Nitrapyrin

Endpoint summary

Description of key information

ORAL

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

NOEL = 3 mg/kg bw/day (male/female); 1-year (dog); EPA OPP 83-1; Barna-Lloyd et al. (1989)

NOEL = 27 mg/kg bw/day (male/female); 90-day (rat); similar to OECD 408; Anon. (1962)  

INHALATION

No study available.

DERMAL

NOEL = 1000 mg/kg bw/day, 21 day (rabbit); OECD 410, EPA OPP 82-2, MAFF, EEC Directive 67/548/EEC; Cosse et al. (1992)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

A 2 year study is available (in rats), a 1 year study is available (in dogs) and two 90-day studies are available (in rats and mice). The 2 year rat study, 1 year dog study and 90-day mouse study were all conducted under GLP conditions and in accordance with standardised guidelines. The three studies were each assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The 90 day rat study was conducted to sound scientific principles; since the methodology was similar to that which is outlined in a standardised guideline it was assigned a reliability score of 2 in line with the criteria of Klimisch et al. (1997). Overall the quality of the database is high.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study was conducted under GLP conditions and in accordance with standardised guidelines; it was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the database is high.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2.2 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study was conducted under GLP conditions and in accordance with standardised guidelines; it was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the database is high.

Additional information

Oral

In the key study (Szabo et al., 1989) the two year repeated dose toxicity of the test material, via the oral route, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines 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.

The No-Observed-Effect-Level (NOEL) for all parameters in males was 5 mg/kg bw/day and for females was 20 mg/kg bw/day. 

Numerous supporting studies are also available.

 

The first supporting study was reported by Barna-Lloyd et al. (1989). The repeated dose toxicity of the test material was investigated, via the oral route, in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPP 83-1.

During the study, test material was fed in the diet to beagle dogs (4/sex/group) at dose levels of 0 (control), 0.5, 3, or 15 mg/kg body weight/day, for a period of one year. Study parameters were overall health status, body weights and gains from baseline, feed consumption, haematology, clinical chemistry, urinalysis, gross lesions at necropsy, and histopathologic findings.

Changes resulting from the administration of test material involved primarily the liver and were seen only in the animals, both male and female, given the highest dose level (15 mg/kg bw/day). These changes were increased absolute and relative liver weights and diffuse enlargement of hepatocytes. Serum alkaline phosphatase activity, judged to be associated with the liver enlargement, and cholesterol levels were also increased in the high dose level animals. No other study parameters were affected by treatment in the high dose level animals; no study parameters were affected by treatment in the middle and low dose level animals.

Under the conditions of the study the No Observed Effect Level for chronic dietary administration of test material was determined to be 3 mg/kg bw/day. 

 

Further supporting information is available in the form of two 90-day studies. The first was conducted with rats (Anonymous, 1962). The repeated dose toxicity of the test material was investigated via the oral route. The methodology followed was similar to that which is outlined in the standardised guideline OECD 408.

During the study test material was fed, as a part of the diet, to groups of male and female rats for a period of 94 days. A dietary level of 0.03%, that was calculated to be an average dose of 27 mg/kg body weight/day, was tolerated without evidence of adverse effect as judged by general appearance and behaviour, mortality, food consumption records, serum urea nitrogen and alkaline phosphatase determinations, haematological determinations, final average body and organ weights, and gross and microscopic examination of vital organs.

At 0.3%, that was calculated to be an average dose of 270 mg/kg body weight/day, growth was retarded in both sexes but especially the male. The organ/body weight ratio in males was increased for the liver, kidney, spleen, lung and testes. In females the organ/body weight ratio was increased for the liver, kidney and spleen. There were both gross and microscopic pathological effects in the livers and kidneys of both sexes.

A dietary level of 0.1%, that was calculated to be an average dose of 90 mg/kg body weight/day, appeared to be about the borderline for toxic effects to be observed. Growth rates for males were lower than for the controls although the difference had no statistical significance. There was a statistically significant lowering of the growth rate for females. The statistically significant differences in organ/body weight ratios were only with the liver for the females and the liver, kidney and testes for the males. There were both gross and microscopic pathological effects observed in the liver and kidney although milder in effect than those occurring at the 0.3% level.

There were no clear differences in the blood and serum biochemical determinations that could be attributed to treatment with the test material, even at the 0.3% level.

 

The second 90-day study was conducted with mice and was reported by Daly in 1995. The study was conducted under GLP conditions and in accordance with the standardised guideline EPA OPP 82-1.

During the study, test material was administered orally, via dietary admixture to 80 B6C3F1 mice (10/sex/group) at dose levels of 0, 200, 300, 400 and 600 mg/kg bw/day for the males and 0, 200, 400, 600 and 800 mg/kg bw/day for the females for a period of up to 3 months.

However, based on the finding of increased absolute and relative liver weights and hepatocellular hypertrophy in mice of both sexes given test material in the diet at 200 mg/kg/ day for 95 or 96 days, a no-observed-effect level (NOEL) was not determined.

In a further supporting study (Barna-Lloyd et al., 1988), the repeated dose toxicity of the test material was investigated in a probe study conducted under GLP conditions. B6C3F1 mice were exposed to the test material in the diet for 2 weeks. Two sets of dose groups (A and B) were treated non-concurrently. Five mice per sex per dose were exposed to the test material at concentrations of 0, 5, 25, 75 and 150 mg/kg bw/day (Group A) and 0, 200, 400 and 600 mg/kg bw/day (Group B).

The 600 mg/kg bw/day dose level was lethal to male mice in 10-12 days. The following differences from control were also attributed to treatment: food consumption, body weight and body weight gain from baseline were decreased in males and females of the 600 mg/kg bw/day dose level; food consumption and weight gain were also decreased in males of the 400 mg/kg bw/day group.

Alanine transaminase was increased in males of the 400 mg/kg bw/day group and in females at 600 and 400 mg/kg bw/day. Absolute and relative liver weights were increased in males at 400 and 200 mg/kg bw/day and in females at 600, 400, 200 and 150 mg/kg bw/day. Pale, enlarged livers were found in both males and females at 400 and 600 mg/kg bw/day. Microscopically, this change consisted of dose related hepatocellular hypertrophy and vacuolation. Severe scattered necrosis of individual hepatocytes was seen in some of the highest dose level males. Less marked microscopic changes were found in the male and female livers at 200 mg/kg bw/day.

Absolute and relative kidney weights were decreased in males at 200 and 400 mg/kg bw/day and females at 600 mg/kg bw/day. Necrosis in the descending portion of the proximal tubules was seen in males only of the 600 mg/kg bw/day group.

Under the conditions of this study, based on the histopathologic liver lesion, the no-observed-effect level (NOEL) in both sexes was 150 mg/kg bw/day. Based on increased absolute and relative liver weights, the NOEL in females was 75 mg/kg bw/day.

In an additional supporting study (Yano and McFadden, 1996), hepatocellular proliferation was assessed retrospectively in the livers of male and female mice given 200 or 400 mg/kg/day of the test material in the diet for two weeks (Barna-Lloyd et al., 1988) by counting hepatocytes stained for proliferating cell nuclear antigen (PCNA).

The purpose of this study was to retrospectively evaluate, by immunohistochemical techniques, whether alterations in hepatocyte proliferation and/ or apoptosis were associated with liver weight increases in male and female mice given the test material in the diet. The study was conducted under GLP conditions.

Hepatocytes were evaluated by immunohistochemical techniques for proliferation and apoptosis from a previously conducted two-week study in which male and female B6C3F1 mice were given 0, 200 or 400 mg/kg/ day of the test material. PCNA was used as an endogenous marker of hepatocellular proliferation and in situ end labelling (ISEL) was utilised as an endogenous marker of hepatocellular apoptosis.

A significant degree of hepatocellular proliferation as determined by PCNA immunohistochemical staining occurred in male mice given 200 or 400 mg/kg/ day for two weeks and to a much lesser degree in female mice given 400 mg/kg/day. Proliferation occurred primarily in the centrilobular regions of the hepatic lobule in males and in the periportal regions of females. Hepatocellular proliferation was not accompanied by alterations in the rate of hepatocellular apoptosis, but was associated with hepatocellular hypertrophy, altered cytoplasmic staining and individual hepatocyte necrosis (males only).

These data suggest that hepatocellular proliferation represents a compensatory response to replace dying hepatocytes.

In the final supporting study (LeBaron, 2009), the proliferative status of hepatocytes, as measured by immunohistochemical staining, was retrospectively evaluated in liver tissue of control and test material-treated mice from a subchronic, 3-month dietary study (Daly, 1995). Liver samples from the reference study that had been previously fixed in formalin and embedded in paraffin were analysed by Ki-67 immunohistochemistry.

Both male and female B6C3F1 mice were analysed (10 animals/sex/dose), which had been treated with 0, 200 or 400 mg/kg bw/day. In the test material-treated male mice there was a dose-related trend of increased hepatocellular proliferation in the 200 and 400 mg/kg bw/day animals when compared to control levels of Ki-67 staining. Statistically identified increases in proliferation (percent Ki-67 positive hepatocytes) were noted in high-dose males in the centrilobular and periportal hepatolobular regions, as well as the total (panlobular) count. The hepatocytes of treated female mice at both doses exhibited increased proliferation, but to a lesser degree than males and did not exhibit a consistent, dose-related trend of increased proliferation.

These data indicate that test material treatment increased hepatocyte mitogenesis in both sexes and these increases were interpreted to be secondary to treatment-related single cell necrosis of hepatocytes.

 

Inhalation

In accordance with Column 1 of REACH Annex VIII, the short-term repeated dose toxicity study (28 days) required under information point 8.6.1 should be conducted using the most appropriate route of administration. Inhalation exposure of the substance is considered to be minimal during manufacture and use.

Furthermore, repeated dose toxicity testing is considered to be adequately addressed with robust, and reliable, data from a number of repeated dose toxicity studies conducted via the oral route. The oral route of exposure is considered to be more relevant for human exposure to the substance.

 

Dermal

The repeated dose toxicity of the test material, when administered via the dermal route, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 410, EPA OPP 82-2, MAFF and EEC Directive 67/548/EEC.

During the study groups of ten New Zealand White rabbits (5/sex/dose) received 15 applications of 0 (control), 100, 500 or 1000 mg test material/kg/day. Data were collected on the following: clinical appearance and behaviour; in-life and terminal body weights; dermal irritation at the test site; clinical chemistry and haematologic parameters; organ weights; and gross and histopathologic appearance of tissues.

There were no indications of systemic toxicity based on in-life clinical observations, body weights, liver, kidneys, and testes weights, clinical pathology parameters, gross pathology or histopathology of the liver and kidneys. During the in-life portion of the study, treatment-related dermal effects consisting of very slight (barely perceptible) to well-defined erythema and/or very slight oedema were observed. One male and two females in the 500 mg/kg/day group had slight to moderate scaling at the site of application.

Treatment-related histopathologic changes at the dermal test site were present in both sexes of rabbits administered 100, 500 or 1000 mg/kg/day. The changes were characterised by very slight to moderate epidermal hyperplasia, focal or multifocal epidermal pustules, multifocal or diffuse hyperkeratosis, multifocal parakeratosis, very slight to moderate dermal inflammation, and multifocal dermal oedema. In males, there generally was a dose-related increase in the incidence and/or severity of effect in the skin at all dose levels. In females there was a dose-related increase in the incidence and/or severity of skin changes when comparing the 100 and 500 mg/kg/day animals, whereas, the effects in 500 and 1000 mg/kg/day females were similar.

Under the conditions of this study, the repeated dermal administration of test material resulted in local dermal irritancy in rabbits administered 100, 500 or 1000 mg/kg/ day. For systemic toxicity, the no observed-effect level (NOEL) was 1000 mg/kg/day, the accepted limit test level for male and female New Zealand White rabbits following dermal administration.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Szabo et al. (1989) was selected as the key study since it was conducted under GLP conditions, to standardised guidelines and used a common rodent species. Furthermore, the exposure duration was the longest of all available studies.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Only one study is available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

Only one study is available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

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 should be classified as STOT RE Category 2 H373 with the signal word "Warning".

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC, the substance should be classified as R48/22, with the symbol Xn.