 1956a).

Groups of 45 male and 45 female mice (from a local dealer, strain and age not specified) were given 2-naphthylamine (BDH, purified by distillation and crystallization via subcutaneous injection (0.1 mL of a freshly prepared or four-week-old 30-mg/ml suspension in arachis oil) twice weekly for 52 weeks (total dose, 312 mg per mouse). A total of 12 mice had subcutaneous sarcomas, seven mice had hepatomas, and two mice had intestinal tumours (one benign, one malignant) (Bonser et al., 1956b). [The Working Group noted the lack of concurrent controls.]

Groups of 10 male and 10 female inbred A/St mice, 6–8 weeks of age, received intraperitoneal injections of 2-naphthylamine in tricaprylin. The injections were given three times per week for eight weeks. Doses per injection were 62.5, 125 or 250 mg/kg bw. At 24 weeks after the first injection the surviving mice were killed and the lungs were excised. The average number of lung tumours per mouse (1.38 ± 0.30) was statistically different (P<0.01) from that in the corresponding solvent control (0.19 ± 0.10) in the mice treated with the highest dose of 2-naphthylamine (total dose 6 g/kg bw). The tumour yield in mice treated with 2-naphthylamine was not significantly different from that in the controls (Theiss et al., 1981).

Three groups of 16 females and 16 males A/J mice, 6–8 weeks of age, received 2-naphthylamine (purity not specified) in tricaprylin by intraperitoneal injection three times per week for eight weeks, resulting, respectively, in total doses per animal of 120, 300 and 600 mg/kg bw. At 24 weeks, the incidence of lung tumours varied from 27% to 47%, which was not significantly different from the lung tumour rate in the vehicle controls (Stoner et al., 1986).

A group of 91 newborn BALB/c mice received a single 0.02-mL subcutaneous injection containing 50 µg 2-naphthylamine (purity unspecified) in 1% aqueous gelatine on the first day of life. Among 71 surviving mice that were killed between the 36^th and 43^rd weeks, 15 (21%) had lung tumours [not significant], and one had a hepatoma. In a concurrent control group, 2/21 (10%) mice developed lung tumours (Roe et al., 1963).

Twelve groups of 50–60 newborn BALB/c mice received subcutaneous injections of 100 µg 2-naphthylamine in 20 µL arachis oil once within 24 hours after birth, or once daily on the first five days of life. After 40 weeks, the numbers of pulmonary adenomas in the surviving mice in the two groups were not statistically different from those in the controls. In a second experiment, newborn BALB/c mice (number not specified) received subcutaneous injections of 100 µg 2-naphthylamine in 20 µl 3% aqueous gelatin once daily on the first five days of life. Surviving mice were killed at 50 weeks of age. Lung adenomas were seen in 9/41 (22%) surviving mice and in 4/48 (8%) controls, which was not significant. No other tumours were found. The failure of 2-naphthylamine to induce tumours in newborn mice was ascribed to immaturity of their metabolic system (Walters et al., 1967).

A group of 68 newborn Swiss mice (sex unspecified) (n = 68) were given a single subcutaneous injection of 30 µL commercial., recrystallized 2-naphthylamine as 3% suspension in gelatin [purity unspecified] within 24 hours after birth. Ten months later, bronchogenic adenomas were found in six of 63 (10%) autopsied mice [not significant], and no tumours in 57 controls (Radomski et al., 1971).

A group of 28 males and 23 females newborn Swiss mice were given subcutaneous injections of 30 µL commercial., recrystallized 2-naphthylamine as 3.3-mg/mL suspension in 3% gelatin (100 µg per dose) on the first, third and fifth day of life. Twelve months later, 2/26 (8%) autopsied male mice had hepatomas, and one of the 21 (5%) autopsied females had a lymphosarcoma. Among the controls (20 males, 30 females) one male mouse had a lymphosarcoma (Radomski et al., 1971).

3.2.2. Rat

A group of 16 male Chester Beatty random inbred albino rats, weighing 200 g, received intraperitoneal injections of 50 mg/kg bw 2-naphthylamine freshly suspended in arachis oil twice weekly for three months. Thereafter, the rats were maintained until tumours were palpable or until death (survival period, 123–622 days). Of 14 rats examined, three had tumours: two (14%) had sarcomas, one (7%) had a salivary gland tumour (Boyland et al., 1963). [The Working Group noted the absence of control rats.]

3.3.1. Mouse

A group of twenty-five IF mice (sex not specified), weighing on average 25 g, received a dorsal dose of a saturated solution of 2-naphthylamine in benzene (applied by skin-painting; no details given) once a week for 99 weeks. No skin tumours or liver tumours were found (Bonser et al., 1952). [The Working group noted the absence of control mice.]

3.4.1. Mouse

A group of 89 albino mice received an intravesicular implant of 2-naphthylamine (dose unclear) in paraffin wax. Forty-one mice received the compound after purification as in Bonser (1943), fourty-eight mice were given the compound after purification by gradient sublimation (Henson et al., 1954). After 40 weeks, eight mice (9%) had developed bladder carcinomas, four of which were invasive. This tumour yield was not significantly different from that in the control mice implanted with paraffin wax alone, but, according to the authors, the tumours were better established histologically (Bonser et al., 1956c). In contrast, among 74 stock mice that survived 40 weeks after having received an intravesicular implant of a stearic-acid pellet containing 2-naphthylamine (dose unclear), no bladder adenomas, papillomas or carcinomas were found (Boyland et al., 1964).

3.4.2. Dog

Four beagle dogs (age and sex unspecified) were given 10 mg commercial 2-naphthylamine (recrystallized before use) dissolved in 5 ml DMSO once every two weeks for 30 months. The solution was instilled by catheter, directly into the bladder lumen. No tumours were seen when the dogs were sacrificed at 45 months (Radomski et al., 1971).