Nitrapyrin

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 1986 to 27 December 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline, and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

solid: crystalline

Details on test material:

- Appearance: white crystalline solid
- Odour: Chlorine-like

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Parental animals were 4 weeks old when sourced. Females were specified to be nulliparous and non-pregnant.
- Weight at study day -1 (group means): (F0) Males: 118.3 - 120.3 g; Females: 104.7 - 105.5 g
- Weight at study day 1 (group means): (F1) Males: 120.9 - 144.8 g; Females: 97.7 - 113.2 g
- Housing: During acclimation, premating, postmating (males) and postweaning (females), the rats were housed singly in suspended, stainless steel cages with wire mesh floors. Catch pans under the cages were lined with absorbent, antibiotic-impregnated paper. Mating was done in clear plastic cages (48 x 38 x 20 cm) with heat-treated, hardwood chip bedding. Bedding was changed approximately weekly. Females were housed in the plastic cages during gestation, lactation and weaning. After weaning, the parental F1 rats were placed in the wire mesh cages until mating. During acclimation, premating treatment and mating, the males and females were housed in the same room. Following mating, the males were segregated to a separate room.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- The animal rooms were designed to maintain adequate environmental conditions concerning temperature, humidity, air flow and lighting.

IN-LIFE DATES: From: 07 October 1986

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST DIETS
- PREPARATION
The test diets were prepared weekly through most of the study; however, during the weaning stages, the diets were prepared more frequently. The stability of test material in rodent chow was known, by the test laboratory, to be acceptable up to 14 days. Diet preparation was done according to the following scheme. A premix (concentration of test material approximately 10 times that of the highest dietary concentration) was prepared by ball-milling the test material with rodent chow overnight. Test diets were prepared by serial dilution beginning with the premix. Paddle-type mixers were used to blend the diets.

SAMPLING
Reference samples of the diets and premix (1/dose level/sex/preparation date) were collected and stored at ambient temperature. Adequate homogeneity of the dietary formulation was confirmed using an intermediate dose level from the first diet preparation.

DELIVERY
- Premating: The test diets were fed to the animals in glass feed crocks. The F0 and F1 generation animals were maintained on their appropriate test diets for approximately 10 and 12 weeks, respectively. The concentration of test material in these test diets was adjusted weekly for body weights and feed consumption to maintain the targeted dose levels.
- Mating: To avoid potential overdosing of the females during the mating period, males and females cohoused for breeding received test diets with the test material concentration provided to the females for the week prior to cohabitation.
- Postmating (Males): Following the end of mating until their scheduled necropsy, the males were placed on their appropriate premating diets. These diets were again adjusted weekly for feed consumption and body weight.
- Gestation, Lactation and Weaning: During gestation, lactation, weaning and after weaning until their scheduled necropsy, the dams continued to receive the same diets fed during the mating phase. The diets were not adjusted for feed consumption or body weight.
- Postweaning (Pups): Weanlings separated from their dam received the same dietary concentration of test material that their dam was given during weaning until all pups were weaned. Weanlings designated for necropsy or scheduled termination were maintained on these diets until sacrificed. After all pups were weaned, the rats designated to be the F1 generation were fed the same dietary concentration as were fed to the F0 animals during the first week of exposure to the test material. Subsequent diets were adjusted for body weight and feed consumption for the next 11 weeks.

Details on mating procedure:

- M/F ratio per cage: 1 male:1 female of the respective treatment group
- Length of cohabitation: 7 days
- Proof of pregnancy: observation of a vaginal plug, or detection of sperm in a vaginal smear, was referred to as the first day of pregnancy.
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes, a third co-habitation period was used, as necessary.
- After successful mating each pregnant female was caged (how): Sperm positive females were separated from the males. Females were housed in the plastic cages during gestation, lactation and weaning.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the diets and premix for analytical confirmation of test material concentration were taken 3 times per generation. The concentration of test material was determined by a gas chromatographic method using an electron capture detector, following extraction from the rodent chow and a partial clean-up procedure.

Duration of treatment / exposure:

Treatment over 2 generations, 43 weeks in total. See Table 1 for details.

Frequency of treatment:

Continuous (in diet)

Details on study schedule:

EXPERIMENTAL DESIGN
Groups of 30 male and 30 female rats (F0) were maintained on test diets containing sufficient concentrations of test material to achieve the targeted dose levels of 0 (control), 5, 20 or 75 mg/kg bw/day. After being fed diets, containing test material, for approximately 10 weeks, the F0 rats were mated, one male and one female of the respective treatment group to produce the F1 litters. Following weaning of the F1 generation (approximately 4 weeks of age), 30 males and 30 females of the F1 animals from each dose group were selected randomly and designated as the parental animals for the next generation (F2). After the weaning stage the F0 adults and 10 F1 weanlings/sex/dose group were submitted for gross pathologic and histopathologic examination. The remaining F1 weanlings were terminated and discarded. After approximately 12 weeks of receiving test diets (after weaning), the F1 adults were mated, one male to one female of their respective treatment groups, to produce the F2 generations. At the end of this weaning stage, the F1 adults and 10 F2 weanlings/sex/ dose group were submitted for gross pathologic and histopathologic examination. The remaining F2 weanlings were terminated and discarded.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels for this study were selected to be 0 (control), 5, 20 and 75 mg/kg bw/day. The 75 mg/kg bw/day dose level was anticipated to produce parental toxicity, based on the results of findings of available subchronic studies. The intermediate dose level of 20 mg/kg bw/day was selected to aid in demonstrating a dose response. The low dose level, 5 mg/kg bw/day, was anticipated to be a no effect level.
- Rationale for animal assignment: A computer based stratification/randomisation procedure was used to allocate animals into dose groups (30 rats/sex/dose level). Initially the rats were ranked according to pre-test body weight and those rats farthest from the mean were removed from the population until only the number of rats required for the study remained. The remaining rats were assigned to the exposure groups using the stratification (by weight) randomisation procedure. This procedure increases the probability of uniform group mean body weights and associated standard deviations at the initiation of the study. Rats not allocated to a dose group were sacrificed. A computerised randomisation procedure (without stratification by weight) that maintained dose group integrity was used to select animals to be the F1 parental group.

Positive control:

not applicable.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily for signs of treatment-related effects and changes in demeanour.
- Cage side observations included, but were not limited to, observing the animal's ambulatory ability, respiration and peripheral circulation. Availability of feed and water, the presence of faeces and urine and excessive feed wastage were checked. Only deviations from normal were recorded. Records of mortality were maintained. Rats found dead were submitted for gross pathologic examination; those found after normal working hours were refrigerated and examined as soon as practical.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All F0 and F1 adult male rats were weighed on the first day of exposure and weekly thereafter throughout the duration of the study, except for the F1 adult males during mating. F0 and F1 adult female rats were weighed on the first day of exposure and weekly thereafter during the premating periods. Sperm positive females were weighed on days 1 (the day designated as sperm positive), 7, 14 and 21 of gestation. Lactating females were weighed on days 1, 4, 7, 14, 21 and 28 postpartum. Non-sperm positive females without litters were not weighed during the gestation or lactation periods

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption for all F0 and F1 males was measured weekly throughout the study except during the 3-week mating periods. Feed consumption for all F0 and F1 females was measured weekly during the premating period. During gestation, feed consumption was measured weekly in sperm positive females. After parturition, feed consumption of the females with pups was measured weekly for about the first 2 - 3 weeks of lactation and then at 2 - 4 day intervals until weaning was completed. The increased frequency of weighing and feeding was necessary to account for feed ingestion by the pups and the increased consumption of the dam during lactation. Feed consumption was not measured for the weanlings separated from the dam and feeding was done as needed.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Histologic examination of reproductive tissues (uterus, ovaries, cervix, oviducts and vagina) was performed on the control and high dose groups

Sperm parameters (parental animals):

Parameters examined in [F0/F1] male parental generations: Histologic examination of reproductive tissues (epididymis, prostate, testes, seminal vesicles and coagulating glands) was performed on the control and high dose groups.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To reduce the variation in growth of the pups, the F1 and F2 litters were culled on day 4 postpartum to 8 pups (4 males and 4 females, if possible).
Litters with 8 or fewer pups were not culled. Pups to be culled were randomly selected using a table of random numbers. Preferential culling of runts was not performed. All pups selected for culling were asphyxiated with carbon dioxide, examined for gross external and internal alterations and discarded (no tissues were saved).
Weaning of all litters was done on day 28 postpartum. Any weanlings not selected for the next generation of adult animals or selected for gross necropsy were sacrificed without any further examination.

PARAMETERS EXAMINED
All litters were examined as soon as possible after delivery.
The following parameters were recorded for each litter during lactation:
(1) the date of parturition;
(2) litter size;
(3) the number of live and dead pups on day 1, 4, 7, 14, 21 and 28 postpartum;
(4) the sex of the pups on day 1, 4 and 28 postpartum;
(5) the weight of each litter on days 1, 4 (prior to and after culling), 7, 14 and 21; and
(6) on day 28 postpartum individual pup weights. Any visible physical abnormalities or changes in demeanour in the neonates during the lactation period were noted.

GROSS EXAMINATION OF DEAD PUPS: Yes
Pups selected for culling were examined for external and internal alterations.

Postmortem examinations (parental animals):

All F0 and F1 adults were examined for gross pathologic alterations by a veterinary pathologist after the last litter of pups had been weaned for the respective generations. The adult rats were fasted overnight, weighed and anaesthetised with methoxyflurane. The trachea was clamped to prevent aspiration of blood prior to decapitation. The eyes were examined in in situ by gently pressing a wet glass slide against the cornea and observing the eye under fluorescent illumination. Liver and kidney weights were measured and organ/body weight ratios were calculated to aid in assessing parental toxicity. Tissues were collected, saved and preserved in neutral, phosphate-buffered 10 % formalin. The lungs were infused with formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the nasal tissues. Histologic examination of target (liver and kidneys) and reproductive (epididymis, prostate, testes, seminal vesicles, coagulating glands in males and uterus, ovaries, cervix, oviducts, vagina in females) tissues was performed on the control and high dose groups. Examination of tissues from the low and intermediate dose groups was limited to those tissues which demonstrated treatment-related alterations in the high dose group; namely kidneys and liver. All organs and tissues exhibiting gross pathologic alterations were also included for histologic examination.

Postmortem examinations (offspring):

Pathology (Weanling Rats)
At the time of weaning, 10 pups/sex/dose level were randomly selected for a complete gross necropsy examination by a veterinary pathologist. The pups were anaesthetised with methoxyflurane, the trachea clamped to prevent aspiration of blood and then decapitated. Gross pathologic examination was done as described for the adult rats and tissues were collected and preserved in neutral, phosphate-buffered 10 % formalin. Livers were examined for histopathologic alterations.

Statistics:

Body weights, body weight gains and organ weights (absolute and relative) were evaluated by Bartlett's test for homogeneity of variances. Based on the outcome of the Bartlett's test, exploratory data analysis was performed by a parametric or non-parametric analysis of variance (ANOVA), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons if the ANOVA was significant. Statistical outliers were identified by a sequential test, but were not routinely excluded from further analysis. Gestation length and litter size were analysed by the Wilcoxon Rank-Sum test with Bonferroni's correction.
Descriptive statistics (means and standard deviations) were calculated and reported for feed consumption. The fertility indices (mating, conception and gestation) were analysed by Fisher's exact probability test. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Survival indices and other incidence data among neonates were analysed using the litter as the experimental unit by the Wilcoxon test.

Reproductive indices:

Mating index, conception index and gestation index

Offspring viability indices:

Survival index and neonatal sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results (parental animals)" for information

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See "Details on results (parental animals)" for information

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results (parental animals)" for information

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No changes recorded were related to treatment with the test material but were incidental to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
- F0 Males premating: Body weight data collected beginning the day prior to the start of test diet administration (Test Day -1) through Test Day 70 showed no treatment-related effects at any dose level.
- F0 Males Mating: No adverse treatment-related changes were observed in any of the dose groups during the 3 weeks of mating. However, the male intermediate dose group had a higher mean body weight than the control group on Test Day 91 that was statistically significant.
- F0 Males postmating: The low dose group males showed higher mean body weights and weight gains than the control group on Test Day 133 and 140 that were statistically significant. The intermediate dose group males also had higher mean body weights and weight gains that were statistically different from the controls that began on Test Day 91 and continued on Test Day 98 and through Test Day 140. The mean body weights and weight gains of the male high dose group were significantly higher than the control values that began on Test Day 112 and persisted through Test Day 140. These changes were relatively small, in all cases they were less than 10 % different from control, and were not dose-related.
- F0 females Premating: During the premating phase (Test Days -1 - 70), the 75 mg/kg bw/day dose group had statistically significant lower body weight gains than the control group on Test Days 7, 14, 28, 35, 42, 49, 63 and 70. The body weights of the high dose group did not show a statistically significant difference from control until Test Day 70, even though their body weight gains were lower beginning on Test Day 7. The lower rate of body weight gain in the female high dose group was judged to be treatment-related but was not considered excessive and did not adversely affect the ability of the rats to bear litters. Body weights and weight gains of the low and intermediate female dose groups were not different from control during the premating phase of this study.
- F0 Females Gestation: The body weights of the 75 mg/kg bw/day dose group were statistically significantly lower than that of the control group on Gestation Day 1 and remained lower on Gestation Days 7 and 14. On Gestation Day 21 the mean body weight of the 75 mg/kg bw/day dose group was lower than the control but not statistically different. The body weights and weight gains of the low and intermediate dose groups were not different from control during gestation.
- F0 Females Lactation: Following parturition, the mean body weight of the 75 mg/kg bw/day dose group was statistically significantly lower than that of the control group (Day 1 Postpartum) and remained so through the 4 weeks of lactation (Day 28 Postpartum), except on Day 14 Postpartum. During the lactation period, the body weight gain of these females was not different from the control group with the exception of Day 21 Postpartum. The observed differences were considered relatively minor and did not affect the ability of the rat to successfully rear its young through the weaning process. The body weights and weight gains of the low and intermediate dose group were not different from control during the 4 weeks following parturition.

- F1 Males Premating: At the beginning of the 12-week premating period, the F1 adult male high dose group had a significantly lower mean body weight as compared to the controls. Their body weights remained lower than control throughout the entire 12-week premating period; however, their weight gains were not different from control indicating that their growth rate was not affected by test material administration during this phase of the study. No significant alteration of body weight or body weight gain was noted in the 5 or 20 mg/kg bw/day F1 dose groups.
- F1 Males Postmating: Results show that the male high dose group had lower body weights as compared to control from Test Day 112 through the end of the study (Test Day 161), while body weights of the low and intermediate dose levels were not affected by treatment. Body weight gain of the high dose groups as well as that of the low and intermediate dose groups was not different from control. These data are consistent with that obtained during the premating phase. The effect of test material on body weights at 75 mg/kg bw/day appeared be the result of effects that were initiated in the early stages of growth and development since the mean average weight of the F1 pups were lower than control. By the beginning of the premating phase for the F1 animals, there was no evidence that test material treatment affected growth rate since body weight gain was comparable to that of the control group.
- F1 Females Premating: As observed in the male high dose group, the 75 mg/kg bw/day F1 adult females had lower body weights at the start of the 12-week premating period. This difference was apparent throughout the 12 weeks. Body weight gain of the high dose F1 females was not different from controls suggesting that the effect of test material on their body weight occurred early in their growth and development as was discussed in relation to the F1 males. No significant treatment-related alterations of body weights or body weight gains were observed in the 5 or 20 mg/kg bw/day F1 adult females.
- F1 Females Gestation: The female high dose group had a significantly lower mean body weight than the control group throughout gestation. This was consistent with the difference observed during the premating phase. Body weight gains were not different from control. No significant differences in body weights or weight gains were observed between the low or intermediate dose groups and the control group.
- F1 Females Lactation: The mean body weights and weight gains for the low and intermediate dose groups were not different from that of the control group, with exception of the intermediate dose group on Day 21 Postpartum. Although the mean body weight of the intermediate dose group on Day 21 Postpartum was statistically significant, the difference appeared to be due to random variability. On Day 1 Postpartum the mean body weight of high dose females was significantly lower than the control group and remained lower throughout the 4-week period. These data are consistent with the premating and gestation body weight data for these animals and that of the F0 females after parturition. The high dose group females showed a decrease in mean body weight gain on Day 4 Postpartum that was significantly different than control and on Day 7 Postpartum the animals did gain weight; however, the weight gain value was still significantly lower than the control group. By Day 14 and through Day 28 Postpartum the high dose group had weight gains that were not different from control.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 Males Premating: No marked differences in feed consumption values were observed at any dose level.
- F0 Males Postmating: After the mating phase, feed consumption of the F0 males was recorded beginning on Study Day 99 and through Test Day 141. All of the male dose groups showed slightly higher feed consumption values than the control group throughout the postmating period (Study Days 99 - 141).
- F0 Females Premating: No marked differences in feed consumption values were observed at any dose level.
- F0 Females Gestation: Feed consumption for the first week of gestation was not recorded for most of the animals; therefore, no meaningful comparisons can be made. No marked differences in feed consumption values were observed at any dose level during the second or third weeks of gestation. The feed consumption values for all treatment groups increased over the gestation period which is normal for pregnant females.
- F0 Females Lactation: No marked differences in feed consumption values were observed at any dose level during the 4-week lactation period. All treatment groups exhibited an increase in consumption values over the 4 weeks.

- F1 Males Premating: No marked differences in feed consumption were observed between any of the groups receiving test material and the control group.
- F1 Males Postmating: The F1 males receiving test material (all dose levels) had slightly higher feed consumption values than the controls after mating. Even though these changes appeared treatment-related, they are of questionable significance and were judged not to be adverse toxicologic effects.
- F1 Females Premating: No marked differences in feed consumption were observed between any of the groups receiving test material and the control group during the premating period.
- F1 Females Gestation: No marked differences in feed consumption were observed between any of the groups receiving test material and the control group. All groups showed a normal increase in feed consumption over the 3 weeks of gestation which is consistent with pregnancy.
- F1 Females Lactation: The high dose group had slightly lower feed consumption values than the control group throughout the 4-week lactation period. No marked differences in feed consumption were observed between the low and intermediate dose groups and control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant treatment-related effects on mating, conception gestation and pup survival indices, sex ratio or gestation length were observed at any dose level for either the F0 or F1 animals.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0 adults: The F0 males of the 5, 20 and 75 mg/kg bw/day dose levels had body weights that were statistically higher than the control group. These changes were judged not to be treatment-related and not considered adverse toxicologic effects because the changes were relatively minor (<10 %), not dose-related and not consistent with previous results. Male rats of the 20 and 75 mg/kg bw/day dose groups had statistically increased kidney and liver weights (absolute and relative). The effects in the livers at both dose levels and the kidney of the high dose level were unequivocally treatment-related based on histopathologic alterations observed in these tissues. The increased kidney weights of the male 20 mg/kg bw/day animals was not associated with any histopathologic changes. The absolute and relative liver weights of the male 5 mg/kg bw/day dose group were statistically increased over that of the controls. The changes in liver weights at the low dose level were judged not to be toxicologically significant since the changes were not associated with any histopathologic alterations and were minor in magnitude.
The F0 females of the 75 mg/kg bw/day dose level had a significantly lower mean fasted body weight than the controls that appeared treatment-related. Also the liver weights (absolute and relative) of these animals were increased significantly as compared to control. These changes were judged treatment-related and were associated with histopathologic alterations. The kidney weights (absolute and relative) of the 75 mg/kg bw/day group were statistically increased with respect to control values but no histopathologic changes were associated with the weight increases. The relative kidney weight of the 20 mg/kg bw/day dose group was statistically increased when compared to that of the control group; however, the increase was not associated with any histopathologic changes. The fasted bodyweight and organ weights (absolute and relative) of the 5 mg/kg bw/day dose group was unaffected by treatment.

- F1 adults: The mean fasted body weight of the 75 mg/kg bw/day dose group was significantly lower than that of the controls. The lower body weight appeared treatment-related but was attributable to effects that occurred early in the growth and development of the F1 pups. Liver and kidney weights (absolute and relative) of the 20 and 75 mg/kg bw/day dose groups were statistically greater when compared to the control group. Histopathologic alterations confirmed treatment-related effects in the livers of these animals but not in the kidneys. The slight increase noted in relative liver weight of the 5 mg/kg bw/day dose group was attributed to random variability since the increase was minor and not associated with histopathologic alterations.
The F1 females of the 75 mg/kg bw/day dose level had a significantly lower mean fasted body weight than the controls that was also attributed to effects that occurred early in their growth and development. The liver weight changes (absolute and relative) observed in the 20 and 75 mg/kg bw/day animals were attributed to treatment based on associated histopathologic alterations. Changes in kidney weights (absolute and relative) in these animals were not associated with histopathologic alterations. No treatment-related effects were observed in the F1 female 5 mg/kg bw/day dose group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- F0 and F1 adults: The males (27/30) of the F0 75 mg/kg bw/day dose group and all males (30/30) of the F1 75 mg/kg bw/day dose group had a generalised increase in the size of the liver that was attributed to the administration of test material. No treatment-related alteration were observed in the male or female rats of the 5 and 20 mg/kg bw/day dose groups or the female rats of the 75 mg/kg bw/day dose group. Gross pathologic lesions judged to be spontaneous and incidental to treatment consisted of strangulated or necrotic fat, accessory spleens, cloudy cornea, hiatal hernias, decreased testes size and ovarian cysts.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- F0 adults: Histologic alterations attributable to test material administration were found in the liver of male and female rats receiving 20 or 75 mg/kg bw/day and in the kidneys of the male rats of the 75 mg/kg bw/day dose group. The liver changes in these animals consisted of hypertrophy of centrilobular hepatocytes and lipid vacuolation. The centrilobular hepatocellular hypertrophy was dose-related. Lipid vacuolation was centrilobular in animals receiving 20 mg/kg bw/day and extended into the midzonal region of the hepatic lobule in animals receiving 75 mg/kg bw/day. The male rats showed a greater degree of hepatocellular hypertrophy and a more pronounced accumulation of lipid vacuoles than the female rats. The kidney changes found in the F0 males consisted of necrosis of the intratubular epithelium (30/30). Intratubular mineralisation was also noted in these animals (20/30) but was dystrophic (i.e., salts deposited in necrotic debris) and judged not to be a direct effect of test material administration. Other normally occurring, miscellaneous degenerative alterations were also present in the kidneys of the adult rats consisting of degeneration/regeneration of renal tubules and dilation of tubules with proteinaceous casts that were not associated with test material administration. These are common, early features of the chronic renal disease which occurs naturally in this strain of rat. The low dose male and female rats did not exhibit any lesions attributed to treatment with test material. A female rat from the 5 mg/kg bw/day dose group was killed moribund. Morbidity was caused by a large, locally infiltrative squamous cell carcinoma of the head that was not associated with test material administration.

- F1 adults: Alterations attributed to treatment with test material were the same as those observed in the F0 adults that consisted of changes in the liver of male and female rats receiving 20 or 75 mg/kg bw/day and in the kidneys of the male rats of the 75 mg/kg bw/day dose group. The histopathologic liver changes in these animals were characterised by hypertrophy of centrilobular hepatocytes and lipid vacuolation. Centrilobular hepatocellular hypertrophy was dose-related. Lipid vacuolation was centrilobular in animals receiving 20 mg/kg bw/day and extended into the midzonal region of the hepatic lobule in animals receiving 75 mg/kg bw/day. As with the high dose F0 males the high dose F1 male rats showed a greater degree of hepatocellular hypertrophy and a more pronounced accumulation of lipid vacuoles than the female rats. For an undetermined reason, intratubular mineralisation was a common feature of the kidneys of both male and females at all dose levels including controls. While this change appeared to be associated with tubular necrosis in the F0 males, in the F1 males the changes were considered incidental to treatment. The miscellaneous degenerative changes found in F1 adult rats were similar to those observed in the F0 adult animals consisting of degeneration/ regeneration of renal tubules and dilation of tubules with proteinaceous casts that were not associated with test material administration. These are common, early features of the chronic renal disease which occurs naturally in this strain of rat. The low dose male and female rats did not exhibit any histopathologic alterations attributed to treatment with test material. A female (F1) rat of the 75 mg/kg bw/day dose group was found dead during the mating phase of the study. The cause of death was judged to be an ascending urinary tract infection with necrosis and bacterial colonisation on the urinary bladder. Urolith formation, possibly resulting in blockage of the urethra, was judged to be the most likely cause of death. The death of this rat was judged not to have been a result of test material administration.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

(parental toxicity)

Effect level:

5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F0 and F1

2

Dose descriptor:

NOEL

Remarks:

(neonatal toxicity)

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lower average pup weights at 75 mg/kg bw/day. Hepatocellular hypertrophy and pronounced accumulation of lipid vacuoles in F1 and F2 male and female pups at 75 mg/kg bw/day

Remarks on result:

other: Generation: F1 and F2

3

Dose descriptor:

NOEL

Remarks:

(reproduction and neonatal survival)

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect observed on fertility parameters and pup survival.

Remarks on result:

other: Generation: F0, F1 and F2

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results (offspring)" for information

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

"Details on results (offspring)" for information

Details on results (F1)

VIABILITY (OFFSPRING)
- First generation (F1): No significant treatment-related effects on pup survival indices or sex ratio were observed at any dose level.
No significant treatment-related effects on litter size including the number of pups born alive or dead were observed at any dose level.
- Second generation (F2): No significant treatment-related effects on pup survival indices or sex ratio were observed at any dose level. The Day 4 Survival Index for the 75 mg/kg bw/day dose group (97.2 %) was statistically different from the control value (99.6 %) but was judged to be due to random variability rather than a significant toxicologic effect. This is supported by the results obtained from the first generation in which the Day 4 Survival Index for the control group was 97.8 % indicating that 97.2 % is well within the normal range of expected survival and that the overall survival of the F2 control group (95 % or 257/271) was not different from that of the F2 high dose group (96 % or 246/256) over the 4 days after parturition.
The low and high dose groups had slightly smaller litter sizes than the control group. These differences were judged to be due to random variability within the population of rats and not treatment-related because a dose-response relationship was not evident and in fact the low dose group had a smaller mean litter size than the high dose group.

CLINICAL SIGNS (OFFSPRING)
No changes recorded for either F1 or F2 pups were related to administration of test material but were considered incidental to treatment.

BODY WEIGHT (OFFSPRING)
- F1 Pup Weights: The mean average pup weight of the 75 mg/kg bw/day dose group was slightly lower than that of the control group beginning on Day 1 Postpartum and was statistically significant on Day 4 Postpartum and through Day 21 Postpartum. On Day 28 Postpartum the pups were weighted individually. The mean average weight of the male pups and that of the female pups from the 75 mg/kg bw/day dose group was significantly lower than their corresponding controls. The pup body weights of the 5 and 20 mg/kg bw/day dose levels were not affected by treatment.
- F2 Pup Weights: The low dose group had slightly higher average pup weights than the control group on Day 1, 4 (before and after culling), 7 and 21 that were statistically significant and the average male pup weight on Day 28 Postpartum was also significantly higher than that of male control pups. The intermediate dose level average pup weights over the 4-week period were not different from control values. The high dose group average pup weights were significantly lower than that of the controls at all time-points. The lower pup body weights of the high dose group appeared to be treatment-related and were consistent with the results observed in the F1 pups.

GROSS PATHOLOGY (OFFSPRING)
- F1 Weanlings: No treatment-related gross alterations were observed at any dose level tested. Gross pathologic lesions judged to be spontaneous and incidental to treatment were similar to those found in the F0 adults.
- F2 Weanlings: All females (10/10) and 7/10 males of the 75 mg/kg bw/day dose group had a generalised increase in the size of the liver that was attributed to the administration of test material. No treatment-related alterations were observed in the male or female rats of the 5 and 20 mg/kg bw/day dose groups. Gross pathologic changes judged to be spontaneous and incidental to treatment were similar to those found in the F1 adults.

HISTOPATHOLOGY (OFFSPRING)
- F1 Weanlings: Histopathologic examination of the F1 weanlings was limited to the liver. The treatment-related histopathologic alterations observed in the 75 mg/kg bw/day dose group consisted of centrilobular vacuolation that was considered slight in the males (8/10) and slight (5/10) to very slight (2/10) in the females. One male of the 75 mg/kg bw/day dose group had architectural alterations of the liver secondary to diaphragmatic hernia that was unassociated with test material administration. No treatment-related hepatic changes were observed in the 5 or 20 mg/kg bw/day dose groups.
- F2 Weanlings: Histopathologic examination of the F2 weanlings was limited to the liver. The treatment-related histopathlogic alterations observed in the 75 mg/kg bw/day dose group consisted of centrilobular vacuolation that was considered slight in the males (9/10) and in the females (10/10). One female of the 20 mg/kg bw/day dose group had architectural alterations of the liver secondary to diaphragmatic hernia that was unassociated with test material administration. No treatment-related, histopathologic changes were observed in the livers of 5 or 20 mg/kg bw/day dose groups.

EXTERNAL AND INTERNAL EXAMINATION
- F1 Pups: Pups selected for culling were examined for external and internal alterations. There appeared to be more runts identified in the high dose group as compared to the other dose groups. Since runts were identified by visual inspection alone, the higher number of runts in the 75 mg/kg bw/day dose group may have been a reflection of the general decreased size (i.e., lower average body weights) of the pups. No other treatment-related abnormalities (external or internal) were observed in any of the treatment groups.
- F2 Pups: No treatment-related external alterations of the F2 pups were noted. The only internal observation was that a single F2 pup of the 75 mg/kg bw/day dose group had a pale liver.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: No adverse effects were observed in their development to sexually mature rats and their subsequent reproductive performance at any dose level.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analytical

- First generation data

Homogeneity of Test Diets

The male 20 mg/kg bw/day dose level diet prepared on Test Day 1 was sampled to determine the homogeneity of the test material in the diet formulation. The results showed that the mixing procedure produced a homogeneous dispersion of the test material in the feed. The slight differences between the various sample sites were due to variability in the analytical method.

Concentration Verification of F0 Test Diets

During the first generation, diets were analysed on Test Days 1, 43 and 113 to verify the concentration of test material in the diets and premixes. The results showed that the observed concentration of test material in the diets was in reasonable accord with the targeted concentration.

- Second generation data

Concentration Verification of F1 Test Diets

The observed concentration of test material in these diets was found to be in reasonable accord with the targeted concentrations. Based on the results of the diet analyses done over the course of this study (Test Days 1, 57 and 120), the diets provided to the test animals contained sufficient amount of test material to deliver the targeted dose levels of 0, 5, 20 and 75 mg/kg bw/day.

Table 2: Verification of Test Material in Test Diets

Target dose (mg/kg bw/day)	F0 generation						F1 generation
	Test day 1 (µg/g chow)		Test day 43 (µg/g chow)		Test day 113 (µg/g chow)		Test day 1 (µg/g chow)		Test day 57 (µg/g chow)		Test day 120 (µg/g chow)
	Target	Observed	Target	Observed	Target	Observed	Target	Observed	Target	Observed	Target	Observed
Premix	5000	5930	7500	7650	10000	10400	10000	11600	10000	10800	10000	11800
Male
75	763	821	1170	1180	1638	1750	763	695	1305	1570	1633	1760
20	204	231	312	331	422	446	204	191	371	436	469	529
5	51	59.4	77	90.4	108	134	51	50.4	92	109	111	132
Female
75	761	870	958	999	953	987	761	724	1064	1200	1158	1220
20	202	222	256	271	266	270	202	182	293	308	329	401
5	51	59	64	68.3	66	79.1	51	49.4	72	68.6	82	92.2
Male and Female
0 (control)	0	ND	0	ND	0	ND	0	ND	0	ND	0	ND

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the No Observable Effect Level (NOEL) for general toxicity in the adult rats of this study was 5 mg/kg bw/day. The NOEL for neonatal toxicity for both males and females in this study was 20 mg/kg bw /day.
Although test material administration resulted in parental toxicity in the 20 and 75 mg/kg bw/day dose levels, no adverse treatment-related effects were demonstrated at any dose level tested on reproductive performance; specifically the mating, conception and gestation indices, neonatal survival, litter size or gestation duration. The NOEL for reproduction and neonatal survival was 75 mg/kg bw/day, the highest dose level tested.

Executive summary:

The toxicity of the test material to reproductive capability and neonatal survival and growth in rats was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPP 83-4.

During the study groups of 30 male and 30 female Fischer 344 rats (F0 generation) were exposed to test material at dose levels of 0, 5, 20 or 75 mg/kg bw/day in the diet. Following 10 weeks of exposure the F0 rats were mated to produce F1 litters. After weaning, 30 F1 pups/sex/dose level were randomly selected to become the F1 adults. Following 12 weeks of exposure to test material these animals were mated to produce the F2 litters. Body weights, feed consumption, liver and kidney weights and gross pathologic and histopathologic examinations were evaluated for evidence of toxicity. Reproductive performance and neonatal survival and development were evaluated to assess the reproductive effects of treatment.

Test material administration produced parental toxicity in both the F0 and F1 adult male and female rats at 20 and 75 mg/kg bw/day. Body weights of both the F0 and F1 adult females and the F1 males receiving test material at 75 mg/kg bw/day were significantly lower when compared to their respective control groups. Liver and kidney weights (absolute and relative) were significantly increased in the F0 and F1 males and F1 females receiving the test material at 20 and 75 mg/kg bw/day relative to their respective control groups. The liver and kidney weights (absolute and relative) of the 75 mg/kg bw/day F0 females were significantly increased relative to their controls. The increased liver weights were associated with histopathologic alterations that were dose-related. The hepatic changes were characterised by hypertrophy of centrilobular hepatocytes and lipid vacuolation. In both the F0 and F1 animals the male rats showed a greater degree of hepatocellular hypertrophy and a more pronounced accumulation of lipid vacuoles than the female rats. The kidney weight changes were unassociated with histopathologic changes except for the F0 male 75 mg/kg bw/day dose group. These histopathologic changes were characterised by slight necrosis of the intratubular epithelium in the F0 high dose males.

The average pup body weights (F1 and F2) of the 75 mg/kg bw/day dose level were significantly lower than their respective controls on Day 4 Postpartum for the F1 pups and on Day 1 Postpartum for the F2 pups. Their body weights remained lower through the end of the study. Treatment-related histopathologic changes were observed in the livers of the F1 and F2 male and female weanling rats at the 75 mg/kg bw/day dose level. These changes were similar to those observed in the adult animals and were characterised by centrilobular vacuolation. Even though the F1 pups had lower body weights that persisted through their adult life and histopathologic hepatic alterations that were evident at weaning as well as at the terminal necropsy, no adverse effects were observed in their development to sexually mature rats and their subsequent reproductive performance.

Although test material administration resulted in parental toxicity in the 20 and 75 mg/kg bw/day dose levels, no adverse treatment-related effects were observed on the reproductive parameters; specifically the mating, conception and gestation indices, neonatal survival, litter size or gestation duration.

Test material, when fed via the diet, produced parental toxicity in both male and female Fischer 344 rats at dose levels of 20 and 75 mg/kg bw/day. The No Observable Effect Level (NOEL) for toxicity in adult rats was 5 mg/kg bw/day. Neonatal rats (males and females) of the 75 mg/kg bw/day dose group had histopathologic liver alterations and decreased body weights. The NOEL for neonatal toxicity was 20 mg/kg bw/day. No adverse effects on reproduction or neonatal survival were demonstrated.

The NOEL for reproduction and neonatal survival was 75 mg/kg bw/day, the highest dose level tested.