Registration Dossier
Registration Dossier
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EC number: 212-634-7 | CAS number: 834-12-8
Toxicological information
Toxicity to reproduction
Currently viewing:
Administrative data
- Endpoint:
- two-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Well documented, according to accepted guidelines
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 999
- Report date:
- 1999
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- Ametryn
- EC Number:
- 212-634-7
- EC Name:
- Ametryn
- Cas Number:
- 834-12-8
- Molecular formula:
- C9H17N5S
- IUPAC Name:
- N2-ethyl-6-(methylsulfanyl)-N4-(propan-2-yl)-1,3,5-triazine-2,4-diamine
- Details on test material:
- - Name of test material (as cited in study report): Ametryn Technical
- Physical state: Solid, white powder
- Analytical purity: 96%
- Lot/batch No.: S 351
- Expiration date of the lot/batch: October 2000
- Storage condition of test material: Ambient (+ 18 to 36°C)
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxicology Department, Rallis Research Center, Bangalore 560 058, India
- Age at study initiation: 4 weeks
- Weight at study initiation: Males: 73-75 g; Females: 65-69 g;
- Fasting period before study: not reported
- Housing: Three rats per sex per cage in sterilized standard polypropylene cages with stainless steel top grill. During the cohabitation period, two animals were housed per cage (one male and one female). After cohabitation period, the '0' day pregnant rats were housed individually throughout gestation and lactation periods until sacrifice.
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g. ad libitum): Ssniff/R-Z (rat food-breeding) powdered food low in germs manufactured by M/s Ssniff Spezialdiaten GmBH., Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany ad libitum
- Water (e.g. ad libitum): Deep borewell water passed through activated charcoal filter and exposed to UV rays (Aquaguard on-line water filter cum purifier manufactured by MIS Eureka Forbes Ltd, Bombay 400 001, INDIA in collaboration with Electrolux of Sweden) was provided in glass bottles ad libitum.
- Acclimation period: Five days under experimental conditions after veterinary examination
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 14-15 filtered air changes per hr
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: feed
- Type of inhalation exposure (if applicable):
- other: N/A
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- TREATMENT:
1. MALE RATS:
Different test groups of rats were provided the test substance mixed food specifically prepared for the group, ad libitum daily at least for 10 weeks prior to the mating period and the treatment was continued during the mating periods until the parental males were sacrificed, which was done after the mating process.
2. FEMALE RATS:
As in males, females received the test substance mixed food specifically prepared for the group, ad libitum daily at least for 10 weeks prior to the mating period and the treatment was continued through mating, pregnancy and up to weaning of F1 offspring, after which, parental females were sacrificed. Selected F1 generation offspring's were treated from weaning until they were sacrificed after obtaining F2 weanlings. - Details on mating procedure:
- MATING PROCEDURE AND VAGINAL SMEARS:
After the scheduled 10 week period of treatment, females were paired with males in a 1:1 ratio. The morning after pairing and every morning thereafter, a vaginal smear was taken from each female and examined for the presence of spermatozoa. Females were also examined for presence of vaginal plug. The day of presence of sperms and/or vaginal plug, was designated as day '0' of gestation. Once mating was confirmed, the mated females were separated and housed individually throughout the gestation and lactation periods.
Females not mated within seven days of pairing with males were removed and placed with another male (that was previously found to be successful in impregnation) of the same group and if the presence of sperms in the vaginal smear and/or vaginal plug were not confirmed within 7 days of pairing the female was again cohabited with yet another male (that was previously found to be successful in impregnation) of the same group for 7 days. Sibling matings were avoided in each generation by pairing males and females of different litters.
The same mating procedure was adopted in the F1 parental animals after the 12-14 week treatment regimen. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Ametryn Technical concentration in experimental food was determined after extraction with acetone by employing gas liquid chromatography with Nitrogen Phosphorous Detector.
- Duration of treatment / exposure:
- MALE RATS:
Different test groups of rats were provided the test substance mixed food specifically prepared for the group, ad libitum daily at least for 10 weeks prior to the mating period and the treatment was continued during the mating periods until the parental males were sacrificed, which was done after the mating process.
FEMALE RATS:
As in males, females received the test substance mixed food specifically prepared for the group, ad libitum daily at least for 10 weeks prior to the mating period and the treatment was continued through mating, pregnancy and up to weaning of F1 offspring, after which, parental females were sacrificed. Selected F1 generation offspring's were treated from weaning until they were sacrificed after obtaining F2 weanlings. - Frequency of treatment:
- ad libitum daily
- Details on study schedule:
- After the scheduled 10 week period of treatment, females were paired with males in a 1:1 ratio. The morning after pairing and every morning thereafter, a vaginal smear was taken from each female and examined for the presence of spermatozoa. Females were also examined for presence of vaginal plug. The day of presence of sperms and/or vaginal plug, was designated as day '0' of gestation. Once mating was confirmed, the mated females were separated and housed individually throughout the gestation and lactation periods. Females not mated within seven days of pairing with males were removed and placed with another male (that was previously found to be successful in impregnation) of the same group and if the presence of sperms in the vaginal smear and/or vaginal plug were not confirmed within 7 days of pairing the female was again cohabited with yet another male (that was previously found to be successful in impregnation) of the same group for 7 days. Sibling mating were avoided in each generation by pairing males and females of different litters. The same mating procedure was adopted in the F1 parental animals after the 12-14 week treatment regimen.
Doses / concentrations
- Remarks:
- Doses / Concentrations:
75, 300, and 1200 ppm
Basis:
nominal in diet
- No. of animals per sex per dose:
- 30/sex/dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
Three dose levels were selected after completion of One Generation Reproduction Toxicity Study with Ametryn Technical in wistar rats (Study No. 21 04/96) and in consultation with the sponsor of the study.
Examinations
- Parental animals: Observations and examinations:
- CLINICAL SIGNS:
All animals were examined once daily throughout the experimental period and the visible signs if any, were recorded. Veterinary/physical examination was carried out before grouping, after grouping, and at the end of each month of experimental period. Ophthalmological examination of all animals was carried out with an ophthalmoscope for parental generation and at sacrifice of each generation using 1% Tropicamide solution and observations were recorded.
PRE-TERMINAL DEATHS:
All animals were observed once daily for pre-terminal deaths and observations were recorded.
BODY WEIGHT:
In each generation male rats were weighed weekly until terminal sacrifice and female rats were weighed weekly until cohabitation with males. Female rats were also weighed on days 0, 5, 10, 15, and 20 of the gestation period and on days 1, 4, 7, 14, and 21 of the lactation period.
FOOD INTAKE:
In each generation, the cagewise food intakes for both sexes (measured by weight) were recorded weekly until the beginning of the cohabitation period. The food intake was measured during the cohabitation period (week 11-13) for the males which have impregnated the females, however the values have not been presented in the report as the no. of values were not uniform between the groups. At the end of the cohabitation period, food intake formales continued to be recorded until terminal sacrifice. For females, the food intake was also recorded individually for the following period: 0-5,
5-10, 10-15 and 15-20 days of the gestation period and 1-4, 4-7, 7-11, 11 -1 4 and 14-18 and 18-21 days of lactation period. - Litter observations:
- OBSERVATIONS ON DAY 1:
All litters were examined within 24 hours after birth and the following parameters were recorded for each litter:
a. Number of pups born (live and dead)
b. Individual sex
c. Sex wise litter weight
d. Observations of individual pups
PRE-TERMINAL DEATHS AND LITTER SIZE:
Daily records of pre-terminal deaths and subsequent changes in litter size were maintained. On day 4 after birth, the pups were culled to reduce the litter size to eight. For standardization of litter size to eight pups, equal number of pups from each sex or any combination of numbers of pups from each sex was adopted. Wherever the litter size was less than eight, no adjustment was made. Selection was at random for each sex. Pups culled on day 4 or those found dead were examined externally and internally and specimens of abnormal tissues, if any, were preserved in 10% neutral buffered formalin.
BODY WEIGHTS:
The pups of each litter were weighed sex wise on days 1, 4 (after culling), 7, 14, and 21 after birth. The individual pup body weights were recorded on the day of weaning i.e. at 21 days after birth.
PHYSICAL DEVELOPMENT:
Any abnormal physical development observed in each pup was recorded. - Postmortem examinations (parental animals):
- TERMINAL SACRIFICE:
All parents (P and F1) and weanlings not selected from F1 generation and all pups from F2 generation were subjected to complete necropsy at terminal sacrifice. Tissue collection was done from all dams and sires and tissues were preserved in 10% buffered neutral formalin. The corpora lutea and implantation sites were counted for each dam. Males of P and F1 parents were sacrificed at an age of 20 - 21, and 21-24 weeks, respectively, while the females were sacrificed on weaning of its litter. Females failing to mate within 21 days of initial pairing were sacrificed after a further observation period of 21 days and examined for macroscopic abnormalities. Specimens of any tissues considered abnormal were processed for histopathological examination. Females failing to produce a viable litter by day 25 of gestation period were sacrificed and examined for macroscopic abnormalities and the presence of corpora lutea, implantations and resorptions. Abnormal tissues and reproductive organs were examined for histopathological changes. Males failing to mate within 7 days of pairing were examined microscopically for any abnormalities of reproductive organs.
HISTOPATHOLOGY:
The organs listed below were collected from all parents of P and F1 generations and preserved in 10% buffered neutral formalin. The gross lesions from all the animals and all collected tissues from males and females suspected for infertility in all the groups were processed by paraffin embedding technique and hematoxylin and eosin stained sections were examined for histopathological changes.
a) Ovaries
b) Uterus with cervix and vagina
c) Epididymes
d) Seminal vesicles
e) Prostate
f) Testes
g) Adrenals
h) Coagulating glands
i) Liver
j) Kidney
k) Pituitary - Postmortem examinations (offspring):
- See information in above section (Parental animals)
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
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- no effects observed
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)
RESULTS OF P GENERATION
P. 1 Clinical Signs, Ophthalmological Examinations, Parturition Performance (in female rats) and Pre-Terminal Deaths:
Veterinary examination was carried out on all the animals, of parental generation, before and after grouping and at the end of each month of the experimental period. In the parental generation, there were no clinical signs attributable to treatment. Incidences of partial cannibalism of pups were observed in 5, 2 and 5 dams of control, low and mid dose groups, respectively while no incidences were observed at the high dose. Ophthalmological examination carried out on all the animals, before grouping and at the terminal sacrifice of parental animals did not reveal any abnormalities in the eyes of the experimental animals.
Parturition performance in female rats:
Parturition performance was unaffected by the treatment at all of the doses tested. One female animal (Ra 3064) at the mid dose, shown signs of dystokia, however, since it was an isolated incident, it was considered to be incidental and not related to the treatment.
Six dams in the control, nine dams each in the low and mid dose groups and seven dams in the high dose group did not litter. There were no treatment related pre-terminal deaths at all of the doses tested. The death of one dam (Ra 3064) at the mid dose was considered to be incidental not related to the treatment.
P. 2 Body Weight:
Male Rats:
At the high dose, the mean body weights and the net body weight gains were significantly lower compared to control. The mean body weights and the net body weight gains of the animals at the low and mid doses were unaffected by the treatment and were not significantly different from the control. The decreases in the mean body weights observed during different weeks of the experiment were within the historical control data except for the first two weeks which were less than the historical control data.
Female Rats:
The mean body weights and the net body weight gains were unaffected by the treatment at all of the doses tested compared to the control except for decreased mean body weights on week 1 at the high dose. However, at the mid dose, significantly higher mean body weights on most weeks of the experiment and net body weight gains prior to co-habitation with males were considered to be incidental and not related to treatment as the same changes were not seen at the low and high doses and the values were well within the range of our historical control data, (except on week 2). The increase/decrease in the mean body weights observed during different weeks of the experiment was within the historical control data except for the first two weeks of the experiment.
P. 3 Food Intake:
Male Rats:
The treatment resulted in lower food intake on weeks 1-2, 4-10 and 14-15 at the high dose. The treatment did not alter the food intake at the low and mid doses. However the lower food intake on week 1 at the mid dose and on week 7 at the low dose and the higher food intake on week 3 at the low and the mid doses were considered to be incidental and not related to treatment. The increase/decrease in the food intake recorded during the different weeks of experiment were within the historical control data except on first week of the experiment which was less than the historical control data.
The food intake was measured during the co-habitation period (weeks 11 -13), for the males which have impregnated the females. However the number of values were not uniform between the groups hence have not been presented in the report.
Female Rats:
The treatment resulted in lower food intake on weeks 5 - 8 and on week 10 prior to cohabitation with males. The treatment did not alter the food intake at the low and mid doses prior to co-habitation with males (except on week 8 at the mid dose). The increase in the food intake on weeks 3 and 10 at the low dose were considered incidental and not related to treatment. The decrease in the food intake was dose correlated on week 8. The increase/decrease in the food intake recorded during the different weeks of the experiment were within the historical control data except on weeks 3, 5 and 6 which were less than the historical control data.
P. 4 Food and Test substance Intake:
The mean daily food consumption (g/kg Bwt/day) in the treated groups did not significantly different from the control group.
P. 5 Maternal Body Weights and Weight Change during Gestation Period:
At the high dose, although the mean maternal body weights were unaffected, the maternal body weight change during 0-5, 10-15 and 0-20 days of the gestation period were significantly lower compared to control. At the mid dose, significantly higher maternal body weights during different intervals of gestation period and the weight change during 5 - 10 days of gestation period were considered to be incidental and not related to treatment as the body weight of animals at the mid dose prior to the cohabitation with males were significantly higher, the same trend was continued even during gestation period and the same changes were not seen at the low and high doses and the values were well within the range of historical control data. There were no statistically significant differences in the maternal body weights and the weight changes during different intervals of gestation period in the low dose group compared to the control group.
The decrease in the maternal body weight changes during 10-15 and 0-20 days of the gestation period were less than the historical control data.
P. 6 Maternal Food Intake during Gestation Period:
At the high dose, the total food intake and the daily food intake (derived from the total food intake) recorded at different intervals during the gestation period were significantly lower compared to the control. There were no statistically significant differences in the total food intake and the daily food intake recorded at different intervals during the gestation period at the low and mid doses compared to control.
The decrease in the total food intake and the daily food intake recorded during different intervals of the gestation period were less than the historical control data.
P. 7 Litter Data at First Observation (F1):
The test substance at the doses tested had no significant effect on the number of pregnancies, number littered, number of live litters, mean litter size, number of pups dead at birth, number of live pups on day one and observations for external abnormalities of live and dead pups.
P. 8 Maternal Body Weights and Weight Change during Lactation Period:
At the high dose, the body weight of dams on lactation days 1 and 4 were significantly lower compared to control. Increased body weight of dams at the mid dose (statistically not significant on lactation days 1 and 4) compared to control was considered to be incidental as the body weights of dams prior to mating period and during gestation period were significantly higher compared to control, the same trend was continued even during lactation period and the same changes were not seen at the low and high doses.
The maternal body weight change during lactation period 1-21 at the mid dose and during lactation period 14-21 and 1-21 at the high dose were significantly higher compared to control. The maternal body weight of dams recorded at different intervals of lactation period at the low dose were unaffected by the treatment. The body weight of dams on lactation days 7, 14 and 21 and the weight change during lactation period 14-21 were more than the historical control data.
P. 9 Maternal Food Intake during Lactation Period:
There were no statistically significant differences in the total food intake and calculated daily food intake of dams recorded during the different intervals of lactation period between the treatment groups compared to control.
P. 10 Number of Pups during Lactation Period (F1):
The mean no. of male and female pups and mean number of pups for combined sex during the different intervals of lactation period in the treatment groups were not different from the control group, statistically.
P. 11 Body Weight of Pups during Lactation Period (F1):
At the high dose, the mean weight of male pups and mean weight of pups for combined sex on lactation days 7, 14 and 21 and mean weight of female pups on lactation day 21 were significantly lower compared to control. There were no statistical differences in the mean weight of male and female pups and the mean weight of pups for combined sex at the low and mid doses recorded during different intervals of lactation period compared to control. The decrease in the mean weight of male and female pups and the mean weight of pups for combined sex recorded during different intervals of lactation period remained within the range of historical control data except on 7th day of lactation for combined sex which was more than the historical control data.
P.12 Survival Data of Pups:
Treatment had no significant effects on the number of live litters, mean litter size index, number of pups dead at birth, number of pups dead/cannibalized up to day 4 and from 5-7, 8-14 and 15-21, and number of pups alive on day 1, 2, 4, 7, 14 and 21 and their associated survival indices at all of the doses tested. However, incidentally higher survival indices on lactation days 4 and 21 at the high dose were observed, since more number of pups found alive on these days of lactation (statistically not significant on lactation day 4) and as there were no pups found dead/cannibalized from lactation days 2 to 4 and 4 to 21, respectively and the values were within the historical control data except day 4 survival index at the high dose which was more than the historical control data.
P.13 Pup observation and Necropsy findings (F1):
No treatment related abnormalities were recorded at all of the doses tested.
All the findings were within the historical control data.
P.14 Fertility Index:
The treatment had no significant effect on fertility indices of sires and dams, fecundity index, mean no. of corpora lutea and implantation, percentage of implantations and pre-implantation loss. The lower percentages of live pups born which were correlated to the higher percentages of post-implantation embryonic loss in all the treated groups were considered incidental and not attributable to treatment. The changes were inconsistent and not in a dose dependent manner. These effects were not seen in F1 generation and in the preliminary one generation study carried out at much higher dose levels (carried out at 250, 750 and 2250 ppm). Incidentally, there was a lower percentage of pre-implantation loss at the high dose which was correlated to the higher mean no. of corpora lutea and implantations and was within the historical control data.
Higher percentages of male fertility index at the high dose was considered incidental and not related to the treatment. Decreased percentages of live pups born and increased percentage of post-implantation loss were not within the historical data range at the mid dose.
P.15 Necropsy Findings of Parents (P):
The incidence of dilatation of pelvis in kidneys in males was 1, 0, 2 and 4 in control, low, mid and high dose groups, respectively. It was considered incidental as the same change has been recorded in our previous studies. Incidences of few other changes that were present in various groups were low and hence considered incidental.
P.16 Histopathological Findings:
Gross lesions from all the animals and all collected tissues from males and females suspected of infertility were examined. Among the males suspected of infertility, Ra 2883 in the control group showed diffuse and severe atrophy of the seminiferous tubules in one testis and associated aspermia in one epididymes. Ra2904 in low dose group had moderate and diffuse degenerative changes in the seminiferous tubules of one testis and aspermia in one epididymes. These changes were considered to be the cause of infertility in the above mentioned animals. Other animals did not show any major histopathological changes. Among infertile females, two animals in the control group (Ra 3010 and Ra 3018) had dilatation of uterus. This was considered to be physiological as there were no changes in the ovaries and hence not the cause of infertility. One animal in the mid dose group (Ra 3075) had a luteal cyst in the ovary and moderate dilatation of uterus and one in the high dose group (Ra 3096) had luteal cysts in the ovaries. These changes were considered to be the cause of infertility in these animals. The cause of infertility in other animals could not be ascertained. More frequently observed histopathological changes were:
Pituitary: Dilated Rathke's cleft.
Kidneys: Dilatation of pelvis and basophilic tubules
Dilatation of pelvis in kidneys and dilated Rathke's cleft in pituitary were more in high dose females, but these changes have been recorded in our previous studies and hence considered incidental.
RESULTS OF F1 GENERATION
F1. 1 Clinical Signs, Ophthalmological Examinations, Parturition Performance (in female rats) and Pre-Terminal Deaths:
Veterinary examination was carried for clinical signs on all the animals of F1 generation at the end of each month of experimental period. In the F1 generation, there were no clinical signs attributable to treatment. The incidence of partial cannibalism of pups were observed in 8, 7, 3, and 6 dams of the control, low, mid and high dose groups, respectively. The incidence of all pups dead/cannibalism was noticed in one dam of mid dose group.
Ophthalmological examination carried out on all the animals, at terminal sacrifice of F1 parents, did not reveal any abnormalities in the eyes of the experimental animals.
Parturition performance in female rats:
Parturition performance was unaffected by the treatment at all of the doses tested and there were no signs of dystokia in any of the groups tested. Three dams in the control group, two dams each in the low and mid dose groups and one dam in the high dose group did not litter. There were no pre-terminal deaths at all of the doses tested.
Fl. 2 Body Weight:
Male Rats:
At the high dose, the mean body weights and the net weight gains of the animals were significantly lower compared to control. The mean body weights were significantly lower on weeks 5, 7, 10, and 15 at the mid dose compared to the control. The mean body weights at the low dose and the net weight gains at the low, mid doses were unaffected by the treatment and were not significantly different from the control. The decrease in the mean body weights was dose correlated on weeks 5, 7, 10, and 15. The decreases in the mean body weights were within the historical control data except on weeks 5, 10 and 15 at the mid dose which were more than the historical control data.
Female Rats:
The mean body weights were significantly lower on all the weeks at the high dose and for the first 9 weeks at the mid dose prior to co-habitation with males. The mean body weights at the low dose and the net weight gains at all of the doses tested were unaffected by the treatment. The decrease in the mean body weight on week 4 at the low dose was considered incidental as it was an isolated incident at this dose. The decrease in the mean body weights was dose correlated up to 4 weeks and on week 9 prior to cohabitation with males. The decrease in the mean body weights were within the historical control data.
F1. 3 Food Intake:
Male Rats:
Treatment resulted in lower food intake on all the weeks (except on week 3) at the high dose and on weeks 2-8 and on week 11 at the mid dose. The treatment did not alter the food intake at the low dose. The decrease in the food intake was dose correlated on weeks 7 and 11. The decrease in the food intake recorded during the different weeks of experiment were within the historical control data except on week 14 which was slightly more than the historical control data. The food intake was measured during the co habitation period (weeks 15-17) for the males which have impregnated the females, however, the number of values were not uniform between the groups, hence were not presented in the report.
Female Rats:
The treatment resulted in lower food intake on weeks 5-11 and on week 13 at the high dose and on weeks 5 and 8 at the mid dose prior to co-habitation with males. The treatment did not alter the food intake at the low dose prior to co-habitation with males.
The decrease in the food intake was dose correlated on week 8. The decrease in the food intake recorded during different weeks of the experiment were within the historical control data except on weeks 8, 9, and 13 which were less than the historical control data.
F1. 4 Food and Test substance Intake:
The mean daily food consumption (g/kg Bwt/day) in the treated groups did not significantly different from the control group.
F1. 5 Maternal Body Weights and Weight Change during Gestation Period:
At the high dose, the maternal body weights recorded during different intervals of gestation period and the weight change during 15-20 days of gestation were significantly lower compared to control and were less than the historical control data. There were no statistically significant differences in the maternal body weights and the weight changes during different intervals of gestation period at the low and mid doses compared to control.
F1. 6 Maternal Food Intake during Gestation Period:
The total feed intake and the daily food intake recorded during 0-5, 15-20, and 0-20 days of gestation period at the high dose and during 15-20 days of gestation period at the mid dose were significantly lower compared to control. There were no statistically significant differences in the total food intake and the daily food intake recorded at different intervals during the gestation period at the low dose compared to control. The decrease in the daily food intake was dose correlated during 15-20 days of gestation period.
The decrease in the total food intake and the daily food intake recorded during different intervals of the gestation period were within the historical control data except for the total food intake and the daily food intake during 0-20 days of gestation period which were less than the historical control data.
F1. 7 Litter Data at First Observation (F2):
The test substance at the doses tested had no significant effect on the number of dams that littered, no. of live litters, mean litter size, no. of pups dead at birth, no. of live pups on day one, and observations for external abnormalities of live and dead pups. However, significantly lower no. of live pups on day one at the mid dose was considered to be related to the higher no. of dead pups at birth, these findings were considered to be incidental as the same findings were not seen at the low and mid doses and the findings were within the historical control data.
F1. 8 Maternal Body Weights and Weight Change during Lactation Period:
At the high dose, the maternal body weights of dams recorded during different intervals of lactation period were significantly lower compared to control. The maternal body weights at the low and mid doses and the weight changes at all of the doses tested recorded during different intervals of lactation period were unaffected by the treatment.
The decrease in the maternal body weights recorded during different intervals of lactation period was within the historical control data.
F1. 9 Maternal Food Intake during Lactation Period:
The total food intake and the daily food intake during different intervals of the lactation period at the high dose and during 7-11, 14-18 and 1-21 days of lactation period at the mid dose were significantly lower compared to control. There were no statistically significant differences in the total food intake and the daily food intake recorded during different intervals of lactation period at the low dose compared to control. The decrease in the total food intake and the daily food intake was dose correlated during 14-18 days of lactation period. The decrease in the total food intake and the daily food intake recorded during 1-4 and 4-7 days of lactation period were within the historical control data but during 1-21 days of lactation period was more than the historical control data.
F1. 10 Number of Pups during Lactation Period (F2):
The mean no. of male and female pups and mean number of pups for combined sex during different intervals of lactation period in the treatment groups were not different from the control group, statistically. However, the decreased mean no. of male pups and mean no. of pups for combined sex at the mid dose on lactation day 1 was considered incidental and not related to the treatment and was within the historical control data.
F1. 11 Body Weight of Pups during Lactation Period (F2):
At the high dose, the mean weight of male pups and the mean weight of pups for combined sex on lactation days 14 and 21 and the mean weight of female pups on lactation days 7, 14, and 21 were significantly lower compared to control.
There were no statistically significant differences in the mean weight of male and female pups and the mean weight of pups for combined sex recorded during different intervals of lactation period in the low and mid dose groups compared to control group. However, significantly higher mean weight of female pups on lactation day 7 at the low and mid dose was considered incidental and not related to the treatment. The decrease in the mean weight of male and female pups and the mean weight of pups for combined sex recorded on lactation days 14 and 21 were within the range of historical control data except on lactation day 14 for combined sex which was more than the historical control data.
F1. 12 Survival Data of Pups:
Treatment had no significant effects on the number of live litters, mean litter size index, number of pups dead at birth, umber of pups dead/cannibalized up to day 4 and from 5-7, 8-14, and 15-21, and number of pups alive on day 1, 2, 4, 7, 14, and 21 and their associated survival indices at all of the doses tested. However, at the mid dose, lower no. of live pups on lactation days 1 and 2 were correlated to the higher no. of dead pups at birth, lower no. of pups discarded was considered to be related to the higher no. of pups dead/cannibalized up to day 4 and lower number of live pups at birth which resulted in lower live birth index. These findings were considered incidental as the same effects were not seen at the mid and high doses and the findings were within the historical control data.
F1. 13 Pup Observation and Necropsy findings (F2):
No treatment related abnormalities were recorded at all of the doses tested. All the findings were within the historical control data.
F1. 14 Fertility Index:
Treatment had no significant effects on the male and female fertility indices, fecundity index, mean no. of corpora lutea and implantations, percentage of implantations, parturition percentage, percentage of live pups born and percentages of pre and post-implantations losses. However, at the mid dose, significantly higher pre-implantation loss resulted in lower percentage of implantation and higher percentage of post implantation embryonic loss resulted into lower percentage of live pups born. These findings were considered to be incidental and not related to the treatment as the same effects were not seen at the low and high doses and were within the historical control data.
F1. 15 Necropsy Findings of Parents (F1):
Few gross pathological changes that were present in various groups were considered incidental as their incidences were low. One animal in the control group (Ra 3111) had an enlarged prostate but was fertile. Microscopically this change was found to be associated with moderate and diffuse suppurative inflammation.
Fl. 16 Histopathological Findings:
Gross lesions from all the animals and all collected tissues from males and females suspected of infertility were examined. Various changes in several organs were considered incidental as their frequency was low. The cause of infertility in any of the above mentioned animals could not be ascertained except for Ra 3260 (Control group), which had a follicular cyst in the ovary and Ra 3343 (high dose group), which had a luteal cyst in the ovary.
P. 1 Clinical Signs, Ophthalmological Examinations, Parturition Performance (in female rats) and Pre-Terminal Deaths:
Veterinary examination was carried out on all the animals, of parental generation, before and after grouping and at the end of each month of the experimental period. In the parental generation, there were no clinical signs attributable to treatment. Incidences of partial cannibalism of pups were observed in 5, 2 and 5 dams of control, low and mid dose groups, respectively while no incidences were observed at the high dose. Ophthalmological examination carried out on all the animals, before grouping and at the terminal sacrifice of parental animals did not reveal any abnormalities in the eyes of the experimental animals.
Parturition performance in female rats:
Parturition performance was unaffected by the treatment at all of the doses tested. One female animal (Ra 3064) at the mid dose, shown signs of dystokia, however, since it was an isolated incident, it was considered to be incidental and not related to the treatment.
Six dams in the control, nine dams each in the low and mid dose groups and seven dams in the high dose group did not litter. There were no treatment related pre-terminal deaths at all of the doses tested. The death of one dam (Ra 3064) at the mid dose was considered to be incidental not related to the treatment.
P. 2 Body Weight:
Male Rats:
At the high dose, the mean body weights and the net body weight gains were significantly lower compared to control. The mean body weights and the net body weight gains of the animals at the low and mid doses were unaffected by the treatment and were not significantly different from the control. The decreases in the mean body weights observed during different weeks of the experiment were within the historical control data except for the first two weeks which were less than the historical control data.
Female Rats:
The mean body weights and the net body weight gains were unaffected by the treatment at all of the doses tested compared to the control except for decreased mean body weights on week 1 at the high dose. However, at the mid dose, significantly higher mean body weights on most weeks of the experiment and net body weight gains prior to co-habitation with males were considered to be incidental and not related to treatment as the same changes were not seen at the low and high doses and the values were well within the range of our historical control data, (except on week 2). The increase/decrease in the mean body weights observed during different weeks of the experiment was within the historical control data except for the first two weeks of the experiment.
P. 3 Food Intake:
Male Rats:
The treatment resulted in lower food intake on weeks 1-2, 4-10 and 14-15 at the high dose. The treatment did not alter the food intake at the low and mid doses. However the lower food intake on week 1 at the mid dose and on week 7 at the low dose and the higher food intake on week 3 at the low and the mid doses were considered to be incidental and not related to treatment. The increase/decrease in the food intake recorded during the different weeks of experiment were within the historical control data except on first week of the experiment which was less than the historical control data.
The food intake was measured during the co-habitation period (weeks 11 -13), for the males which have impregnated the females. However the number of values were not uniform between the groups hence have not been presented in the report.
Female Rats:
The treatment resulted in lower food intake on weeks 5 - 8 and on week 10 prior to cohabitation with males. The treatment did not alter the food intake at the low and mid doses prior to co-habitation with males (except on week 8 at the mid dose). The increase in the food intake on weeks 3 and 10 at the low dose were considered incidental and not related to treatment. The decrease in the food intake was dose correlated on week 8. The increase/decrease in the food intake recorded during the different weeks of the experiment were within the historical control data except on weeks 3, 5 and 6 which were less than the historical control data.
P. 4 Food and Test substance Intake:
The mean daily food consumption (g/kg Bwt/day) in the treated groups did not significantly different from the control group.
P. 5 Maternal Body Weights and Weight Change during Gestation Period:
At the high dose, although the mean maternal body weights were unaffected, the maternal body weight change during 0-5, 10-15 and 0-20 days of the gestation period were significantly lower compared to control. At the mid dose, significantly higher maternal body weights during different intervals of gestation period and the weight change during 5 - 10 days of gestation period were considered to be incidental and not related to treatment as the body weight of animals at the mid dose prior to the cohabitation with males were significantly higher, the same trend was continued even during gestation period and the same changes were not seen at the low and high doses and the values were well within the range of historical control data. There were no statistically significant differences in the maternal body weights and the weight changes during different intervals of gestation period in the low dose group compared to the control group.
The decrease in the maternal body weight changes during 10-15 and 0-20 days of the gestation period were less than the historical control data.
P. 6 Maternal Food Intake during Gestation Period:
At the high dose, the total food intake and the daily food intake (derived from the total food intake) recorded at different intervals during the gestation period were significantly lower compared to the control. There were no statistically significant differences in the total food intake and the daily food intake recorded at different intervals during the gestation period at the low and mid doses compared to control.
The decrease in the total food intake and the daily food intake recorded during different intervals of the gestation period were less than the historical control data.
P. 7 Litter Data at First Observation (F1):
The test substance at the doses tested had no significant effect on the number of pregnancies, number littered, number of live litters, mean litter size, number of pups dead at birth, number of live pups on day one and observations for external abnormalities of live and dead pups.
P. 8 Maternal Body Weights and Weight Change during Lactation Period:
At the high dose, the body weight of dams on lactation days 1 and 4 were significantly lower compared to control. Increased body weight of dams at the mid dose (statistically not significant on lactation days 1 and 4) compared to control was considered to be incidental as the body weights of dams prior to mating period and during gestation period were significantly higher compared to control, the same trend was continued even during lactation period and the same changes were not seen at the low and high doses.
The maternal body weight change during lactation period 1-21 at the mid dose and during lactation period 14-21 and 1-21 at the high dose were significantly higher compared to control. The maternal body weight of dams recorded at different intervals of lactation period at the low dose were unaffected by the treatment. The body weight of dams on lactation days 7, 14 and 21 and the weight change during lactation period 14-21 were more than the historical control data.
P. 9 Maternal Food Intake during Lactation Period:
There were no statistically significant differences in the total food intake and calculated daily food intake of dams recorded during the different intervals of lactation period between the treatment groups compared to control.
P. 10 Number of Pups during Lactation Period (F1):
The mean no. of male and female pups and mean number of pups for combined sex during the different intervals of lactation period in the treatment groups were not different from the control group, statistically.
P. 11 Body Weight of Pups during Lactation Period (F1):
At the high dose, the mean weight of male pups and mean weight of pups for combined sex on lactation days 7, 14 and 21 and mean weight of female pups on lactation day 21 were significantly lower compared to control. There were no statistical differences in the mean weight of male and female pups and the mean weight of pups for combined sex at the low and mid doses recorded during different intervals of lactation period compared to control. The decrease in the mean weight of male and female pups and the mean weight of pups for combined sex recorded during different intervals of lactation period remained within the range of historical control data except on 7th day of lactation for combined sex which was more than the historical control data.
P.12 Survival Data of Pups:
Treatment had no significant effects on the number of live litters, mean litter size index, number of pups dead at birth, number of pups dead/cannibalized up to day 4 and from 5-7, 8-14 and 15-21, and number of pups alive on day 1, 2, 4, 7, 14 and 21 and their associated survival indices at all of the doses tested. However, incidentally higher survival indices on lactation days 4 and 21 at the high dose were observed, since more number of pups found alive on these days of lactation (statistically not significant on lactation day 4) and as there were no pups found dead/cannibalized from lactation days 2 to 4 and 4 to 21, respectively and the values were within the historical control data except day 4 survival index at the high dose which was more than the historical control data.
P.13 Pup observation and Necropsy findings (F1):
No treatment related abnormalities were recorded at all of the doses tested.
All the findings were within the historical control data.
P.14 Fertility Index:
The treatment had no significant effect on fertility indices of sires and dams, fecundity index, mean no. of corpora lutea and implantation, percentage of implantations and pre-implantation loss. The lower percentages of live pups born which were correlated to the higher percentages of post-implantation embryonic loss in all the treated groups were considered incidental and not attributable to treatment. The changes were inconsistent and not in a dose dependent manner. These effects were not seen in F1 generation and in the preliminary one generation study carried out at much higher dose levels (carried out at 250, 750 and 2250 ppm). Incidentally, there was a lower percentage of pre-implantation loss at the high dose which was correlated to the higher mean no. of corpora lutea and implantations and was within the historical control data.
Higher percentages of male fertility index at the high dose was considered incidental and not related to the treatment. Decreased percentages of live pups born and increased percentage of post-implantation loss were not within the historical data range at the mid dose.
P.15 Necropsy Findings of Parents (P):
The incidence of dilatation of pelvis in kidneys in males was 1, 0, 2 and 4 in control, low, mid and high dose groups, respectively. It was considered incidental as the same change has been recorded in our previous studies. Incidences of few other changes that were present in various groups were low and hence considered incidental.
P.16 Histopathological Findings:
Gross lesions from all the animals and all collected tissues from males and females suspected of infertility were examined. Among the males suspected of infertility, Ra 2883 in the control group showed diffuse and severe atrophy of the seminiferous tubules in one testis and associated aspermia in one epididymes. Ra2904 in low dose group had moderate and diffuse degenerative changes in the seminiferous tubules of one testis and aspermia in one epididymes. These changes were considered to be the cause of infertility in the above mentioned animals. Other animals did not show any major histopathological changes. Among infertile females, two animals in the control group (Ra 3010 and Ra 3018) had dilatation of uterus. This was considered to be physiological as there were no changes in the ovaries and hence not the cause of infertility. One animal in the mid dose group (Ra 3075) had a luteal cyst in the ovary and moderate dilatation of uterus and one in the high dose group (Ra 3096) had luteal cysts in the ovaries. These changes were considered to be the cause of infertility in these animals. The cause of infertility in other animals could not be ascertained. More frequently observed histopathological changes were:
Pituitary: Dilated Rathke's cleft.
Kidneys: Dilatation of pelvis and basophilic tubules
Dilatation of pelvis in kidneys and dilated Rathke's cleft in pituitary were more in high dose females, but these changes have been recorded in our previous studies and hence considered incidental.
RESULTS OF F1 GENERATION
F1. 1 Clinical Signs, Ophthalmological Examinations, Parturition Performance (in female rats) and Pre-Terminal Deaths:
Veterinary examination was carried for clinical signs on all the animals of F1 generation at the end of each month of experimental period. In the F1 generation, there were no clinical signs attributable to treatment. The incidence of partial cannibalism of pups were observed in 8, 7, 3, and 6 dams of the control, low, mid and high dose groups, respectively. The incidence of all pups dead/cannibalism was noticed in one dam of mid dose group.
Ophthalmological examination carried out on all the animals, at terminal sacrifice of F1 parents, did not reveal any abnormalities in the eyes of the experimental animals.
Parturition performance in female rats:
Parturition performance was unaffected by the treatment at all of the doses tested and there were no signs of dystokia in any of the groups tested. Three dams in the control group, two dams each in the low and mid dose groups and one dam in the high dose group did not litter. There were no pre-terminal deaths at all of the doses tested.
Fl. 2 Body Weight:
Male Rats:
At the high dose, the mean body weights and the net weight gains of the animals were significantly lower compared to control. The mean body weights were significantly lower on weeks 5, 7, 10, and 15 at the mid dose compared to the control. The mean body weights at the low dose and the net weight gains at the low, mid doses were unaffected by the treatment and were not significantly different from the control. The decrease in the mean body weights was dose correlated on weeks 5, 7, 10, and 15. The decreases in the mean body weights were within the historical control data except on weeks 5, 10 and 15 at the mid dose which were more than the historical control data.
Female Rats:
The mean body weights were significantly lower on all the weeks at the high dose and for the first 9 weeks at the mid dose prior to co-habitation with males. The mean body weights at the low dose and the net weight gains at all of the doses tested were unaffected by the treatment. The decrease in the mean body weight on week 4 at the low dose was considered incidental as it was an isolated incident at this dose. The decrease in the mean body weights was dose correlated up to 4 weeks and on week 9 prior to cohabitation with males. The decrease in the mean body weights were within the historical control data.
F1. 3 Food Intake:
Male Rats:
Treatment resulted in lower food intake on all the weeks (except on week 3) at the high dose and on weeks 2-8 and on week 11 at the mid dose. The treatment did not alter the food intake at the low dose. The decrease in the food intake was dose correlated on weeks 7 and 11. The decrease in the food intake recorded during the different weeks of experiment were within the historical control data except on week 14 which was slightly more than the historical control data. The food intake was measured during the co habitation period (weeks 15-17) for the males which have impregnated the females, however, the number of values were not uniform between the groups, hence were not presented in the report.
Female Rats:
The treatment resulted in lower food intake on weeks 5-11 and on week 13 at the high dose and on weeks 5 and 8 at the mid dose prior to co-habitation with males. The treatment did not alter the food intake at the low dose prior to co-habitation with males.
The decrease in the food intake was dose correlated on week 8. The decrease in the food intake recorded during different weeks of the experiment were within the historical control data except on weeks 8, 9, and 13 which were less than the historical control data.
F1. 4 Food and Test substance Intake:
The mean daily food consumption (g/kg Bwt/day) in the treated groups did not significantly different from the control group.
F1. 5 Maternal Body Weights and Weight Change during Gestation Period:
At the high dose, the maternal body weights recorded during different intervals of gestation period and the weight change during 15-20 days of gestation were significantly lower compared to control and were less than the historical control data. There were no statistically significant differences in the maternal body weights and the weight changes during different intervals of gestation period at the low and mid doses compared to control.
F1. 6 Maternal Food Intake during Gestation Period:
The total feed intake and the daily food intake recorded during 0-5, 15-20, and 0-20 days of gestation period at the high dose and during 15-20 days of gestation period at the mid dose were significantly lower compared to control. There were no statistically significant differences in the total food intake and the daily food intake recorded at different intervals during the gestation period at the low dose compared to control. The decrease in the daily food intake was dose correlated during 15-20 days of gestation period.
The decrease in the total food intake and the daily food intake recorded during different intervals of the gestation period were within the historical control data except for the total food intake and the daily food intake during 0-20 days of gestation period which were less than the historical control data.
F1. 7 Litter Data at First Observation (F2):
The test substance at the doses tested had no significant effect on the number of dams that littered, no. of live litters, mean litter size, no. of pups dead at birth, no. of live pups on day one, and observations for external abnormalities of live and dead pups. However, significantly lower no. of live pups on day one at the mid dose was considered to be related to the higher no. of dead pups at birth, these findings were considered to be incidental as the same findings were not seen at the low and mid doses and the findings were within the historical control data.
F1. 8 Maternal Body Weights and Weight Change during Lactation Period:
At the high dose, the maternal body weights of dams recorded during different intervals of lactation period were significantly lower compared to control. The maternal body weights at the low and mid doses and the weight changes at all of the doses tested recorded during different intervals of lactation period were unaffected by the treatment.
The decrease in the maternal body weights recorded during different intervals of lactation period was within the historical control data.
F1. 9 Maternal Food Intake during Lactation Period:
The total food intake and the daily food intake during different intervals of the lactation period at the high dose and during 7-11, 14-18 and 1-21 days of lactation period at the mid dose were significantly lower compared to control. There were no statistically significant differences in the total food intake and the daily food intake recorded during different intervals of lactation period at the low dose compared to control. The decrease in the total food intake and the daily food intake was dose correlated during 14-18 days of lactation period. The decrease in the total food intake and the daily food intake recorded during 1-4 and 4-7 days of lactation period were within the historical control data but during 1-21 days of lactation period was more than the historical control data.
F1. 10 Number of Pups during Lactation Period (F2):
The mean no. of male and female pups and mean number of pups for combined sex during different intervals of lactation period in the treatment groups were not different from the control group, statistically. However, the decreased mean no. of male pups and mean no. of pups for combined sex at the mid dose on lactation day 1 was considered incidental and not related to the treatment and was within the historical control data.
F1. 11 Body Weight of Pups during Lactation Period (F2):
At the high dose, the mean weight of male pups and the mean weight of pups for combined sex on lactation days 14 and 21 and the mean weight of female pups on lactation days 7, 14, and 21 were significantly lower compared to control.
There were no statistically significant differences in the mean weight of male and female pups and the mean weight of pups for combined sex recorded during different intervals of lactation period in the low and mid dose groups compared to control group. However, significantly higher mean weight of female pups on lactation day 7 at the low and mid dose was considered incidental and not related to the treatment. The decrease in the mean weight of male and female pups and the mean weight of pups for combined sex recorded on lactation days 14 and 21 were within the range of historical control data except on lactation day 14 for combined sex which was more than the historical control data.
F1. 12 Survival Data of Pups:
Treatment had no significant effects on the number of live litters, mean litter size index, number of pups dead at birth, umber of pups dead/cannibalized up to day 4 and from 5-7, 8-14, and 15-21, and number of pups alive on day 1, 2, 4, 7, 14, and 21 and their associated survival indices at all of the doses tested. However, at the mid dose, lower no. of live pups on lactation days 1 and 2 were correlated to the higher no. of dead pups at birth, lower no. of pups discarded was considered to be related to the higher no. of pups dead/cannibalized up to day 4 and lower number of live pups at birth which resulted in lower live birth index. These findings were considered incidental as the same effects were not seen at the mid and high doses and the findings were within the historical control data.
F1. 13 Pup Observation and Necropsy findings (F2):
No treatment related abnormalities were recorded at all of the doses tested. All the findings were within the historical control data.
F1. 14 Fertility Index:
Treatment had no significant effects on the male and female fertility indices, fecundity index, mean no. of corpora lutea and implantations, percentage of implantations, parturition percentage, percentage of live pups born and percentages of pre and post-implantations losses. However, at the mid dose, significantly higher pre-implantation loss resulted in lower percentage of implantation and higher percentage of post implantation embryonic loss resulted into lower percentage of live pups born. These findings were considered to be incidental and not related to the treatment as the same effects were not seen at the low and high doses and were within the historical control data.
F1. 15 Necropsy Findings of Parents (F1):
Few gross pathological changes that were present in various groups were considered incidental as their incidences were low. One animal in the control group (Ra 3111) had an enlarged prostate but was fertile. Microscopically this change was found to be associated with moderate and diffuse suppurative inflammation.
Fl. 16 Histopathological Findings:
Gross lesions from all the animals and all collected tissues from males and females suspected of infertility were examined. Various changes in several organs were considered incidental as their frequency was low. The cause of infertility in any of the above mentioned animals could not be ascertained except for Ra 3260 (Control group), which had a follicular cyst in the ovary and Ra 3343 (high dose group), which had a luteal cyst in the ovary.
Effect levels (P0)
- Dose descriptor:
- NOEL
- Effect level:
- 75 ppm
- Sex:
- male/female
- Basis for effect level:
- other: Significant effects on body weights and food consumption at higher dose levels.
- Remarks on result:
- other: Generation: P and F1 generations (migrated information)
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
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
Details on results (F1)
See details on results (parental animals) for information.
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
Applicant's summary and conclusion
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