Abstract

Formaldehyde inhalation is linked to nasal cancer and leukemia in humans. Formaldehyde-induced DNA-protein crosslinks and enhanced cell proliferation are important in the pathogenesis of nasal cancer and, potentially, leukemia. Mutations in the tumor suppressor gene Trp53 have been associated with formaldehyde-induced nasal tumors and might be a key mechanistic event in formaldehyde-induced leukemia. The objective of this study was to evaluate the potential role of the Trp53 gene in formaldehyde-induced nasal carcinogenicity, leukemia or lymphohematopoietic cancer, and potentially other neoplasms in genetically susceptible mice. Male Trp53 haploinsufficient (Trp53⁺) mouse strains (B6.129-Trp53^tm1Brd and C3B6.129F1-Trp53^tm1Brd) were exposed to 0-, 7.5- or 15-ppm formaldehyde (25/group) 6 h/d, 5 d/wk for 8 wk, and then held for 32 wk. Blood was collected for hematology, and major tissues and gross lesions were collected for histopathology. The primary formaldehyde-related finding was squamous metaplasia of the respiratory epithelium of the nose. Inhalation of a maximum tolerated dose of formaldehyde caused significant injury to the nasal mucosa and cell proliferation, but did not cause nasal tumors or an increased prevalence of leukemia or lymphohematopoietic cancer in Trp53⁺ mice. All observed neoplasms were considered background lesions for these mouse strains. The results of this short-term carcinogenicity study do not support a role for Trp53 in formaldehyde-induced neoplasia.

Acknowledgments

This research was supported by the Intramural Research Program of the NIH National Institute of Environmental Health Sciences. These studies were conducted at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. The authors thank Dr. Grace Kissling for statistical analysis and Drs. Michelle Hooth and Gordon Flake for critical review of this manuscript.

Authors

• Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
  Daniel L. Morgan, Ph.D.
  Darlene Dixon, Ph.D., D.V.M.
  Debra H. King
  Greg S. Travlos, D.V.M., D.A.C.V.P.
  Ronald A. Herbert, Ph.D., D.V.M.
  John E. French, Ph.D.
  Erik J. Tokar, Ph.D.
  Michael P. Waalkes, Ph.D.
• Integrated Laboratory Systems, Research Triangle Park, North Carolina, USA
  Michael P. Jokinen, D.V.M.

Peer Review

The draft research report on the study of formaldehyde exposure to T53 haploinsufficient mice was evaluated by the reviewers listed below. These reviewers served as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, reviewers determined if the design and conditions of these NTP studies were appropriate and ensured that this NTP Research Report presented the experimental results and conclusions fully and clearly.

Publication Details

Publisher: National Toxicology Program

Publishing Location: Research Triangle Park, North Carolina

ISSN: 2473-4756

DOI: https://doi.org/10.22427/NTP-RR-3

Report Series: NTP Research Report Series

Report Series Number: 3

Official Citation: Morgan, DL, Dixon, D, King, DH, Travlos, GS, Herbert, RA, French, JE, Tokar, EJ, Waalkes, MP, Jokinen, MP. 2017. NTP Research Report on Absence of Formaldehyde-Induced Neoplasia in Trp53 Haploinsufficient Mice Exposed by Inhalation. NTP RR 3. Research Triangle Park, NC: National Toxicology Program (3): 1-29.

Introduction

Formaldehyde is recognized as a nasal carcinogen in humans¹ and laboratory rats.^2-4 Exposure of rats to formaldehyde results in development of squamous cell carcinoma (SCC) in the anterior and posterior lateral meatus of the nasal cavity.² DNA-protein crosslinks (DPC) and cell proliferation are prominent at sites of SCC formation in the rat nasal cavity,^4-6 suggesting that incomplete repair of DPC results in mutations within the proliferating cell population and might lead to neoplasia. Considerable evidence shows that formaldehyde-induced mutations in the tumor suppressor gene Trp53 are important in the pathogenesis of nasal cancer.^7,8 Mutations in Trp53 were identified in formaldehyde-induced nasal SCC in rats,⁹ and abnormal Trp53 protein was shown to accumulate in the nasal tissue of formaldehyde-exposed rats.¹⁰ Mutations in Trp53 result in loss of tumor suppression functions including RNA repair, cell cycle arrest, senescence, and apoptosis,¹¹ resulting in a loss of normal growth control and clonal expansion of mutated cells.^12-14

Formaldehyde also has been reported to cause myeloid leukemia in humans,¹⁵ although the mechanism is unknown. Mutations in the Trp53 gene could be important in the pathogenesis of leukemia or lymphohematopoietic cancer. Zhang et al.¹⁶ proposed that hematopoietic stem cells in the nasal epithelium or in circulation undergo formaldehyde-induced mutations that result in loss of Trp53 and acquisition of the capacity for self-renewal. The Trp53 gene is involved in regulating the self-renewal of hematopoietic stem cells.^17-20 In addition, disruption of the Trp53 pathway has been shown to enhance production of induced pluripotent stem cells capable of self-renewal.²¹ Acquisition of self-renewal capacity is one of the initial steps in cancer development.²² Formaldehyde-induced loss of Trp53 would be rare, however, and indeed an increased incidence of lymphohematopoietic cancer has not been observed in formaldehyde-exposed wild type rats and mice.³

Currently no good animal model is available for investigating the mechanism(s) by which formaldehyde causes leukemia. Because considerable evidence suggests a role of Trp53 in formaldehyde-induced nasal SCC, we evaluated two Trp53-haploinsufficient (Trp53⁺) mouse strains in this study. We hypothesized that formaldehyde-induced loss of Trp53 would be increased in Trp53⁺ mice, resulting in an increased incidence of SCC of the nose and leukemia or lymphohematopoietic cancer, and potentially neoplasms at other sites.

Materials and Methods

Animals

Two mouse strains heterozygous for the null and wildtype Trp53 allele were used to evaluate the potential role of Trp53 in formaldehyde-induced lymphohematopoietic cancer. The inbred B6.129-Trp53^tm1Brd (Model P53N12-M) mouse strain was selected for this study because of its previous use as a model for lymphohematopoietic tumors in short-term cancer bioassays.^23-29 The C3B6.129F1-Trp53^tm1Brd strain also was selected based on the observation of chemical exposure-dependent lymphohematopoietic tumors of either lymphoid- or myeloid-committed stem cell origin.^30,31 This strain is an outcross between C3H/HeNTac female (Model C3H-F) xB6.129-Trp53^tm1Brd (Model P53N12-M) homozygous Trp53 null allele male to produce the haploinsufficient F1 progeny. Heterozygotes were selected because they have a lower background incidence of sporadic tumors than the homozygotes, and the latency for sporadically occurring tumors is longer than in the homozygotes.^32,33 Although rats appear to be more susceptible than mice to formaldehyde-induced SCC of the nose, a genetically modified rat strain was not available at the time of this study.

Male C3B6.129F1-Trp53^tm1Brd mice were 9 weeks old on arrival and male B6.129-Trp53^tm1Brd haploinsufficient mice (Taconic, Hudson, NY) were 8–10 weeks old on arrival. After a 3- (B6.129-Trp53^tm1Brd) to 4-week (C3B6.129F1-Trp53^tm1Brd) quarantine period, mice were weighed and randomly assigned to three exposure groups (Table 1). Five mice per strain were assigned to sentinel groups. All mice were acclimated to the Hazleton 2000 exposure chambers for 3 days prior to exposure. Food (NIH-31) was removed during the 6-hour exposure periods; and water was always available. Body weights were recorded weekly.

Table 1

Mortality in Trp53⁺ Mice Exposed to Formaldehyde.

Results

Body and Organ Weights

Body weights of C3B6F1.129-Trp53^tm1Brd mice exposed to 7.5-ppm formaldehyde were significantly less than controls (p < 0.05) from week 1 to week 11, and at weeks 37 and 38. Body weights of mice exposed to 15-ppm formaldehyde were significantly less than controls (p < 0.05) from week 1 to week 24, and at weeks 37 and 38 (Figure 1). Absolute and relative liver weights were significantly decreased 11% and 8%, respectively, in mice exposed to 15-ppm formaldehyde. Lung, kidney, spleen, and thymus weights of formaldehyde-exposed mice were not significantly different from controls (data not shown).

Figure 1

Body Weights of Trp53⁺ Mice Exposed to Formaldehyde

Body weights of B6.129-Trp53^tm1Brd mice exposed to formaldehyde were not significantly different from controls at any time point (Figure 1). Absolute and relative liver, lung, kidney, spleen, and thymus weights of formaldehyde-exposed B6.129-Trp53^tm1Brd mice were not significantly different from controls (data not shown).

Hematology

Hematological parameters for formaldehyde-exposed C3B6F1.129-Trp53^tm1Brd (Table 2) and B6.129-Trp53^tm1Brd (Table 3) mice were not significantly different from their respective controls. Neither strain showed any indication of treatment-related hematotoxicity, leukemia, or lymphoma.

Table 2

Hematological Parameters of C3B6.129F1-Trp53^tm1Brd Mice Exposed to Formaldehyde.

Table 3

Hematological Parameters of B6.129-Trp53^tm1Brd Mice Exposed to Formaldehyde.

Histopathology

Nasal Cavity

Formaldehyde exposure-related non-neoplastic lesions were observed primarily in the nasal cavity of C3B6F1.129-Trp53^tm1Brd and B6.129-Trp53^tm1Brd mice. These nasal lesions were quantitatively and qualitatively similar in both strains. Nasal cavity lesions considered related to formaldehyde exposure are summarized in Table 4. The number of animals examined per group includes the early-death animals.

Table 4

Nasal Lesions in Trp53⁺ Mice Exposed to Formaldehyde.

The only nasal cavity lesion observed in the control C3B6F1.129-Trp53^tm1Brd and B6.129-Trp53^tm1Brd animals was minimal hyaline degeneration of the respiratory epithelium, consisting of one or more small foci of epithelial cells containing intra-cytoplasmic accumulation of brightly stained eosinophilic material. This minimal lesion, observed in many of the control animals, is considered a spontaneous background finding.

Squamous metaplasia of the respiratory epithelium was observed in the nasal cavity of many C3B6F1.129-Trp53^tm1Brd and B6.129-Trp53^tm1Brd mice exposed to 7.5- and 15-ppm formaldehyde. The incidence and average severity (minimal to mild) of squamous metaplasia were greater in the 15-ppm group animals as compared with the 7.5-ppm animals (Table 4). Squamous metaplasia was observed consistently in Level 1, presumably because this level of the nose was exposed to the highest concentration of formaldehyde, and occasionally in Level 2, especially in the 15-ppm exposure groups (Figure 2). This lesion was not observed in Level 3. Squamous metaplasia was observed most commonly on the medial surface of the maxilloturbinates, facing the nasal septum. Other affected sites included the lateral wall, tips of the nasoturbinates, and, occasionally, the dorsal surface of the dorsal meatus. Squamous metaplasia was characterized microscopically by areas of replacement of the normal respiratory epithelium by a thin to occasionally moderately thick layer of stratified squamous epithelium. In some cases, the squamous epithelium was quite thin and was recognized as squamous epithelium by the presence of a layer of thin, laterally flattened cells on the epithelial surface typical of squamous epithelium. Often, a thin to moderately thick layer of keratin, sometimes containing cell debris, was present on the epithelial surface of turbinates in exposed (Figure 3B) but not control (Figure 3A) mice. In a few noses, accumulated keratin was present in the space behind a turbinate scroll, and in some cases filled the space behind the scroll (Figure 3C).

Figure 2

Squamous Metaplasia of the Nasal Cavity in Mice Exposed to Formaldehyde

Figure 3

Keratin Accumulation and Osteogenesis in Nasal Cavity of Mice Exposed to Formaldehyde

Osteogenesis, proliferation of new bone, was observed in the turbinate bone in a few C3B6.129F1-Trp53^tm1Brd and B6.129-Trp53^tm1Brd animals exposed to formaldehyde (Table 4). Microscopically, the osteogenesis had a similar appearance in the two strains and in each animal consisted of a small focus of proliferating bone within the lamina propria of the affected turbinate characterized by eosinophilic osteoid containing numerous large plump osteoblast nuclei (Figure 3D).

Neoplasms in C3B6.129F1-Trp53^tm1Brd Mice

Several neoplasms were observed in control and formaldehyde-exposed C3B6.129F1-Trp53^tm1Brd mice, and none was considered caused by formaldehyde exposure (Table 5). Three lymphomas were found in the early-death C3B6.129F1-Trp53^tm1Brd animals: one in the 7.5-ppm group involving the thymus and two lymph nodes, and the remaining two in the 15-ppm group involving only the thymus. No lymphomas were observed in the control group. The incidences of lymphoma in the formaldehyde-treated groups were not statistically significant (p > 0.05) relative to controls. The lymphomas all had a typical and similar morphological appearance, and consisted of diffuse sheets of relatively large lymphocytes with large, moderately pleomorphic basophilic, granular nuclei with one or more prominent nucleoli and a scant amount of eosinophilic cytoplasm (Figure A-1).

Table 5

Neoplasms in C3B6.129F1-Trp53^tm1Brd Mice Exposed to Formaldehyde.

Figure A-1

Thymus, Cortex, Lymphoma of C3B6.129F1-Trp53^tm1Brd Mice Exposed to Formaldehyde

Five osteosarcomas were found in bone and skeletal muscle of C3B6.129F1-Trp53^tm1Brd mice (Table 5). Osteosarcomas were present in two scheduled sacrifice animals (one control and one 7.5-ppm animal), and both had metastasized to the lung. The three remaining osteosarcomas occurred in the early-death control animals. Microscopically, the osteosarcomas were discrete masses composed of irregular intersecting trabeculae of eosinophilic osteoid, the matrix material for bone, admixed with variable numbers of highly pleomorphic neoplastic spindle-shaped cells (Figure 4A). Although osteosarcomas are considered to originate from bone, three osteosarcomas (from the early-death control animals) had skeletal muscle attached to them but no bone. We assumed they had originated in bone and had invaded adjacent skeletal muscle, but that the bony origins of these neoplasms were not included in the microscopic sections. Osteosarcomas stained positively for vimentin, an intermediate filament typically present in cytoplasm of non-epithelial cells (Figure 4B), and stained negatively for smooth muscle actin, myogenin, and F4/80.

Figure 4

Neoplastic Lesions in Trp53⁺ Mice Exposed to Formaldehyde

In addition to osteosarcomas, an undifferentiated sarcoma was also observed in the skeletal muscle of a 7.5-ppm C3B6.129F1-Trp53^tm1Brd animal. This neoplasm was a large, discrete mass consisting of numerous large, round to ovoid to elongated cells with abundant eosinophilic cytoplasm and small basophilic nuclei. Hepatocellular adenoma and carcinoma, histiocytic sarcoma, and hemangiosarcoma also were observed in some animals, but the incidences were not significantly increased or concentration related (Table 5).

Osteosarcomas in two control mice and a hemangiosarcoma in one 7.5-ppm animal were in contact with or had enveloped the microchip transponders used for animal identification. Sarcomas, osteosarcomas, and histiocytic sarcoma in eight C3B6.129F1-Trp53^tm1Brd animals were located remotely from the microchips.

Neoplasms in B6.129-Trp53^tm1Brd Mice

A variety of observed neoplasms in formaldehyde-exposed B6.129-Trp53^tm1Brd mice were not considered chemical related (Table 6). Lymphomas were present in two formaldehyde-exposed B6.129-Trp53^tm1Brd animals, one in the 7.5-ppm group and one in the 15-ppm group. The lymphoma in the 7.5-ppm animal, which appeared to have originated in the thymus, had spread widely, being present in nearly every tissue examined. In contrast, the lymphoma in the 15-ppm animal appeared to have originated in the mesenteric lymph node with a small amount of involvement of the bronchial lymph node. The lymphomas appeared morphologically similar to one another and to the lymphomas observed in the C3B6.129F1-Trp53^tm1Brd animals (Figure A-1).

Table 6

Neoplasms in B6.129-Trp53^tm1Brd Mice Exposed to Formaldehyde.

Skeletal muscle rhabdomyosarcomas were observed in five formaldehyde-exposed B6.129-Trp53^tm1Brd animals, one in the 7.5-ppm group and four in the 15-ppm group (includes early death animals) (Table 6). Rhabdomyosarcomas were not observed in B6.129-Trp53^tm1Brd control animals. Although a statistically significant trend (p = 0.042) was found, the incidence of rhabdomyosarcomas in the 15-ppm group was not statistically significant (p = 0.133) relative to controls. Microscopically, the neoplasms were large masses composed of solid foci and interlacing bands of neoplastic cells generally admixed with varying amounts of dense fibrous tissue stroma (Figure 4C). The neoplasms were composed primarily of variably sized pleomorphic polygonal to fusiform cells, with large ovoid to fusiform, moderately basophilic to vesicular nuclei with one to several prominent nucleoli and small to moderate and occasionally large amounts of eosinophilic cytoplasm with indistinct borders. Few to numerous mitotic figures were present, and varying numbers of large multinucleated cells resembling rhabdomyoblasts were observed. Cross striations were not observed; however, the tumors stained positively for vimentin, a marker for cells of mesenchymal origin including skeletal muscle, and myogenin, a nuclear transcription factor essential for skeletal muscle development and repair, and a specific marker for skeletal muscle (Figure 4D).

Various other neoplasms occurred at low incidences in control and formaldehyde-exposed B6.129-Trp53^tm1Brd animals, all of which were considered background lesions unrelated to formaldehyde exposure (Table 6). Microchip transponders were found in contact with, or enveloped by, tumors in five B6.129-Trp53^tm1Brd animals exposed to formaldehyde. Sarcomas in 10 B6.129-Trp53^tm1Brd animals were not associated with microchip transponders.

Discussion

Formaldehyde-induced injury to the nasal epithelium, regenerative proliferation, and mutations in these proliferating cells are key steps in the pathogenesis of formaldehyde-induced nasal SCC.^4,6 Mutations in the tumor suppressor gene Trp53 have been detected in formaldehyde-induced nasal tumors and in preneoplastic hyperkeratotic plaques, indicating that Trp53 mutations are involved in the pathogenesis of SCC.^9,10 Based on these data, Trp53⁺ mice were expected to be highly susceptible to formaldehyde-induced nasal SCC, and possibly leukemia and other neoplasms. Under the conditions of this short-term study, however, formaldehyde exposure did not result in SCC of the nasal cavity or in an increased incidence of leukemia or lymphohematopoietic neoplasms in two genetically susceptible Trp53⁺ mouse strains. Formaldehyde-induced loss heterozygosity (LOH) in Trp53 and loss of tumor suppressor function may have been insufficient to induce neoplasia. Although mutations to Trp53 can result in a dysfunctional protein, some mutations to Trp53 can result in a gain of function (GOF) that promotes tumor development.^36,37 Formaldehyde-induced mutations of Trp53 that result in GOF may be necessary for induction of neoplasia.

Although rats are more susceptible than mice to formaldehyde-induced nasal tumors,³ a Trp53⁺ rat strain was not available at the time of this study. Mice are reportedly less susceptible than rats because of their greater ability to reduce their minute ventilation upon repeated formaldehyde exposures,^38,39 thereby reducing tissue damage and cell turnover in the nasal mucosa. Spontaneous SCC of the nasal cavity is extremely rare in rats and mice. Tumors of the nasal cavity have not been observed in NTP historical controls for B6C3F1/N or B6.129-Trp53^tm1Brd mice. Background nasal tumor incidences for the C3B6.129F1-Trp53^tm1Brd were not available. Induction of SCC in wild type mice by formaldehyde is also extremely rare. Exposure of C57BL/6 x C3HF1 mice to 14.3-ppm formaldehyde for 2 years followed by a 6-month holding period resulted in only two mice with nasal tumors.³

In this study, mice were exposed to concentrations of formaldehyde that caused significant cell injury, inflammation, and regeneration in the nasal mucosa, which are recognized as key events in nasal SCC development.⁵ Although significant regenerative cell proliferation and squamous metaplasia were observed in exposed mice, a higher formaldehyde concentration, a longer exposure duration, or a longer holding period might be necessary for accumulation of genetic alterations in Trp53 and development of SCC in mice. The severity of nasal lesions in both mouse strains, the significantly reduced body weights of C3B6.129F1-Trp53^tm1Brd mice, and the lack of body weight gain in both strains, however, indicated that exposure to 15 ppm for 8 weeks represented a maximum tolerated dose. An 8-week exposure duration was considered sufficient because the doubling time for hematopoietic stem cells is reported to be between 2 and 4 weeks with the entire hematopoietic stem cell pool turning over every 8 weeks.^40,41 Although exposed mice could have been held longer, 50 weeks of age was selected because, at approximately 70 weeks of age, about one-half of Trp53⁺ mice reportedly develop background tumors.³³

Formaldehyde exposure did not significantly increase the incidence of leukemia or lymphohematopoietic neoplasms in either B6.129-Trp53^tm1Brd or C3B6.129F1-Trp53^tm1Brd mice. The formaldehyde exposure concentrations were observed to cause disruption of the nasal epithelium, potentially allowing exposure of the underlying nasal cavity stem cells and circulating hematopoietic stem cells in the nasal vasculature. Maximal exposure of stem cells would be expected to occur following disruption (ulceration, necrosis) of the epithelium. As noted in this study, however, the accessibility of the nasal stem cells to inhaled formaldehyde could decrease as the damaged nasal epithelium is rapidly replaced by the more resistant squamous epithelium. In addition, the squamous epithelium produced multiple layers of keratin that might also protect underlying cells from inhaled formaldehyde. Whether a similar phenomenon occurs in exposed rats is unclear. Lymphoma was observed in several formaldehyde-exposed mice and was absent in control groups for both mouse strains. The low incidence of lymphoma in formaldehyde-exposed mice, however, was not statistically significant. Lymphoma was not considered exposure related because of the low incidence, the lack of statistical significance, and because lymphoma is one of the most common spontaneous tumors reported in Trp53⁺ mice.^32,42

Sarcomas were the most prevalent tumor observed in B6.129-Trp53^tm1Brd mice and were the most common cause of early death due to the rapid growth of these tumors. The use of subcutaneous microchip transponders for animal identification might have influenced the incidence of sarcomas in B6.129-Trp53^tm1Brd. Sarcomas can occur in B6.129-Trp53^tm1Brd mice in association with subcutaneous microchips but can also occur spontaneously and in reaction to other treatments.⁴³ Although the mechanism for a synergistic or additive effect of subcutaneous microchip implantation and formaldehyde exposure on skeletal muscle sarcoma development in B6.129-Trp53^tm1Brd is not clear, evidence is insufficient to rule out such an effect.

Osteosarcoma was a common spontaneous lesion in C3B6.129F1-Trp53^tm1Brd mice, occurring in about 15% of controls and in only one animal exposed to formaldehyde (7.5 ppm). Osteosarcomas are generally uncommon neoplasms in mice but appear to be more common in Trp53⁺ mice. Microchips were found within or in contact with tumors in two of the four control mice with osteosarcoma, and in one mouse in the 7.5-ppm group, the microchip was in contact with a hemangiosarcoma. As noted with the skeletal muscle sarcomas in B6.129-Trp53^tm1Brd mice, immunostaining of osteosarcomas associated with, or remote from, microchips showed no differences.

Conclusion

Under the conditions of this study, inhalation exposure to a maximum tolerated dose of formaldehyde did not cause an increased incidence of nasal SCC, leukemia, or lymphohematopoietic tumors in Trp53⁺ mice. Both mouse strains developed a variety of neoplasms, but all were considered spontaneous background lesions and not a result of formaldehyde exposure. The results of this short-term carcinogenicity study do not support a role for Trp53 in formaldehyde-induced neoplasia.

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National Toxicology ProgramNTP report on the toxicology studies of aspartame (CAS No. 22839-47-0) in genetically modified (FVB Tg.AC hemizygous) and B6.129-Cdkn2atm1Rdp (N2) deficient mice and carcinogenicity studies of aspartame in genetically modified [B6.129-Trp53tm1Brd (N5) haploinsufficient] mice (feed studies). Natl Toxicol Program Genet Modif Model Rep. 2005 Oct;(1):1–222. [Natl Toxicol Program Genet Modif Model Rep.].https://www​.ncbi.nlm​.nih.gov/entrez/query​.fcgi?cmd=Retrieve&db​=PubMed&list_uids​=18685711&dopt​=Abstract18685711 [PMC free article: PMC8935287] [PubMed: 18685711]

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National Toxicology ProgramNTP toxicology studies of acesulfame potassium (CAS No. 55589-62-3) in genetically modified (FVB Tg.AC Hemizygous) mice and carcinogenicity studies of acesulfame potassium in genetically modified [B6.129-Trp53(tm1Brd) (N5) Haploinsufficient] mice (feed studies)mice. Natl Toxicol Program Genet Modif Model Rep. 2005 Oct;(2):1–113. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8935293] [PubMed: 18784762]

25.

National Toxicology ProgramToxicology studies of sodium bromate (CAS No. 7789-38-0) in genetically modified (FVB Tg.AC Hemizygous) mice (dermal and drinking water studies) and carcinogenicity studies of sodium bromate in genetically modified [B6.129-Trp53tm1Brd (N5) haploinsufficient] mice (drinking water studies). Natl Toxicol Program Genet Modif Model Rep. 2007 Mar;(6):1–169. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8935282] [PubMed: 18784759]

26.

National Toxicology ProgramNTP report on the toxicology studies of dichloroacetic acid (CAS No. 79-43-6) in genetically modified (FVB Tg.AC hemizygous) mice (dermal and drinking water studies) and carcinogenicity studies of dichloroacetic acid in genetically modified [B6.129-Trp53(tm1Brd) (N5) haploinsufficient] mice (drinking water studies). Natl Toxicol Program Genet Modif Model Rep. 2007 Apr;(11):1–168. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8935288] [PubMed: 18784768]

27.

National Toxicology ProgramNTP report on the toxicology studies of dicyclohexylcarbodiimide (CAS No. 538-75-0) in F344/N rats, B6C3F 1 mice, and genetically modified (FVB Tg.AC hemizygous) mice and carcinogenicity study of dicyclohexylcarbodiimide in genetically modified [B6.129-Trp53 tm1Brd (N5) haploinsufficient] mice (dermal studies). Natl Toxicol Program Genet Modif Model Rep. 2007 Sep;(9):1–138. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8935299] [PubMed: 18784765]

28.

National Toxicology ProgramToxicology studies of bromodichloromethane (CAS No. 75-27-4) in genetically modified (FVB Tg.AC Hemizygous) mice (dermal, drinking water, and gavage studies) and carcinogenicity studies of bromodichloromethane in genetically modified [B6.129-Trp53(tm1Brd) (N5) haploinsufficient] mice (drinking water and gavage studies). Natl Toxicol Program Genet Modif Model Rep. 2007 May;(5):1–227. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8950459] [PubMed: 18784761]

29.

National Toxicology ProgramToxicology studies of allyl bromide (CAS No. 106-95-6) in genetically modified (FVB Tg.AC hemizygous) mice and carcinogenicity studies of allyl bromide in genetically modified [B6.129-Trp53tm1Brd (N5) Haploinsufficient] mice (dermal and gavage studies). Natl Toxicol Program Genet Modif Model Rep. 2008 Apr;(7):1–122. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8935283] [PubMed: 18784760]

30.

NTP Toxicology and Carcinogenesis Studies of Mixtures of 3'-Azido-3'-Deoxythymidine (AZT), Lamivudine (3TC), and Nevirapine (NVP) (CAS Nos. 30516-87-1, 134678-17-4, 129618-40-2) in Genetically Modified C3B6.129F1-Trp53(tm1Brd) N12 Haploinsufficient Mice (in utero and postnatal gavage studies). Natl Toxicol Program Genet Modif Model Rep. 2013; (16):1-236. Natl Toxicol Program Genet Modif Model Rep. [PMC free article: PMC8935284] [PubMed: 24503698]

31.

National Toxicology ProgramToxicology study of senna (CAS No. 8013-11-4) in C57BL/6NTAC Mice and toxicology and carcinogenesis study of senna in genetically modified C3B6.129F1/Tac-Trp53tm1Brd haploinsufficient mice (Feed Studies). Natl Toxicol Program Genet Modif Model Rep. 2012 Apr;(15):1–114. [Natl Toxicol Program Genet Modif Model Rep.]. [PMC free article: PMC8935298] [PubMed: 23001288]

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Appendix A. Supplemental Material