Nitrapyrin

Carcinogenicity

Description of key information

NOEL (toxicity) = 5 mg/kg bw/day (males); NOEL (toxicity) = 20 mg/kg bw/day (females); NOEL (carcinogenicity) = 20 mg/kg bw/day (males); NOEL (carcinogenicity) = 60 mg/kg bw/day (females); 2 year (rat); EPA OPP 83-1, 83-2, Szabo et al. (1989)

NOEL (toxicity) = 25 mg/kg bw/day (male/female); NOEL (carcinogenicity) = 75 mg/kg bw/day (male/female) - no oncogenic response observed; 2 year (mouse); EPA OPP 83-5, Quast et al. (1990)

No NOEL identified (excessive toxicity at 125 and 250 mg/kg bw/day); 2 year (mouse); EPA OPP 83-2, OECD 451, EEC Directive 87/302/EEC, Part b, MAFF 1985; Stebbins & Cosse (1997)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

chronic

Species:

other: rat

Quality of whole database:

Three 2-year studies, on rats and mice, are available to address the carcinogenicity endpoint. All three studies were conducted under GLP conditions and in accordance with standardised guidelines and were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the database is high.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to carcinogenicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to carcinogenicity.

Additional information

Three studies are available to help assess the carcinogenic potential of the test material. All three studies were conducted under GLP conditions and the methods followed standardised guidelines. All three studies were thereby assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997) and the mouse studies are considered together in a weight of evidence approach. Further information on the mouse studies is also provided in the form of a summary document written by LaRocca et al. (2015).

In the first study (Szabo et al., 1989) the oncogenicity, and chronic repeated dose toxicity, of the test material was investigated under GLP conditions and in accordance with EPA OPP 83-1 and 83-2 in Fischer 344 rats.

During the study sixty rats/sex/dose level were fed test material in their diets at concentrations formulated to provide 0 (control), 5, 20, or 60 mg/kg bw/day. Ten rats/sex/dose level were randomly designated at the start of the study for an interim sacrifice at 12 months. The remaining animals were continued on test until 24 months or they died or were killed moribund. Parameters measured during the study were clinical observations; body weights; feed consumption; haematology; clinical chemistries and electrolytes; urinalyses; and gross and histopathologic examination.

The primary effects of administration of 60 mg test material/kg bw/day to male rats for up to 2 years were a decrease in body weight gain relative to controls in the second year of the study, a statistically significant increase in early mortality, increased relative and absolute kidney weights (24-month sacrifice only), histopathologic changes in the kidneys characterised at the 12-month sacrifice as protein droplet accumulation (α2u-globulin) in the epithelial cells of the proximal convoluted tubules and at the 24-month sacrifice as an increase in the severity of chronic progressive glomerulonephropathy and the presence of primary renal tumours, increased relative and absolute liver weights, and histopathologic changes in the liver characterised as hypertrophy and fatty vacuolation of centrilobular hepatocytes and an increased incidence of biliary hyperplasia. Other effects of the treatment regimen, which may be secondary to the aforementioned changes, included lower alanine transaminase and aspartate transaminase activity and increased total serum bilirubin concentration during the first 12-months; increased blood urea nitrogen (all sampling periods); decreased albumin and total protein concentrations; decreased urine specific gravity; increased absolute and relative adrenal gland weight (24-month sacrifice only); and coalescence of thyroid follicles. Mechanistically, the altered clinical chemistry parameters reflect the presence of toxicologic lesions in the liver and kidneys of these animals. Adrenal gland and thyroid gland changes most likely are physiologic responses to stress in animals with severe renal disease and ongoing hepatic alterations.

Altered parameters in females at the high dose level were a decrease in body weight gain relative to control within the first 50 days on study, increased absolute and relative kidney weights (12-month sacrifice only), histopathologic changes in the kidney consisting of an increase in the severity of the chronic progressive glomerulonephropathy which occurs in this strain of rat, increased absolute and relative liver weights, and histopathologic changes in the liver characterised as hypertrophy and fatty vacuolation of centrilobular hepatocytes. The severity and incidence of these lesions were generally less in females than in males. As in male rats, several clinical chemistry parameters were elevated in relation with the histopathologic identified organ toxicity, however this occurred only at the 24-month sampling period. Survival in high dose level female rats was unaffected by test material treatment.

At the 20 mg/kg bw/day treatment level, the effects of treatment seen in males consisted of a decrease in body weight gain relative to control during the final 4 months of this study, increased relative and absolute kidney weights (24-month sacrifice only), histopathologic changes in the kidneys characterised at the 12-month sacrifice as protein droplet accumulation in the epithelial cells of the proximal convoluted tubules, increased relative and absolute liver weights, and histopathologic changes in the liver characterised as hypertrophy and fatty vacuolation of centrilobular hepatocytes and an increased incidence of biliary hyperplasia. Other effects of the treatment regimen, which may be secondary to the aforementioned changes, included: increased blood urea nitrogen (18 and 24-month sampling periods); decreased albumin and total protein concentrations (18 and 24 month sampling periods); and increased absolute and relative adrenal gland weight (24-month sacrifice only). The altered clinical chemistry parameters reflect the presence of toxicologic lesions in the liver and kidneys of these animals. Adrenal gland changes, as in the high dose level male rats, most likely are a physiologic response to stress in animals with ongoing renal and ongoing hepatic disease.

In female rats administered 20 mg/kg bw/day, no effects of treatment were seen in the two years of dietary administration.

The chronic toxicity noted at the high dose in both male and female rats in this study was considered indicative of a sufficient challenge for the assessment of oncogenicity. Male rats administered 60 mg/kg bw/day had a sex-specific incidence of primary renal tumours which were not seen in any other male dose level or in any female dose level. Besides the male renal tumours, there was no increase in tumours of any type in any other organ or tissue at any of the dose levels tested which was attributable to test material administration. This effect is not human relevant; in a two-year chronic rat bioassay with nitrapyrin, there was no indication of treatment-related tumors except for an increase in male rat kidney tumors related to the u2u-globlin mechanism, which is not considered to be relevant to humans.

Under the conditions of the study, the No Observed Effect Level for all parameters in male rats was 5 mg/kg bw/day and for females was 20 mg/kg bw/day. The NOEL for carcinogenicity in male rats was 20 mg/kg bw/day and for females was 60 mg/kg bw/day.

In the second study (Quast et al., 1990) the repeated dose toxicity and oncogenicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPP 83-5.

During the study test material was administered to 50 male and 50 female B6C3F1 mice of each sex at 0, 5, 25 or 75 mg/kg/day for 2 years. A satellite group of 10 mice per sex per dose was included for evaluation after 1 year on test. Parameters evaluated included general appearance and demeanour, mortality, body weights, feed consumption, haematology, clinical chemistry, selected organ weights, gross and histopathologic observations.

The liver and kidneys were affected by treatment in male and female mice given the high dose. A slight increase in liver weight and histopathologic changes occurred in high dose males from the satellite and the oncogenicity groups. In addition, the serum alanine aminotransferase activity (ALT) in the high dose satellite group of male mice was elevated. In the female high dose group, the absolute and relative liver weights were statistically elevated in the oncogenicity study. Kidney weights in high dose male and female mice were generally elevated without a corresponding morphologic alteration.

The treatment-related response of the liver and kidneys in these mice was consistent with the tissues previously identified as target organs in the range-finding and subchronic studies. Treatment-related changes were also observed in the intestinal epithelial cells of the duodenum in male and female middle and high dose mice. These changes were considered physiologic in nature, associated with the presence of the test material within the lumen of the intestinal tract, since the mice were not fasted prior to necropsy.

The tumours present in male and female mice in this oncogenicity study were typical of those observed in control mice from other mouse oncogenicity studies. Variations in tumour incidence between treated and control mice were not statistically identified.

Under the conditions of the study, the No Observed Effect Level in this study was 25 mg/kg/day for toxicity, based upon the liver and kidney effects at 75 mg/kg/day. Dietary administration of 5, 25 or 75 mg test material/kg/ day for 2 years to B6C3F1 mice did not result in an oncogenic response.

In the third study (Stebbins & Cosse, 1997) the carcinogenic properties of the test material were investigated under GLP conditions and in accordance with EPA OPP 83-2, OECD 451, EEC Directive 87/302/EEC, Part b, and MAFF 1985.

During the study test material was administered to 50 male and 50 female B6C3F1 mice at 0, 125 or 250 mg/kg/day for 2 years. An interim group of 10 mice/sex/dose was included in the study for evaluation of chronic toxicity after 12 months. Body weights, feed consumption, clinical appearance, and mortality were monitored throughout the study. Haematological parameters were measured following 12 and 24 months of dosing. Necropsies were conducted at approximately 12 and 24 months, selected organ weights were collected, and gross pathologic and histopathologic examinations were conducted.

The maximum tolerated dose was exceeded in males and females given 125 or 250 mg/kg/day, based on treatment-related hepatocellular necrosis and hepatocellular proliferation. In addition, significant treatment-related depression of body weights occurred in males administered 250 mg/kg/day. Body weights from this group of mice were decreased 3 - 12% relative to controls, and body weight gains were decreased 26 - 33% relative to controls, over much of the dosing period.

Chronic dietary administration of test material caused excessive toxicity of the liver, which included hepatocellular necrosis and increased hepatocellular turnover in both sexes administered 125 or 250 mg/kg/day. These alterations led to the development of an increased incidence of hepatocellular adenomas and/or carcinomas in males administered 250 mg/kg/day, and females administered 125 or 250 mg/kg/day. Males and females administered 125 or 250 mg/kg/day also had an increased incidence of hyperplasia, papillomas, and/or squamous cell carcinomas of the nonglandular mucosa of the stomach.

Other primary treatment-related alterations consisted of vacuolation, hyperplasia and hypertrophy of mucosal epithelial cells of the duodenum and jejunum in males and females of both treatment groups.

In conclusion, dietary administration of test material for 2 years to B6C3F1 mice resulted in tumorigenesis of the liver in males given 250 mg/kg/day and females given 125 or 250 mg/kg/day, and tumorigenesis of the stomach in males and females given 125 or 250 mg/kg/day.

Data from the first two studies, together with available genetic toxicity data, demonstrate that the test material was not genotoxic and that there were no tumours elicited in rats or mice that were relevant for human risk assessment. However, the third study (Stebbins & Cosse 1997), conducted at two substantially higher-dose levels (0, 125 or 250 mg/kg/day) than the original studies (0, 5, 20, or 60 mg/kg bw/day; and 0, 5, 25 or 75 mg/kg/day) identified liver, stomach, epididymal and Harderian gland tumours.

In order to assess the relevance of these findings for human risk assessment, relevant microscopic changes in these tissues were examined and genotoxicity and mechanistic data were examined. The maximum tolerated dose had been exceeded in mice given 125 or 250 mg/kg/day, based on 26 - 33% decreased body weight gains (males - 250 mg/kg/day), hepatocellular necrosis and compensatory hepatocellular proliferation (males and females -125 and 250 mg/kg/day). Increased incidences of proliferative lesions in the forestomach mucosa were believed to be secondary to the irritant effects of the substance. The forestomach effects were interpreted to not be a direct carcinogenic effect.

Higher incidences of Harderian gland adenomas (females) and undifferentiated sarcomas in the epididymis represented normal biological variations in incidence and were unrelated to the test material. Therefore, it is considered that exposure to the substance does not produce target organ toxicity in exposed individuals and would not be expected to increase the risk of cancer.

In the summary document written by LaRocca et al. (2015), several studies were considered to investigate the human relevance of nitrapyrin-induced liver tumours, which were evaluated based upon the Bradford Hill criteria followed by subsequent application in a Human Relevance Framework (HRF). The mode-of-action (MoA) for the observed nitrapyrin-induced liver tumours is characterised by the following key events: 1) constitutive androstane receptor (CAR) nuclear receptor (NR) activation, and 2) increased hepatocellular proliferation, which leads to increased hepatocellular foci and tumour formation (apical endpoint). Several relevant in vivo and in vitro studies were performed in order to generate the necessary data to support these key events. The key events show clear, threshold-based, dose-responsive alterations and provide informative, temporal-specific characterisation of nitrapyrin-induced liver effects. The key events also demonstrate reversibility upon discontinuance of treatment, which is consistent with a CAR-mediated MoA.The data provide convincing evidence that the key events, as well as the hepatocellular tumours, do not occur at, or below, a defined point-of-departure (POD) dose level of 75 mg/kg/day. Additional data, including in vitro studies evaluating the proliferative response of primary mouse and human hepatocytes to nitrapyrin and showing that nitrapyrin exposure induced a clear, dose-responsive increase in DNA synthesis in mouse hepatocytes but no change in DNA synthesis in human hepatocytes at any dose, also support that this MoA, specifically for nitrapyrin, is not relevant for humans. Overall, the data support that nitrapyrin-induced mouse liver tumours are mediated by CAR activation and due to qualitative differences between mice and humans, nitrapyrin is not likely to be carcinogenic to humans. 

Justification for selection of carcinogenicity via oral route endpoint:

Multiple studies have been provided to address the carcinogenicity endpoint. Since all the studies are considered to be reliable without restriction, a single study could not be selected as key over the others.

Carcinogenicity: via oral route (target organ): digestive: liver; digestive: stomach; urogenital: kidneys