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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

- Two-generation reproduction toxicity study in rats, NOAEL based on non-neoplastic liver histopathology in parental animals: 8.4 mg/kg bw, NOAEL for offspring based on reduced body weight at birth: 43.7 mg/kg bw, study comparable to OECD TG 416, Borders 1985

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Mar 1983 to 31 May 1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
1981
Deviations:
yes
Remarks:
oestrus cycle and sperm parameters were not determined, data on sexual maturation were not evaluated, food consumption was only determined during pre-mating period, only brain, ovary and testes weights were determined.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: F0: 35 days
- Weight at study initiation: (P) Males: 104.8 g; Females: 108.4 g; (F1) Males: 36.8 – 48.1 g; Females: 33.7 – 43.1 g
- Housing: Individual, hanging, stainless steel, wire-bottom cages were employed during the quarantine period and all phases of the study, except during the mating, lactation, and gestation periods. Group housing in similarly designed galvanized steel caging was employed during the mating trials. These cages, equipped with solid-bottom stainless steel floorplates and nesting material were used during gestation and lactation. The floorplates and nesting material were supplied to gestating females on approximately the fifteenth day of gestation and were removed from the cages of lactating females when the progeny were approximately 7 days of age.
- Diet: rodent chow, ad libitum
- Water: ad libitum
- Acclimation period: Quarantine: 411 potential F0 parental animals were held in quarantine for 13 days following their arrival at this facility. During this time, the veterinary sciences staff examined the animals for health status. Initially, the animals were group-housed for their adaptation to the feed and the automated watering system. The animals were then housed individually for the remainder of the quarantine period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 25.5
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Mar 1983 to 31 May 1984
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was mixed with the basal diet using Hobart to achieve concentrations (w/w) of either 0, 100, 500, or 2500 ppm (active ingredient) test substance. Fresh diets were prepared at weekly intervals. Sufficient diet was offered to each animal to assure ad libitum feeding. Fresh diets were offered at weekly intervals with one exception: final diet preparation interval were fed 10 days.
Details on mating procedure:
- M/F ratio per cage: one male and two females
- Length of cohabitation: Males were rotated among the females at 10-day intervals. Each female was paired with a maximum of 2 different males.
- Proof of pregnancy: vaginal plug or sperm in vaginal
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually and supplied with nesting material at approximately 15 days of gestation

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The percent (w/w) of test substance in test diet was determined by analytical assay at approximately month 2 of the study and at monthly intervals thereafter, with one exception. During month 4 of the study, it was noted that the test article was crystallizing and had to be heated prior to diet preparation. To avoid heating the entire batch of test article each time the diet was prepared, an aliquot was obtained from the primary container to be maintained for diet preparation. A sample of this aliquot was analyzed to assure its integrity. Homogeneity and stability of prepared test diets were initiated prior to the start of the study. Analyses of the test article concentrations in prepared diets were conducted at monthly intervals. In addition, samples of the test article and the prepared test diets were sent to the Sponsor every 3 months (starting with the first month).
Duration of treatment / exposure:
12 weeks pre-mating and thereafter over two generations
Frequency of treatment:
Continuously
Details on study schedule:
See "Any other information on materials and methods incl. tables" section. Fifteen males and 30 females were selected from the population of progeny weaned from each treatment group to serve ss F1 parental animals. These selections were done randomly employing computer-generated random selection.
Dose / conc.:
100 ppm
Remarks:
Generation F0: males: 14.3 - 5.5 mg/kg bw; females: 15.6 - 6.7 mg/kg bw;
Generation F1: males: 15.8 - 6.0 mg/kg bw; females: 14.4. - 7.3 mg/kg bw;
Dose / conc.:
500 ppm
Remarks:
Generation F0: males: 73.4 - 11.5 mg/kg bw; females: 74.2 - 24.5 mg/kg bw;
Generation F1: males: 76.2 - 30.3 mg/kg bw; females: 73.1 - 39.9 mg/kg bw;
Dose / conc.:
2 500 ppm
Remarks:
Generation F0: males: 297.6 - 144.7 mg/kg bw; females: 304.4 - 173.6 mg/kg bw;
Generation F1: males: 393.4 - 159.9 mg/kg bw; females: 393.4 - 200.4 mg/kg bw;
No. of animals per sex per dose:
15 males and 30 females
Control animals:
yes, concurrent vehicle
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: All animals were observed at least twice each day for mortality, morbidity, and overt signs of toxicity, with exception on two occasions the afternoon checks were inadvertently not recorded. Each female was observed daily during gestation and lactation. Any unusual observations noted during the periods of gestation and lactation were recorded.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Each animal was removed from its cage at least once each week and was thoroughly examined for the detection of physical change.

BODY WEIGHT:
- Time schedule for examinations: All F0 and F1 parental animals were weighed weekly for 12 weeks during the pre-mating period, males were then weighed at monthly intervals until their sacrifice. Those females which failed to conceive, deliver viable progeny, or retain progeny throughout the lactation period were weighed at the monthly intervals along with the males, until their termination. Final body weights were obtained for each animal at their termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations: at weekly intervals for 12 weeks for F0 and F1 parental animals during the pre-mating period
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1a/F1b/F2a/F2b] offspring: The sexes and numbers of pups delivered , delivered viable, delivered stillborn, and found partially cannibalized were recorded for each litter of progeny on the day of parturition. The numbers of pups surviving to lactation days 4, 7, 14, and 21 (weaning) were also determined. Litters in excess of 8 pups were reduced to that number (employing computer-generated random selection for sacrifice) on lactation day 4. When possible, 4 males and 4 females were retained , however if less than 4 of either sex survived to day 4, all survivors of that sex were retained along with the appropriate number of pups of the other sex to obtain a total of 8 progeny.
Individual pup weights and sexes were determined on lactation days 0, 4, 7, 14, and 21 (weaning) for all surviving progeny of each litter obtained.
Each litter of progeny was examined twice daily for mortalities. Each pup was also examined thoroughly for developmental anomalies at birth and again at termination.

GROSS EXAMINATION OF DEAD PUPS:
- All pups found dead during the lactation period were subjected to a gross necropsy examination for the detection of gross malformations.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The surviving F0 parental males were sacrificed at 265 days of age having been fed their respective test/control diets for 230 days prior to sacrifice.
- Maternal animals: All surviving F0 generation females were sacrificed at 261-262 days of age having been fed their respective test/control diets for 226-227 consecutive days prior to sacrifice
- All F1 generation parental males were sacrificed at 232 to 251 days of age. The F1 generation females were 254 to 273 days of age at final sacrifice.

GROSS NECROPSY
- Gross necropsy examinations and organ weight determinations were performed on all parental animals surviving to final sacrifice. Additionally, any parental animals that were sacrificed in a moribund condition or died prior to final sacrifice were subjected to complete gross pathologic examinations.
- Gross necropsy consisted of the external body surface and all orifices, cranial cavity, external and cut surfaces of the brain and spinal cord; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organs; and the carcass.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Histopathologic examinations were performed upon all parental animals (sacrificed , found dead , moribund sacrifice). Slides were prepared for all tissues and organs listed below and a qualified pathologist evaluated these slides and reported all findings noted: liver, testes (with epididymides), ovaries (with corpus), uterus, prostate, vagina, seminal vesicles and all other tissues/organs appearing abnormal.
Absolute organ weights were determined for the brain (including brain stem) and ovaries or testes (with epididymides).
Postmortem examinations (offspring):
SACRIFICE
- 'a' Litter Progeny: Ten males and 10 females per treatment group were selected for gross pathologic study. These selections were done randomly employing computer-generated random selection. Additionally, any ' a' progeny that exhibited untoward developmental anomalies were subjected to pathologic studies. The remaining F1a/F2a pups were sacrificed and discarded.

- 'b' Litter Progeny:
F1b Progeny: Fifteen males and 30 females were selected from the population of progeny weaned from each treatment group to serve as F1 parental animals, selections were done randomly employing computer-generated random selection. Ten males and 10 females from each treatment group were then selected in a similar manner for pathologic study.
F2b Progeny: Ten males and 10 females from each treatment group were randomly selected for pathologic study. In addition, all 'b litter progeny which exhibited an untoward developmental anomaly were subjected to pathologic study. The remaining ' b' litter progeny were sacrificed and discarded.

GROSS NECROPSY
- Gross necropsy consisted of the external body surface and all orifices, cranial cavity, external and cut surfaces of the brain and spinal cord; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organs; and the carcass.

HISTOPATHOLOGY / ORGAN WEIGTHS
Slides were prepared for all tissues and organs listed below and a qualified pathologist evaluated these slides and reported all findings noted: liver, testes (with epididymides), ovaries (with corpus), uterus, prostate, vagina, seminal vesicles and all other tissues/organs appearing abnormal.
Absolute organ weights were determined for the brain (including brain stem) and ovaries or testes (with epididymides).
Statistics:
Quantitative continuous variables i.e., body weights and food consumption, were analyzed by analysis of variance with significant differences described by that treatment further studied by multiple comparison (Tukey's or Scheffe's, dependent upon 'N' values).
Organ weight ratios were studied employing Kruskal-Wallis analyses. Chi-Square analysis and Fisher's Exact Test were performed where appropriate.
Reproductive indices:
Mating Index = (Number of Copulations/Number of Estrus Cycles Utilized) x 100
Fertility Index = (Number of Pregnancies/Number of Copulations) x 100
Gestation Index = (Number of Parturitions/Number of Pregnancies) x 100
Female Fertility Index = (Number of Pregnancies/Number of Females Mated) x 100
Male Fertility Index = (Number of Sires/Number of Males Mated) x 100
Offspring viability indices:
Born Viable = (No. Pups Delivered Alive/Total No. Pups Delivered) x 100
Born Dead = (No. Stillbirths/Total No. Pups Delivered) x 100
Born and Cannibalized = (No. Pups Found Partially Cannibalized/Total No. Pups Delivered) x 100
4 Day = (No. Pups Viable at Lactation Day 4/No. Pups Born Alive) x 100
7 Day = (No. Pups Viable at Lactation Day 7/No. Pups Retained at Lactation Day 4) x 100
14 Day = (No. Pups Viable at Lactation Day 14/No. Pups Retained at Lactation Day 4) x 100
21 Day = (No. Pups Viable at Lactation day 21/No. Pups Retained at Lactation Day 4) x 100
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three F0 parental females died prior to final sacrifice. The first female (100 ppm) was found dead during the pre-mating period at 92 days of age having been fed test diets for 57 days. The second female ( 500 ppm) was found dead during the rest period between the F1a and F1b litters at 175 days of age having been fed test diets for 140 days. The third female (2500 ppm), was found dead after delivering 14 viable and 2 stillborn F1b progeny; this female was 222 days of age and was fed respective test diet for 187 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the the F0 generation, body weight data for males fed diets containing 500 and 2500 ppm were slightly less than the control males. However the extent of these reductions failed to indicate a direct relationship with the level of exposure to the test article and therefore appear to be the result of strain variance.
F0 parental females fed diets containing 2500 ppm exhibited reduced body weights when compared to the control females with statistically significant decreases noted at 13 of the 18 intervals. The pre-mating and total weight gains calculated for these females were also reduced.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males fed diets containing 2500 ppm test substance exhibited reduced food intake at weeks 1 and 7 of the F0 premating period. Food consumption values obtained for the 2500 ppm F0 parental females were reduced in comparison to the control females at 9 of the 13 food consumption intervals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The other treatment-related hepatic lesion was "clear-cell change" and was seen frequently in only the F0 males. The lesion consisted of both individual and small clusters of hepatocytes which had marked cytoplasmic vacuolation. These cells were scattered throughout the liver sections. Very little eosinophilic material remained in the cytoplasm of affected cells. The vacuolation sometimes consisted of numerous discrete cytoplasmic vacuoles, but in other cells nearly all the entire cytoplasm was clear without the presence of vacuoles. There was usually no nuclear displacement. The location of the affected cells varied within the hepatic lobules, and the overall severity of the change ranged from minimal to moderate within the sections examined.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of the F0 generation animals revealed a single benign neoplasm. It was found in the ear of a T-II female rat and was diagnosed as a fibroma. Although it occurred in a treated animal, such neoplasms can occur spontaneously in the ears or subcutaneous tissue of normal rats, and this tumor is not considered to be treatment-related.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Vaginal discharges were noted either during gestation or lactation of the F0 generation for 3 control (0 ppm), 1 T-I (100 ppm), 1 T-II (500 ppm ), and 5 T-III ( 2500 ppm ) females. One control and 1 T-I female resorbed their F1b litters and 2 T-III females resorbed their F1a litters. In addition, during the F1b litter T-III one female was found dead on lactation day 1 after delivering 14 viable and 2 stillborn pups.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
100 ppm
Based on:
test mat.
Remarks:
approx. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No consistent treatment-related ante mortem observations were noted. One male control animal was sacrificed in a moribund condition during the F1b matings.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the F1 generation one male (500 ppm) was sacrificed during the F2b mating trials (205 days of age) in a moribund condition which apparently resulted from a cage injury. Two F1 generation females (2500 ppm) did not survive to final sacrifice. The first was found dead during parturition of its F2a litter (142 days of age) and the second was sacrificed in a moribund condition during parturition of its F2a litter (142 days of age).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the F1 generation, the 2500 ppm males exhibited reduced body weights when compared to the control males. This decrease began at the initiation of the F1 generation (week 0) and continued through the final sacrifice. All other body weight data obtained for the treated groups F1 parental males were comparable to the concurrent control males. F1 parental females fed diets containing 2500 ppm test substance exhibited reduced body weights when compared to the control females. Statistical evaluations of these data revealed significant reductions for 14 of the 19 intervals. In addition, the pre-mating and total weight changes for these females were reduced when compared to the control females. Significant body weight reductions were noted for the F1 generation females fed diets containing 500 ppm at weeks 10 and 11 and a significant reduction in the pre-mating weight gain was noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The F1 generation males fed diets containing 2500 ppm test substance exhibited reduced food intake at weeks 2, 6 and 10 of the pre-mating period. Females fed diets containing 2500 ppm test substance exhibited reduced food intake throughout the F1 pre-mating period. Food consumption data obtained for the 100 and 500 ppm males and females were considered to be comparable to the control animals. Statistical differences which were noted for these groups were sporadic in occurrence and therefore considered the result of strain variance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Three F1 generation animals died or were sacrificed in a moribund condition prior to final sacrifice. One male (500 ppm) was sacrificed in a moribund condition at 205 days of age and exhibited dried crusted blood in the nose and a maxilla that was fractured approximately 1 cm posterior to the incisors. One female (2500 ppm ) was found dead at 142 days of age and necropsy findings were limited to uterine counts. Necropsy findings for another 2500 ppm female that was sacrificed in a moribund condition were severe paleness of all body tissues, pale liver, multiple red areas in the lung, cannibalized pup remains in the stomach, 4 late resorptions in the left uterine horn, one pup involved in a torsion of the right uterine horn which obstructed the vagina, and a 180° left to right torsion of the vagina.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were found in the liver in F1 male and female rats. The most frequent change seen was "cellular swelling". This occurred in both periportal and centrilobular areas and consisted of a mild increase in the size of hepatocytes with an apparent increase in cytoplasmic volume and compression of adjacent hepatic sinusoids. The affected cells did not have the irregular vacuolation typical of glycogen which is frequently seen in normal hepatocytes from animals which had not been fasted. There sometimes was nuclear enlargement also present in these cells. The incidence and severity of cellular swelling increased with dose in both the F1 males and females. The incidence was over 95% in both sexes in the T-III group and the severity mild to moderate in the males and minimal to moderate in the females. The incidence of cellular swelling was also significant in the T-II group in both the males and females at the .05 level using Fisher 's Exact Test, This change was also evident in the liver of the T-III F2b weanlings, where it was present in 10/10 males and 9/ 10 females.
The other treatment-related hepatic lesion was "clear-cell change" and was seen frequently in the F1 males and females. The lesion consisted of both individual and small clusters of hepatocytes which had marked cytoplasmic vacuolation. These cells were scattered throughout the liver sections. Very little eosinophilic material remained in the cytoplasm of affected cells. The vacuolation sometimes consisted of numerous discrete cytoplasmic vacuoles, but in other cells nearly all the entire cytoplasm was clear without the presence of vacuoles. There was usually no nuclear displacement. The location of the affected cells varied within the hepatic lobules, and the overall severity of the change ranged from minimal to moderate within the sections examined. Three F1 animals died or were non-scheduled sacrifices prior to the scheduled sacrifice. One male fractured his maxilla and was sacrificed. One moribund female was sacrificed and found to have a uterine torsion. One female was found dead but the cause of death could not definitely be determined.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Four benign and one malignant neoplasms were found in the F1 generation animals. All these neoplasms occurred in T-I F1 generation rats. A fibroma occurred in one T-I male rat. The other neoplasms were each found in a different T-I female. The deciduoma found in the uterus of one animal consisted of a proliferation and persistence of decidual cells in the decidua basalis of the endometrium at an old implantation site. Such lesions, as well as the other neoplasms observed, do occur spontaneously in rats of this age range and are not considered treatment-related.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
During the F1 generation, vaginal discharges were noted either during gestation or lactation of the F1 generation for 3 control (0 ppm ), 2 T-I (100 ppm), 2 T-II (500 ppm) and 3 T-III (2500 ppm) females. One female from each of the control, T-I and T-II groups resorbed their F2a litters and 1 control, 2 T-I and 2 T-II females resorbed their F2b litters. On lactation days 0 and 1 of the F2b litter, T-III female exhibited pale eyes, ears, and appendages, was listless ( day 0), and had dried brown substance on fur (perianal). This female delivered a single pup that was cannibalized and still exhibited a tan vaginal discharge on lactation day 3 (3 days post parturition). In addition, during the F2a litter one T-III female was found dead after delivering 6 viable pups and another T-III female was sacrificed in a moribund condition on lactation day 1 after delivering 4 viable, 2 stillborn and 1 cannibalized pup.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the F1a litter, progeny obtained from dams exposed to 2500 ppm exhibited reduced body weights in comparison to the control progeny on lactation days 4, 7, 14, and 21. Statistical evaluations, utilizing the individual progeny body weight data, revealed these reductions to be statistically significant when compared to the control progeny; statistical evaluations utilizing the mean litter weight data, revealed significant reductions in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F1b progeny were reduced in comparison to the control progeny on lactation days 14 and 21. Statistically significant body weight reductions were noted for the 2500 ppm F1b progeny at lactation day 14 (analyses utilizing individual body weight data) and day 21 (analyses utilizing individual and mean litter weight data).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
One 2500 ppm F1a anomalous pup (a partially opened left eyelid and a left eye which was smaller than normal), two 2500 ppm F1b anomalous pups (eyes which appeared smaller than normal; a left eye which was enlarged with an opacity) and one 2500 ppm F1b pup with an unopened eyelid (the eye was, however not missing, and apparently of normal size) were obtained from dams treated with the test substance.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared to the control males, the 2500 ppm F1a males exhibited significant reductions in mean body weight, brain weight, and testes with epididymides weight and a significant increase in their brain to body weight ratio. All other body weight and organ weight data obtained for F1a progeny were comparable to the control progeny. Body weight and organ weight data obtained for sacrificed F1b progeny were comparable to the control progeny.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Three F1a and 2 F1b anomalous progeny were obtained from the test substance treated dams. During the F1a litter, female's (100 ppm) pup was anurous and displayed clubbed limbs; another female's (100 ppm) pup had a cleft lip; and a female's (2500 ppm) pup displayed a partially opened left eyelid and a left eye which was smaller than normal was confirmed by necropsy examination.
During the F1b litter, a female's (2500 ppm) pup displayed a domed forehead and eyes which appeared smaller than normal, and another female's (2500 ppm) pup exhibited a left eye which appeared larger than normal and covered with a cloudy film. Gross pathologic examination of another pup revealed the brain was dilated, however no untoward findings were noted for the eyes. The necropsy examination of another pup of the same female revealed that the left eye was enlarged with an opacity. In addition, pup from a different 2500 ppm female exhibited an unopened eyelid , however gross pathologic examination revealed the eye was present and apparently normal in size.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Swelling of hepatocytes was found in the liver of the F1b weanlings. The incidence of this lesion was statistically significant in 2500 ppm males and females
Other effects:
not examined
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
During the F2a litter, the number of pups delivered, delivered viable and surviving to lactation day 4 were statistically reduced for the 2500 ppm group of dams exposed to the test substance when compared to the control dams. Statistically significant reductions were also noted for this group of dams during the F2b litter for progeny surviving to lactation days 7, 14 and 21. No other statistically significant differences were noted for delivery and population data during the F2a and F2b litters.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the F2a litter, progeny obtained from dams exposed to 2,500 ppm exhibited statistically reduced body weights in comparison to the control progeny on lactation days 4, 7, 14 and 21 utilizing the individual progeny body weight data. Statistical evaluations utilizing the mean litter weight data revealed significant reductions for this group in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F2b progeny were statistically reduced in comparison to the control progeny on lactation days 0 through 21 (utilizing individual body weight data) and on lactation days 4 through 21 (utilizing mean litter weight data).
The 500 ppm group' s F2a progeny revealed a statistically significant increase (utilizing individual progeny body weight data) on lactation day 0 and statistically significant reductions for the F2b progeny on lactation days 0, 14 and 21.
In general, statistical analyses of the 100 and 500 ppm progeny body weight data revealed sporadically occurring reductions that were either singular in occurrence or within the range that has been seen at this facility among control progeny. Additionally, although birth weights for the 100 ppm F2b progeny were reduced there was an increase in the mean numbers of pups per litter delivered by 100 ppm dams as compared to the control dams.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared to the control males, the 2500 ppm F2a males exhibited significant reductions in mean final body weight, the testes with epididymides weight, and the testes with epididymides to brain weight ratio and a significant increase in their brain to body weight ratio.
A significant increase in the brain to body weight ratio was noted for the 2500 ppm F2b males and a significant reduction in brain weight was noted for the 2500 ppm F2b females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Two F2a and 2 F2b anomalous progeny were obtained during the F1 generation. During the F2a litter, one control female's (0 ppm) pup displayed a raised area of skin on the abdomen and another female (500 ppm) delivered a stillborn pup which exhibited agnathia and possible exencephaly (dam cannibalized top of head). During the F2b litter, one female's (100 ppm) pup displayed clubbed limbs, shortened torso and a shortened tail, and another female's (500 ppm) pup kept the left eyelid closed. Gross pathologic examination of another pup from the 500 ppm female revealed the left eye was shrunken in size. The remaining anomalies noted were limited to runt pups and pups with hematomas and the incidence of these findings which occurred among the treated groups were similar to the control. Statistical analyses of the progeny anomaly data revealed no significant differences.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were found in the liver in F2b male and female rats. The most frequent change seen was "cellular swelling". This occurred in both periportal and centrilobular areas and consisted of a mild increase in the size of hepatocytes with an apparent increase in cytoplasmic volume and compression of adjacent hepatic sinusoids. The affected cells did not have the irregular vacuolation typical of glycogen which is frequently seen in normal hepatocytes from animals which had not been fasted. There sometimes was nuclear enlargement also present in these cells. The incidence and severity of cellular swelling increased with dose was evident in the liver of the T-III F2b weanlings, where it was present in 10/10 males and 9/ 10 females.
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F2b
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: birth weights
Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
2 500 ppm
System:
male reproductive system
Organ:
testes
other: epididymides
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

Dietary verification of doses

The dietary analyses results were generally within +10% of the theoretical diet concentrations. Homogeneity of the prepared diets and the stability of prepared diets through 14 days (diets stored frozen or ambient) were established by analytical methods.

Table 2. Test article consumption data in a two generation reproduction study in albino rats

Generation

Sex

Group
(ppm)

Test article consumption:
maximum-minimum week mean values
(mg/kg)

F0

F0

F0

F0

F0

F0

M

F

M

F

M

F

100
100
500
500
2500
2500

14.3 - 5.5
15.6 - 6.7
73.4 - 11.5
74.2 - 24.5
297.6 - 144.7
304.4 - 173.6

F1
F1
F1
F1
F1
F1

M

F

M

F

M

F

100
100
500
500
2500
2500

15.8 - 6.0
14.4. - 7.3
76.2 - 30.3
73.1 - 39.9
393.4 - 159.9
393.4 - 200.4

 

Table 3. Body weight gain (g) of parental animal

 

Concentrations of test substance

Males

Females

0 ppm

100 ppm

500 ppm

2500
ppm

0 ppm

100 ppm

500 ppm

2500
ppm

Pre-maiting weight gain (weeks 1-12)

F0

369.0

350.7

339.9

339.7

172.7

173.1

168.4

137.0**

F1

434.3

440.9

439.7

396.1

225.8

220.6

206.2**

191.6**

Total weight gain

F0

509.5

500.9

479.8

481.2

252.0

240.9

254.4

193.4**

F1

593.0

615.5

612.3

550.2

313.4

306.8

293.5

254.3**

** statistically significant difference P<0.01

 

Table 4. Body weight (g) of dams over gestation and lactation periods (week 13 forward)

 

F0

F1

Concentration

0 ppm

100 ppm

500 ppm

2500 ppm

0 ppm

100 ppm

500 ppm

2500 ppm

Week
(approximate)

 

16

296.0

305.4

318.4

269.6

284.3

285.3

286.3

 

20

311.9

308.0

312.5

271.3**

317.7

304.9

301.7

262.3

24

351.8

354.9

365.7

286.3

306.3

329.1

323.3

275.5

28

361.0

371.4

374.1

305.0

327.3

343.3

337.1

280.1*

32

-

-

-

-

336.7

337.7

322.2

283.7**

Final body
weight

360.5

350.1

363.7

301.7**

353.1

350.1

336.6

287.9**

* statistically significant difference P<0.05, ** statistically significant difference P<0.01

 

Table 5. Selected litter data

Litter

Day

Mean number of live pups

Mean pup body weight (g)

Concentration of test substance (ppm)

0

100

500

2500

0

100

500

2500

F1a

0

12.2

11.7

11.2

11.8

6.1

6.0

6.1

6.2

4

11.0

11.4

11.0

11.4

8.9

8.8

9.2

8.1**

7

6.8

7.4

7.5

7.7

14.6

13.0**

15.2

12.3**

14

6.8

7.2

7.5

7.4

28.2

25.4**

26.9*

21.9**

21

6.8

7.2

7.4

7.4

48.0/46.1

44.1/42.5**

47.7/44.9

35.8/34.7**

F1b

0

11.4

12.7

13.2

12.4

6.1

6.0

6.4**

6.1

4

10.3

12.3

12.9

12.1

8.8

9.2

9.5**

8.6

7

6.4

7.7

7.8

7.4

13.4

14.5

14.5*

13.3

14

6.3

7.7

7.7

7.2

25.7

27.7*

28.0**

22.9**

21

6.3

7.7

7.7

7.2

44.3/40.1

46.1/43.5

47.7/43.5

35.6/32.5**

F2a

0

12.5

13.0

10.8

9.2**

5.4

5.3

5.7*

5.4

4

12.0

11.6

10.4

8.1**

8.5

7.9**

8.3

7.5**

7

7.8

7.2

7.2

6.5

13.7

13.3

13.0

10.8**

14

7.8

7.2

7.0

6.2

25.6

25.4

25.2

20.0**

21

7.8

7.1

7.0

6.2

43.8/41.1

42.5/40.4

42.5/39.8

31.5/30.2**

F2b

0

12.8

13.4

12.7

11.3

5.8

5.4**

5.5**

5.4**

4

12.5

13.2

12.5

10.6

8.8

8.6

8.5

7.3**

7

7.7

8.0

7.8

6.2*

14.6

14.7

13.8

10.9**

14

7.7

8.0

7.8

5.7**

29.4

28.7

26.8**

21.9**

21

7.7

8.0

7.8

5.7**

48.8/46.2

48.6/46.6

45.1/42.3**

36.7/33.3**

Day 21 pup body weights are males/females ** statistically significant difference P<0.01,

* P<0.05

Data for lactation days 7 and 14 not shown

 

Table 6. Histopathological changes in the liver of parental animals and progeny

Generation

Concentration of test substance

Males

Females

0 ppm

100 ppm

500 ppm

2500 ppm

0 ppm

100 ppm

500 ppm

2500 ppm

Hypertrophy

F0

7/15

3/15

13/15*

14/15*

4/30

3/29

6/30

29/30*

F1

0/15

1/15

5/15*

15/15*

0/30

2/30

15/30*

29/30*

F1b

2/10

1/10

2/10

10/10*

1/10

1/10

2/10

8/10*

F2b

0/10

0/10

2/10

10/10*

0/10

0/10

1/10

9/10*

 

 

 

 

 

 

 

 

 

F0

0/15

2/15

3/15

14/15*

1/30

1/29

1/30

1/30

F1

2/15

5/15

8/15*

11/15*

2/30

4/30

7/30

10/30*

F1b

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

F2b

0/10

0/10

0/10

1/10

1/10

0/10

0/10

0/10

* statistically significant difference P<0.05

 

Table 7. Mean testes weights (% of control group)

Generation

Dietary concentration of the test substance

Males

0 ppm

100 ppm

500 ppm

2500 ppm

Mean absolute testes weight (with epididymides) (% of control group)

F0

100

101

97.1

109

F1a

100

96.4

93.5

66.2*

F1b

100

107

109

96.7

F2a

100

93.7

94.8

73.0*

F2b

100

106

98.5

84.8

Mean relative testes (with epididymides) / body weight (% of control group)

F0

100

103

102

114

F1a

100

101

99.7

100

F1b

100

108

103

112

F2a

100

94.7

96.2

98.2

F2b

100

107

101

108

* statistically significant difference P<0.05

 

Table 8. Mean brain weights (% of control group) in male rats

Generation

Dietary concentration of the test substance

Males

0 ppm

100 ppm

500 ppm

2500 ppm

Mean absolute brain (% of control group)

F0

100

101

101

101

F1a

100

98.0

99.4

88.9**

F1b

100

98.3

104

97.1

F2a

100

101

99.4

95.0

F2b

100

102

103

96.1

Mean relative brain / body weight (% of control group)

0

100

102

106

106

F1a

100

107

113

137*

F1b

100

103

97.1

116

F2a

100

107

101

129**

F2b

100

103

105

121**

* statistically significant difference P<0.05

** statistically significant difference P<0.01

 

Table 9. Mean brain weights (% of control group) in female rats

Generation

Dietary concentration of the test substance

Females

0 ppm

100 ppm

500 ppm

2500 ppm

Mean absolute brain (% of control group)

F0

100

99.1

101

101

F1a

100

102

102

97.4

F1b

100

102

103

96.3

F2a

100

102

103

95.5

F2b

100

102

101

92.4*

Mean relative brain / body weight (% of control group)

F0

100

102

101

120**

F1a

100

98.9

96.1

122

F1b

100

94.6

89.3

110

F2a

100

117

103

115

F2b

100

95.0

101

108

* statistically significant difference P<0.05

** statistically significant difference P<0.01

Conclusions:
Swelling of hepatocytes in areas of the liver, diagnosed as cellular swelling, was found in the liver of the male and female F0, F1b, F1 and F2b rats in a dose-related manner and appears to be due to the administration of the test substance. The incidence of this lesion was statistically significant at 500 and 2500 ppm in the F0 and F1 parents and at 2500 ppm in the F1b and F2b weanlings. The incidence of clear-cell change in the liver also increased with increasing doses of the test substance in the male F0 rats and the male and female F1 rats and appears also to be related to administration of the test article.
Statistical analysis of the incidence of both hepatocellular swelling and of clear-cell change in rats receiving 100 ppm of the test substance revealed the incidence of these lesions was not significantly different from those of the controls and therefore the lesions in this dose level are not felt to be treatment-related.
Therefore, the NOAEL for parental toxicity in this two generation reproduction study in rats in compliance with GLP and following an method equivalent to OECD 416 guideline used dietary administration of the test substance was 100 ppm (app. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females). The NOAEL for reproduction and offspring toxicity was 500 ppm (48.8 mg/kg bw for males and 43.7 mg/kg bw in females).
Executive summary:

The study was conducted according to draft Guidelines of U.S:FIFRA Subdiv. F § 83-4 and it complies with OECD 416 and directive 87/302 EEC Part B guidelines. The final Guidelines were not yet released when the study was conducted. The study was performed according to the principles of GLP.

A two-generation reproduction study was conducted to ascertain the potential effects of the test substance upon the growth, reproductive performance, and offspring of 2 consecutive generations of albino rats. Ad libitum feeding of diets containing either 0 (VC), 100 (T-I), 500 (T-II), or 2500 (T-III) ppm (active ingredient) the test substance was employed as the route of administration.

The results obtained during this study are summarized as follows. Four F0 generation parental animals died or were sacrificed in a moribund condition: a 0 ppm male was sacrificed in a moribund condition during the F1b mating trials; a 100 ppm female was found dead during the pre-mating period; a 500 ppm female was found dead during the rest period between the F1a and F1b litters; and a 2500 ppm female was found dead during parturition of her F1b litter.

During the second generation, a 500 ppm male was sacrificed in a moribund condition during the F2b mating trials (apparently the result of a cage injury) and two 2500 ppm females died during parturition of their F2a litters (one was found dead and one was sacrificed in a moribund condition ). The remaining parental animals (F0 and F1 generation) survived to final sacrifice.

During the F0 generation, body weight data obtained for the treated males did not reveal any reductions which were considered to be the result of the test substance ingestion. During the F1 generation, the 2500 ppm males exhibited reduced body weight when compared to the control males. All other body weight dataobtained for the treated F1 parental males were comparable to the concurrent control males. F0 and F1 parental females fed diets containing 2500 ppm exhibited reduced body weights throughout their respective generations. In addition, the pre-mating and overall weight gains for the F0 and F1 2500 ppm females were decreased when compared to the concurrent control females. The F1 generation females fed diets containing 500 ppm exhibited significant reductions at 2 of the 12 pre-mating intervals and in the pre-mating weight gain when compared to the control females, other significant body weight reductions were noted. F0 generation males fed diets containing 2500 ppm exhibited reduced food intake at weeks 1 and 7 of the F0 pre-mating period; No F1 generation males fed diets containing 2500 ppm exhibited reduced food intake at weeks 2, 6 and 10 of the pre-mating period. All other food consumption data obtained for parental males during both generations were comparable to the control males during the pre-mating periods. Food consumption values obtained for the 2500 ppm F0 and F1 parental females were reduced in comparison to the control females. Food consumption data obtained for the 100 and 500 ppm females were considered to be comparable to the control females. No consistent antemortem observations were noted which appeared to be the result of the test substance ingestion. Reproductive indices calculated for the groups of rats fed the test substance were comparable to the concurrent control group. Vaginal discharges were noted either during gestation or lactation of the F0 generation for 3 control (0 ppm), 1 T-I ( 100 ppm), 1 T-II ( 500 ppm), and 5 T-III ( 2, 500 ppm) females. One control and 1 T-I female resorbed their F1b litters and 2 T-III females resorbed their Fla litters. In addition, during the F1b litter a T-III female was found dead on lactation day 1 after delivering 14 viable and 2 stillborn pups. During the F1 generation, vaginal discharges were noted either during gestation or lactation of the F1 generation for 3 control (0 ppm), 2 T-I ( 100 ppm), 2 T-II ( 500 ppm) and 3 T-III (2,500 ppm) females. One female from each of the control, T-I and T-II groups resorbed their F2a litters and 1 control, 2 T-I and 2 T-II females resorbed their F2b litters. On lactation days 0 and 1 of the F2b litter, a T-III female exhibited pale eyes, ears, and appendages, was listless ( day 0), had dried brown substance on fur ( perianal). This female delivered a single pup which was cannibalized and still exhibited a vaginal discharge (tan) on lactation day 3 (3 days post parturition). In addition , during the F2a litter a T-III female was found dead after delivering 6 viable pups and a T-III female was sacrificed in a moribund condition on lactation day 1 after delivering 4 viable, 2 stillborn and 1 cannibalized pups. Delivery and population data obtained for the groups of dams exposed to the test substance were comparable to the control dams during both the F1a and F1b litters. the number of pups delivered, delivered viable and surviving to During the F2a litter , lactation day 4 were statistically reduced for the 2500 ppm dams when compared to the control dams. Statistically significant reductions were also noted for this group of dams during the F2b litter for progeny surviving to lactation days 7, 14 and 21. No other significant differences were noted for delivery and population data during the F2a and F2b litters. Calculated indices of progeny survival were considered to be comparable for treated and control groups during the F1a and F1b litters. During the F2b litter, progeny survival indices at lactation days 7, 14 and 21 were statistically reduced for the 2500 ppm group dams. No other significant differences were noted for progeny survival during the F2a and F2b litters. During the F1a litter, progeny obtained from dams exposed to 2500 ppm exhibited reduced body weights in comparison to the control progeny on lactation days 4, 7, 14, and 21. Statistical evaluations, utilizing the individual progeny body weight data, revealed these reductions to be statistically significant when compared to the control progeny; statistical evaluations utilizing the mean litter weight data, revealed significant reductions in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F1b progeny were reduced in comparison to the control progeny on lactation days 14 and 21. Statistically significant body weight reductions were noted for the 2500 ppm F1b progeny at lactation day 14 (analyses utilizing individual body weight data) and day 21 ( analyses utilizing individual and mean litter weight data). During the F2a litter, progeny obtained from dams exposed to 2500 ppm exhibited statistically reduced body weights in comparison to the control progeny on lactation days 4, 7, 14 and 21 utilizing the individual progeny body weight data. Statistical evaluations utilizing the mean litter weight data, revealed significant reductions for this group in comparison to the control progeny beginning at lactation day 7 and continuing through weaning ( lactation day 21). Body weight data obtained for the 2500 ppm F2b progeny were statistically reduced in comparison to the control progeny on lactation days 0 through 21 (utilizing individual body weight data) and on lactation days 4 through 21 (utilizing mean litter weight data). Body weight data obtained for the 100 and the 500 ppm progeny did not reveal any consistent alterations which appeared to be a direct result of the test article. Examinations of progeny structural development did not reveal any aberrant findings which were considered to be related to the test substance exposure. Statistical evaluation of the organ weight data obtained for F0 generation animals revealed the brain to body weight data obtained for the 2500 ppm parental females was increased when compared to control females and significant reductions in mean final body weight, brain weight, and testes with epididymides weight and a significant increase in their brain to body weight ratio for the 2500 ppm F1a male progeny. During the F1 generation, the brain to body weight data obtained for the 2500 ppm parental females was increased when compared to control females. When compared to the control males, the 2500 ppm F2a males exhibited significant reductions in mean final body weight, the testes with epididymides weight, and the testes with epididymides to brain weight ratio and a significant increase in their brain to body weight ratio. Mean final body weight data obtained for the 2500 ppm F2a female progeny were somewhat less than the control females, however, no statistically significant differences were noted. The 2500 ppm F2b male and female progeny exhibited significant reductions in mean final body weights. A significant increase in the brain to body weight ratio was noted for the 2500 ppm F2b males and a significant reduction in brain weight was noted for the 2500 ppm F2b females. No other statistically significant differences were noted in organ weight data obtained for the the test substance animals when compared to their concurrent control. Gross pathologic examination of the parental animals from both generations (sacrifice, found dead, moribund sacrifice) revealed no untoward findings which appeared to be the result of the test substance ingestion. Necropsy examination conducted upon sacrificed progeny and progeny which died during the lactation period revealed no untoward findings which were considered the result of the test substance exposure. The results of the microscopic examinations conducted by the third party laboratory revealed the exposure of male and female albino rats to 100, 500 or 2500 ppm of the test substance in the diet during gestation and for two reproductive cycles did not produce any changes in the reproductive tract that appear to be treatment-related. Swelling of hepatocytes in areas of the liver, diagnosed as cellular swelling, was found in the liver of the male and female F0, F1b, F1 and F2b rats in a dose-related manner and appears to be due to the administration of the test substance. The incidence of this lesion was statistically significant at the .05 level using Fisher's Exact Test, at 500 and 2500 ppm in the F0 and F1 parents and at 2500 ppm in the F1b and F2b weanlings. The incidence of clear-cell change in the liver also increased with increasing doses of the test substance in the male F0 rats and the male and female F1 rats and appears also to be related to administration of the test article. The incidence of clear-cell change was similarly statistically significant for at least one group and one sex of rats at both the 500 and 2500 ppm levels. Statistical analysis of the incidence of both hepatocellular swelling and of clear-cell change in rats receiving 100 ppm of the test substance revealed the incidence of these lesions was not significantly different from those of the controls and therefore the lesions in this dose level are not felt to be treatment-related. No other lesions found in this study were considered treatment-related.

Therefore, the NOAEL for parental toxicity in this two generation reproduction study in rats in compliance with GLP and following an method equivalent to OECD 416 guideline used dietary administration of the test substance was 100 ppm (app. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females). The NOAEL for reproduction and offspring toxicity was 500 ppm (48.8 mg/kg bw for males and 43.7 mg/kg bw in females).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
8.4 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant OECD TG 416
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

All available data was assessed and the study representing the worst-case effects was included as key study. The test substance did not induce toxicity to reproduction in rats.


 


The key study (Borders 1985) is a two-generation toxicity study in rats, comparable to OECD TG 416 and performed according to the principles of GLP. The study was conducted to ascertain the potential effects of the test substance upon the growth, reproductive performance, and offspring of 2 consecutive generations of albino rats. Ad libitum feeding of diets containing either 0, 100, 500, or 2500 ppm (active ingredient) the test substance was employed as the route of administration.  


Four F0 generation parental animals died or were sacrificed in a moribund condition: a 0 ppm male was sacrificed in a moribund condition during the F1b mating trials; a 100 ppm female was found dead during the pre-mating period; a 500 ppm female was found dead during the rest period between the F1a and F1b litters; and a 2500 ppm female was found dead during parturition of her F1b litter. During the second generation, a 500 ppm male was sacrificed in a moribund condition during the F2b mating trials (apparently the result of a cage injury) and two 2500 ppm females died during parturition of their F2a litters (one was found dead and one was sacrificed in a moribund condition). The remaining parental animals (F0 and F1 generation) survived to final sacrifice. Reproductive indices calculated for the groups of rats fed the test substance were comparable to the concurrent control group. Vaginal discharges were noted either during gestation or lactation of the F0 generation for 3 control), 1 female at 100 ppm, 1 female at 500 ppm, and 5 females at 2500 ppm. One control and 1 female at 100 ppm, resorbed their F1b litters and 2 females at 500 ppm resorbed their F1a litters. In addition, during the F1b litter a female at 500 ppm was found dead on lactation day 1 after delivering 14 viable and 2 stillborn pups. During the F1 generation, vaginal discharges were noted either during gestation or lactation of the F1 generation for 3 control (0 ppm), 2 females at 100 ppm, 2 females at 500 ppm and 3 females 2500 ppm. One female from each of the control, 100 ppm and 500 ppm groups resorbed their F2a litters and 1 control, 2 100 ppm and 2 500 ppm females resorbed their F2b litters. On lactation days 0 and 1 of the F2b litter, a female at 2500 ppm exhibited pale eyes, ears, and appendages, was listless (day 0), had dried brown substance on fur (perianal). This female delivered a single pup which was cannibalized and still exhibited a vaginal discharge (tan) on lactation day 3 (3 days post parturition). In addition, during the F2a litter a 2500 ppm female was found dead after delivering 6 viable pups and a 2500 ppm female was sacrificed in a moribund condition on lactation day 1 after delivering 4 viable, 2 stillborn and 1 cannibalised pups. Delivery and population data obtained for the groups of dams exposed to the test substance were comparable to the control dams during both the F1a and F1b litters. the number of pups delivered, delivered viable and surviving to. During the F2a litter, lactation day 4 were statistically reduced for the 2500 ppm dams when compared to the control dams. Statistically significant reductions were also noted for this group of dams during the F2b litter for progeny surviving to lactation days 7, 14 and 21. No other significant differences were noted for delivery and population data during the F2a and F2b litters. Calculated indices of progeny survival were considered to be comparable for treated and control groups during the F1a and F1b litters. During the F2b litter, progeny survival indices at lactation days 7, 14 and 21 were statistically reduced for the 2500 ppm group dams. No other significant differences were noted for progeny survival during the F2a and F2b litters. During the F1a litter, progeny obtained from dams exposed to 2500 ppm exhibited reduced body weights in comparison to the control progeny on lactation days 4, 7, 14, and 21. Statistical evaluations, utilizing the individual progeny body weight data, revealed these reductions to be statistically significant when compared to the control progeny; statistical evaluations utilizing the mean litter weight data, revealed significant reductions in comparison to the control progeny beginning at lactation day 7 and continuing through weaning (lactation day 21). Body weight data obtained for the 2500 ppm F1b progeny were reduced in comparison to the control progeny on lactation days 14 and 21. Statistically significant body weight reductions were noted for the 2500 ppm F1b progeny at lactation day 14 (analyses utilizing individual body weight data) and day 21 ( analyses utilizing individual and mean litter weight data). During the F2a litter, progeny obtained from dams exposed to 2500 ppm exhibited statistically reduced body weights in comparison to the control progeny on lactation days 4, 7, 14 and 21 utilizing the individual progeny body weight data. Statistical evaluations utilising the mean litter weight data, revealed significant reductions for this group in comparison to the control progeny beginning at lactation day 7 and continuing through weaning ( lactation day 21). Body weight data obtained for the 2500 ppm F2b progeny were statistically reduced in comparison to the control progeny on lactation days 0 through 21 (utilising individual body weight data) and on lactation days 4 through 21 (utilising mean litter weight data). Body weight data obtained for the 100 and the 500 ppm progeny did not reveal any consistent alterations which appeared to be a direct result of the test article. Examinations of progeny structural development did not reveal any aberrant findings which were considered to be related to the test substance exposure. Statistical evaluation of the organ weight data obtained for F0 generation animals revealed the brain to body weight data obtained for the 2500 ppm parental females was increased when compared to control females and significant reductions in mean final body weight, brain weight, and testes with epididymides weight and a significant increase in their brain to body weight ratio for the 2500 ppm F1a male progeny. During the F1 generation, the brain to body weight data obtained for the 2500 ppm parental females was increased when compared to control females. When compared to the control males, the 2500 ppm F2a males exhibited significant reductions in mean final body weight, the testes with epididymides weight, and the testes with epididymides to brain weight ratio and a significant increase in their brain to body weight ratio. Mean final body weight data obtained for the 2500 ppm F2a female progeny were somewhat less than the control females, however, no statistically significant differences were noted. The 2500 ppm F2b male and female progeny exhibited significant reductions in mean final body weights. A significant increase in the brain to body weight ratio was noted for the 2500 ppm F2b males and a significant reduction in brain weight was noted for the 2500 ppm F2b females. No other statistically significant differences were noted in organ weight data obtained for the test substance animals when compared to their concurrent control. Gross pathologic examination of the parental animals from both generations (sacrifice, found dead, moribund sacrifice) revealed no untoward findings which appeared to be the result of the test substance ingestion. Necropsy examination conducted upon sacrificed progeny and progeny which died during the lactation period revealed no untoward findings which were considered the result of the test substance exposure. The results of the microscopic examinations conducted by the third party laboratory revealed the exposure of male and female albino rats to 100, 500 or 2500 ppm of the test substance in the diet during gestation and for two reproductive cycles did not produce any changes in the reproductive tract that appear to be treatment-related. Swelling of hepatocytes in areas of the liver, diagnosed as cellular swelling, was found in the liver of the male and female F0, F1b, F1 and F2b rats in a dose-related manner and appears to be due to the administration of the test substance. The incidence of this lesion was statistically significant at the .05 level using Fisher’s Exact Test, at 500 and 2500 ppm in the F0 and F1 parents and at 2500 ppm in the F1b and F2b weanlings. The incidence of clear-cell change in the liver also increased with increasing doses of the test substance in the male F0 rats and the male and female F1 rats and appears also to be related to administration of the test article. The incidence of clear-cell change was similarly statistically significant for at least one group and one sex of rats at both the 500 and 2500 ppm levels. Statistical analysis of the incidence of both hepatocellular swelling and of clear-cell change in rats receiving 100 ppm of the test substance revealed the incidence of these lesions was not significantly different from those of the controls and therefore the lesions in this dose level are not felt to be treatment-related. No other lesions found in this study were considered treatment-related. 


The NOAEL for parental toxicity was 100 ppm (app. 8.4 mg/kg bw/day males and 9.7 mg/kg bw/day females). The NOAEL for reproduction and offspring toxicity was 500 (48.8 mg/kg bw for males and 43.7 mg/kg bw in females). 


 


There is one supporting study for this endpoint (Fritz 1981). This study was conducted according to an EPA guideline and did not followed GLP. Dietary concentrations of 0, 400, 2000 and 5000 ppm were administered to rats for 155 days. The rats were killed after weaning of the F1 and F2 generation, respectively. Autopsy and histopathologal examination were performed on a selected number of F1 and F2 weanlings and F1 adults. The test substance revealed toxicity in the F0 generation at the concentration of 5000 ppm as indicated by a decrease in body weight gain, food consumption and perinatal death of the dams. At 2000 ppm, the F0, F1 and F2 animals reacted to the treatment by a reduction in body weight gain, at 400 ppm only the F1 animals were affected in this respect. A slight but significant increase in the liver to body weight ratio was recorded for several experimental groups. A slight hypertrophy of centrilobular hepatocytes was found in the adults of the 2000 ppm group. All the results recorded for the present study indicated a dose-dependent non-specific toxicity of the test material at the concentrations used. The reproductive parameters were, with the exception of a slight reduced implantation rate in F0 the females of the 5000 ppm group, not affected by the treatment.

Effects on developmental toxicity

Description of key information

- Developmental toxicity in rat, NOAEL for maternal and developmental toxicity: 30 mg/kg bw, EPA OPP 83-3, Giknis 1987


- Developmental toxicity in rabbit, NOAEL for maternal toxicity > 30 mg/kg bw, comparable to OECD TG 414, Raab 1986

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Apr 1987 to 16 May 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:COBS CD (SD)BR VAF/PLUS
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight (at mating): 210-300 g
- Fasting period before study: no
- Housing: Individually in solid bottom cages
- Diet: Certified Chow and water ad libitum
- Water: Ad libitum
- Acclimation period: Approximately 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 14/10

IN-LIFE DATES: 29 Apr 1987 to 16 May 1985
Route of administration:
oral: gavage
Vehicle:
other: 3 % aqueous cornstarch containing 0.50 % Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing preparations of test substance were prepared as a 0.30, 0.90 or 3.60% suspension in 3% aqueous cornstarch containing 0.50% Tween 80.

VEHICLE
- Concentration in vehicle: 0.30, 0.90 or 3.60% suspension in 3% aqueous cornstarch containing 0.50% Tween 80
- Amount of vehicle: 10 mL/kg bw/day (on the sixth day of dosing the dosing volume of the highest dose group was lowered to 8.3 mL/kg bw/day because of severe maternal toxicity)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each of the samples submitted was assayed for the test substance with HPLC method ASM-87 with modifications.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0
Duration of treatment / exposure:
From day 6 to 15 of gestation
Frequency of treatment:
Daily
Duration of test:
Until day 20 of gestation
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 1: low dose
Dose / conc.:
90 mg/kg bw/day (actual dose received)
Remarks:
Group 2: mid dose
Dose / conc.:
360 mg/kg bw/day (nominal)
Remarks:
Group 3. High dose. At the sixth day of dosing the dose level was lowered to 300 mg/kg bw/day.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: The 120 females were randomly distributed into 4 dose groups of 30 animals each and a maximum of 24 sperm positive animals were placed on study within each dose group.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: body weights recorded on gestational days 0, 6, 8, 12, 16 and 20

FOOD CONSUMPTION:
- Food consumption for each animal determined: food consumptions were recorded for a weekly period from day 0 to day 6, and daily thereafter throughout gestation.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead and viable fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes
Statistics:
Body weight, body weight gain and food consumption by One-Way Analysis of Variance (ANOVA) with Bartlett's Test for Homogeneity of Variance and Dunnett's Method of Multiple Comparisons between control and treatment groups. The outcome of the ANOVA are not discussed. The calculations derived from the ANOVA are employed in the subsequent Bartlett's and Dunnett's Tests. The focus of these analyses is on the results of the Dunnett's comparisons between the control and each of the treated groups. When Bartlett's Test results are highly significant (i.e., p<.001), the set of comparisons may be recalculated with the exclusion of outlier(s) or another statistical test may be used.
Foetal weight analysed by the Healy Analysis.
Number of corpora lutea, implantations, resorption sites, viable foetuses; % post-implantation loss, and foetal sex ratios were also analysed statistically.
Indices:
% post-implantation loss
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Severe signs of compound-related maternal toxicity were observed in dams of the high dose group during the first week of dosing, including a statistically significant increase in the incidence of lethargy, ataxia, and salivation when compared to the control group and biologically significant signs of rales, prostration, hypothermia and bradypnea. Due to the severity of these toxic signs, the high dose level was lowered from 360 mg/kg/day to 300 mg/kg/day on the sixth day of dosing. The incidence and severity of these toxic signs decreased immediately following the lowering of the dose level. There were no treatment-related clinical observations at 30 mg/kg/day and only a single observation of rales at 90 mg/kg/day.
Other incidental clinical signs that were observed in the study were scabs, blood around the mouth, and chromodacryorrhea. Alopecia was also observed in all dose groups
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One dam from the control group was found dead on day 20 of gestation in this study. The cause of death was thought to be complications resulting during early labor and delivery. Since this was a control animal, this finding was not compound-related. All other females survived to scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no significant differences in body weight or corrected gestational day 20 body weight between the controls and any treatment group during the course of the study. Statistically significant decreases in maternal weight gain were detected at 90 and 360/300 mg/kg/day for the interval of gestational days 6-8. This interval coincides with the initiation of dosing and also with the observation of severe maternal toxicity in the high dose group. The decrease in weight gain in the high dose group is further substantiated by a statistically significant decrease in weight gain in dams of this group for the interval of gestational days 6-16. There were no statistically significant differences in the corrected body weight change between the control animals and any treatment group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant reduction in food consumption, at 300/360 mg/kg/day from gestational days 7-10, and at 90 mg/kg/day during the intervals of gestational days 8-9 and days 10-11. These intervals correspond to the first few days of dosing, and can be correlated with the severe maternal toxicity observed in the high-dose group and the less pronounced maternal toxicity observed in the intermediate dose group during this same period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Remarks on result:
other: Marked maternal toxicity at 360/300 mg/kg bw/day
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two out of a total of 1141 viable foetuses were grossly malformed. One female at 90 mg/kg/day had a cleft lip and palate, micromelia and a club foot and a female at 360/300 mg/kg/day had anasarca.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant increase in the incidence of minor skeletal variations at 90 and 360/300 mg/kg/day including increases in the incidence of rudimentary ribs and sternabrae not ossified. In the absence of other foetal effects, these skeletal variations are attributed to the maternal effects observed rather than a specific foetal effect.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
One foetus at 90 mg/kg/day a cleft/hare lip. (Total incidence of cleft/hare lip at 90 mg/kg/day = 2 including the one seen on external observation). At 360/300 mg/kg/day, one foetus had cleft palate and one cleft palate, hydromelia and protruding tongue.
There was a statistically significant increase in the incidence of visceral variations at 360/300 mg/kg/day for renal papilla(e) short, renal papilla(e) absent, and dilated ureters. These variations are thought to represent a slight delay in the normal development of the urinary system and were secondary to the maternal toxicity observed in the high-dose groups and were not representative of any direct foetotoxicity.

Although the incidence of cleft palate is not statistically significant in this study, the occurrence of this malformation in 3 foetuses from 3 litters (one intermediate and two high-dose) is remarkable because cleft palate is an uncommon malformation in CD rats. While cleft palate has not been seen in control CD rats at this facility, data from other laboratories show that cleft palate does occur in control animals at incidence of 0-0.35%. In this study, the incidence of cleft palate in the intermediate dose group was 0.33%, while in the high-dose group the incidence was approximately 0.70%. The cleft palate and foetal variations were thought to be secondary to the maternal toxicity.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations
Remarks on result:
other: increased incidence of cleft palate at 360/300 mg/kg bw/day, increased incidences of visceral variation at 360/300 mg/kg bw/day and skeletal variations at 90 and 360/300 mg/kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: face
external: paw
visceral/soft tissue: urinary
Description (incidence and severity):
One female at 90 mg/kg/day had a cleft lip and palate, micromelia and a club foot and a female at 360/300 mg/kg/day had anasarca. At 360/300 mg/kg/day, one foetus had cleft palate and one cleft palate, hydromelia and protruding tongue. There was a statistically significant increase in the incidence of visceral variations at 360/300 mg/kg/day for renal papilla(e) short, renal papilla(e) absent, and dilated ureters.
These effects are a secondary non-specific consequence of maternal toxicity effects.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Table 1. Intergroup comparison of body weight gain (g) – selected times

 

Dose level (mg/kg/day)

Days of gstation

0 (control)

30

90

360/300

0-6

29.05

32.00

32.41

28.09

6-8

7.14

3.76

3.14*

2.68*

12-16

23.09

22.19

23.91

23.27

6-16

46.82

44.38

45.62

40.18*

16-20

49.27

51.33

59.68

59.91

0-20

125.14

127.71

133.05

128.18

0-20#

57.23

52.24

56.11

50.95

* Statistically significant difference from control group mean, p<0.05

# body weight gain excluding gravid uterus weight

Table 2. Intergroup comparison of food consumption (g) – selected times

 

Dose level (mg/kg/day)

Days of gestation

0 (control)

30

90

360/300

6-7

21.00

21.90

18.73

18.82

7-8

21.00

21.43

20.32

16.82

8-9

22.18

22.24

19.68*

18.58*

9-10

21.77

23.19

20.82

16.68*

10-11

22.77

22.38

19.82*

20.36

19-20

25.00

25.62

26.45

26.27

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 3. Caesarean section observations for all pregnant females

Observation

Dose level (mg/kg/day)

0 (control)

30

90

360/300

# Animals Assigned (Mated)

24

24

24

24

# Animals Pregnant

23

21

22

22

   #Intercurrent deaths

1

0

0

0

% Pregnant

95.8

87.5

91.7

95.7

Corpora Lutea/Dam

16.9

16.7

17.3

16.5

Implantations/Dam

13.5

14.2

14.3

14.0

Total # viable foetuses

270

284

302

285

Total # Litters (viable)

22

21

22

22

Live Foetuses/Dam

12.3

13.5

13.7

13.0

Mean number early resorptions

1.1

0.7

0.5

1.0

Mean number late resorptions

0

0

0

0.1

Mean number resorptions (total)

1.1

0.7

0.6

1.1

Mean number pre-natal death

0.08

0.04

0.04

0.08

% dead foetuses

0.3

0

0

0

Mean Foetal Weight (g)

3.5

3.5

3.5

3.5

               Males (g)

3.6

3.7

3.6

3.6

               Females (g)

3.3

3.4

3.4

3.4

Sex Ratio (% Males per litter)

51.9

49.3

48.3

46.0

Table 6. Visceral variations 

Visceral variations

Dose level (mg/kg/day)

0 (control)

30

90

360/300

No. foetuses with renal papilla(e) short

32/141

27/148

40/156

57/148**

No. foetuses with renal papilla(e) absent

4/141

4/148

8/156

16/148**

No. foetuses with dilated ureter(s) short

38/141

21/148

38/156

63/148**

**Statistically different from control at P<0.01

Table 7. Skeletal variations 

Skeletal variations

Dose level (mg/kg/day)

0 (control)

30

90

360/300

No. foetuses with rudimentary ribs

0/129

1/136

4/146

53/137

No. litters with foetuses with rudimentary ribs

0/22

1/21

4/22*

16/22**

No. foetuses with sternebrae not ossified

49/129

54/136

83/146*

99/137**

**Statistically different from control at P<0.05

*Statistically different from control at P<0.01

Conclusions:
In a developmental study performed in compliance with GLP and following an EPA OPP 83-3 guideline, the no observed adverse effect level (NOAEL) for maternal toxicity and developmental toxicity is 30 mg/kg/day. The NOAEL for teratogenicity is 30 mg/kg/day.
Executive summary:

In a developmental study performed in compliance with GLP and following an EPA OPP 83-3 guideline, the test substance was evaluated for its embryotoxic, foetotoxic and/or teratogenic potential in rats. the test substance technical was administered to three groups of 24 pregnant Crl:COBS CD (SD)BR VAF/PLUS rats at doses of 30, 90 or 360/300 mg/kg once daily by gavage during gestational days 6-15. A fourth (control) group received an equivalent volume (10 mL/kg/day) of vehicle only (3% cornstarch in 0.5% Tween 80). On gestational day 20, all dams were sacrificed and a laparohysterectomy was performed followed by an examination of the reproductive tract and its contents.

Severe signs of maternal toxicity were observed at the 360 mg/kg/day dose level. Consequently, the dosage was lowered to 300 mg/kg/day and the severity of the side-effects decreased. However, other signs of treatment-related effects were observed in dams of both the intermediate and high-dose groups and included decreased food consumption and decreased body weight gain.

Administration of the test substance did not have a statistically significant effect on any of the reproductive parameters examined, the incidence of foetal gross, visceral or skeletal malformations, foetal body weights or foetal sex ratios. Cleft palate was observed in three foetuses from three dams, one in an intermediate and two in high-dose group litters. However, historical data from both in-house and other laboratories show that cleft palate does occur sporadically in both untreated rats and in rats treated with non-teratogenic compounds and may be associated with maternal toxicity. The cleft palate was thought to be secondary to the maternal toxicity. Signs of foetotoxicity were observed in both the intermediate and high-dose groups as an increased incidence of minor skeletal and visceral variations. These foetotoxic effects are thought to be secondary to the maternal toxicity rather than due to any direct foetal effects. There was no evidence of any treatment-related embryotoxicity observed in this study. The no-effect level was 30 mg/kg/day in this study for both the foetus and the dam. Evidence of maternal and foetotoxicity was noted at 90 mg/kg/day (lowest effect level). The highest dose, 360 mg/kg/day, was not well tolerated by the dams; lowering the dose to 300 mg/kg/day produced what is considered as the maximum tolerated dose in the study.

The no observed effect level (NOAEL) for maternal toxicity and developmental toxicity is 30 mg/kg/day. The NOAEL for teratogenicity is 30 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Jun 1985 to 12 Jul 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Remarks:
(SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3.14 - 4.15 kg
- Housing: Mesh bottom stainless steel cages that were changed approximately bi-weekly. Paper liners below the cages were changed daily.
- Diet: rabbit chow ad libitum
- Water: ad libitum
- Acclimation period: approximately 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3
- Humidity (%): 50 ± 20
- Photoperiod: 14-hour periods of light and 10-hour periods of darkness

IN-LIFE DATES: From: 17 June 1985 To: 12 July 1985
Route of administration:
oral: gavage
Vehicle:
other: 3% cornstarch containing 0.5% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose solutions were prepared with 2.00%, 5.00% or 8.00% suspension in 3% cornstarch containing 0.5% Tween 80. The volume of suspension of compound or vehicle to be administered to each animal was determined by the animal's body weight recorded on gestational days 7, 10, and 14.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All suspensions were assessed for stability, purity, characterization, and retention of the test substance.
Details on mating procedure:
- Impregnation procedure: artificial insemination

Duration of treatment / exposure:
From Day 7 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
Untill Day 29 of gestation
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2 low dose.
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 3 mid dose.
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
Group 4 high dose.
No. of animals per sex per dose:
19 females per dosing group
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: The does were observed daily for changes in general appearance and behavior. Mortality was checked twice daily.

BODY WEIGHT:
- Time schedule for examinations: Days 0, 7, 10, 14, 20, 24 and 29

FOOD CONSUMPTION:
- Time schedule for examinations: daily for gestational Days 5 to 29

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation Day 29
- At the time of necropsy, all does were examined for gross pathologic changes. Representative maternal gross lesions were excised, stored in formalin, and at the discretion of the Study Director, no tissues were submitted to the Pathology Section for microscopic evaluation.
- Organs examined: thoracic and abdominal cavities(to include a cut and examination of the kidneys), ovaries and uterus

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: amniotic fluid
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
Body weights, body weight gain and food consumption were analysed by one-way ANOVA with Barlett's test for homogeneity and Dunett's test. Reproductive parameters were analysed by a onesided trend test and the chi-square test. The fetal sex ratio was analysed with Mantel's test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant increase in the incidence of stool alterations (decreased/no/soft) and abortions/early deliveries among animals of the high-dose group as compared to controls. This effect was thought to be compound-related. Other non-treatmentrelated clinical observations were: alopecia, blood (in pan/vaginal/forepaws), urine abnormalities (haematuria/anuria), and sores.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two does from the low- and intermediate-dose groups respectively, were found dead while on study. The cause of death in the low dose group animal is unknown, however for the animal from the intermediate-dose group, it was thought to be the result of a dosing accident. Additionally, seven does were sacrificed prior to schedule because they had delivered early or aborted - one from the control group (delivered early), one from the intermediate-dose group (aborted) and five from the high-dose group (4 aborted, and 1 delivered early).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in mean maternal body weight on gestational day 20 was observed for does in the high-dose group, and statistically significant decreases in maternal body weight gain (or actual body weight losses) were detected for gestational days 7-10 and 20-24 in both intermediate- and high-dose group does and for gestational days 10-14 and 14-20 in the high-dose group only. These findings are indicative of compound-related maternal toxicity in the intermediate- and high-dose groups and may be correlated to the decreased feed consumption noted for these two groups above. The statistically significant increases in body weight gain noted on gestational days 20-24 in the intermediate- and high-dose groups are considered to be incidental and unrelated
to the observed maternal toxicity. In the low-dose group the body weight gain on day 20-24 was significantly higher than control. There were no other statistically significant compound-related effects on body weight or weight gain observed in does of the low-dose group. No statistical differences in corrected maternal body weight on day 29 of gestation were detected between control and treated does.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Maternal toxicity was observed in both the intermediate- and high-dose groups as statistically significant decreases in feed consumption as compared to the controls during the dosing period. In the intermediate-dose group, these decreases were observed for the intervals of gestational days 7-8. In the high-dose group, decreases were also noted for the entire dosing period (intervals 7-8 to 19-20). Since these decreases in feed consumption occurred during the dosing period, the maternal toxicity is considered to be compound related. A rebound effect was observed in does of both the intermediate- and high-dose groups as a statistically significant increase in feed consumption during the following intervals: day 24-25 for the intermediate-dose group and; day 27-28 for the high-dose group. Low dose feed consumption values did not differ significantly from Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no statistically significant, compound-related, necropsy observations in this study. Incidental observations made at necropsy included: red, spongy lungs; fluid-filled intestines, stomach ulcerations; pitted kidneys; umbilical hernia, small spleen; and discolored amniotic fluid. Since there were no compound related pathological findings, no maternal tissues were submitted for microscopic examination.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Seven does were sacrificed prior to schedule because they had delivered early or aborted - one from the control group, one from the intermediate-dose group and five from the high-dose group.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increased incidence of resorptions was observed among does of the high-dose group. This observation is thought to be unduly influenced by the total resorption of the litter of one doe in the high-dose group. Statistical significance was lacking when data from this animal was excluded from the analysis.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
17 early resorptions in the high-dose group.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Decreased body weight and food consumption at 250 and 400 mg/kg bw/day
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 470 live fetuses representing 58 litters were examined externally for the presence of external malformations and/or variations. One fetus was observed to have a bilateral cleft lip and an umbilical hernia. This fetus was from the intermediate-dose group and was the only fetus observed to have an external malformation.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no skeletal malformations observed in this study. Various skeletal variations, however, were noted across all study groups. With the exception of a statistically significant increased incidence of fully formed thirteenth ribs among the fetuses of the high-dose group, no other variations were thought to be related to maternal treatment with the test substance. The high frequency of the variation fully formed thirteenth rib, is thought to be a consequence of maternal toxicity in the high-dose group and not a direct fetotoxic response.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of five fetuses, representing four litters, were found on visceral examination to have visceral variations. One control group fetus was observed to have a red area on its left lung. One intermediate-dose group fetus had a thickened, opaque aorta and a small gallbladder, and another fetus from the intermediate-dose group was found to have ovarian cysts. In the high-dose group two fetuses from the same litter had coagulated blood above their bladders. One fetus from the intermediate-dose group was described at visceral examination as a possible lateral hermaphrodite. However, following microscopic examination, the fetus was confirmed to be a male.
The only visceral malformation observed in this study was observed in the intermediate-dose group fetus that was grossly malformed and consisted of hydronephrosis with hydroureter. Since the majority of the visceral observations are limited to a single intermediate-dose group fetus without any dose-response relationship, these observations are considered to have been spontaneous in nature.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
Remarks on result:
other: Increased incidence of resorptions, abortions and early deliveries and increased incidence of fully formed 13th rib at 400 mg/kg bw/day.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: supernumerary rib
Description (incidence and severity):
The high frequency of the variation fully formed thirteenth rib , is thought to be a consequence of maternal toxicity in the high-dose group and not a direct fetotoxic response.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Table 1. Summary of reproductive and fetal findings

 

Dose level (mg/kg bw/day)

Reproductive parameters

0

100

250

400

Number of pregnant does/ no. inseminated

15/19

18/19

17/19

18/19

Found dead/sacrificed

0

1

1

0

Aborted/Delivered early

1a

0

1b

5*c

Viable litters examined

14

17

15

12d

Mean no. Corpora Lutea

11.6

12.6

13.3

13.5

Mean no. implantations

8.4

9.4

10.0

9.2

Early resorptions: per group (mean per dam)

2 (0.1)

6 (0.4)

6 (0.4)

17 e(1.3)

Late resorptions per group (mean per dam)

8 (0.6)

6 (0.4)

5 (0.3)

10 (0.8)

Total resorptions per group (mean per dam)

10 (0.7)

12 (0.7)

11 (0.7)

27* (2.1*)

Viable fetuses per group (mean per dam)

101 (7.2)

146 (8.6)

130 (8.7)

93 (7.2)

Dead fetuses per group (mean per dam)

6 (0.4)

1 (0.1)

9 (0.6)

0 (0)

Fetal sex ratio (% males)

55

49

50

41

Fetal weight (g), males/females

43.0/44.2

44.4/43.1

42.8/41.1

42.8/43.2

Gross malformations

Number of fetuses examined

101

146

130

93

Cleft lip

0

0

1g

0

Umbilical hernia

0

0

1g

0

Visceral malformations

Hydronephrosis with hydroureter

0

0

1g

0

Selected skeletal variations

Fully formed (13th) ribs

35

63

58

63*

Rudimentary ribs

32

20

22

11

Floating rudimentary ribs

0

5

4

2

Wavy ribs

0

0

0

1

*Statistically different from control at P<0.05, a delivery on day 29, b abortion on day 21

c) 3 dams aborted on day 26, one aborted on day 22 and one delivered on day 29

d) one doe was pregnant but had no vible fetuses at terminal sacrifice, e one doe resorbed the whole litter, 10 pups.

g) same fetus cleft lip, umbilical hernia, hydoronephrosis with hydroureter, thick aorta and small gallbladder

Conclusions:
In this study performed in compliance with GLP and following a method comparable to OECD TG 414 guideline, signs of fetotoxicity secondary to maternal toxicity were observed in the high-dose group only, as an increase in the incidence of the skeletal variation, fully formed 13th rib. There was no evidence of teratogenicity in this study. The no-observed-adverse-effect level was estimated to be >250 mg/kg/day for the fetus and >100 mg/kg/day for the doe.
Executive summary:

In this study performed in compliance with GLP and following a method comparable to OECD TG 414 guideline, three dose levels of the test substance (100, 250, or 400 mg/kg/day) were studied for their embryo/fetal toxicity and teratogenic potential in New Zealand White rabbits. The test article was administered as a suspension in 3% cornstarch containing 0.5% Tween 80 (vehicle) once daily from day 7 through day 19 of gestation at a volume of 5.0 mL/kg/day. Control animals received an equivalent volume of vehicle. Signs of maternal toxicity were observed in both the intermediate- and high-dose groups and were thought to be compound-related. These signs included: decreased feed consumption and decreased body weight gain in the high-dose group alone. Increased incidence of stool variations and abortions/early deliveries were also observed in the high-dose group. Administration of the test substance did not have a statistically significant effect on fetal body weights, sex ratios, the incidence of visceral or skeletal malformations, visceral variations or any of the reproductive parameters examined with the exception of a statistically significant increased incidence of resorption in the high-dose group. However , this parameter was believed to have been unduly influenced by the presence of a single litter with total resorption. Signs of fetotoxicity secondary to maternal toxicity were observed in the high-dose group only, as an increase in the incidence of the skeletal variation, fully formed 13th rib. There was no evidence of teratogenicity in this study. The no-observed-adverse-effect level was estimated to be >250 mg/kg/day for the fetus and >100 mg/kg/day for the doe.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant EPA OPP 83-3 study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

All available data was assessed and the study representing the worst-case effects was included as key study. The test substance did not induce developmental toxicity in rats and rabbits.


In the key study (Marcsisin 1987), performed in compliance with GLP and following an EPA OPP 83-3 guideline, the test substance was evaluated for its embryotoxic, foetotoxic and/or teratogenic potential in rats. the test substance technical was administered to three groups of 24 pregnant Crl:COBS CD (SD)BR VAF/PLUS rats at doses of 30, 90 or 360/300 mg/kg once daily by gavage during gestational days 6-15. A fourth (control) group received an equivalent volume (10 mL/kg/day) of vehicle only (3% cornstarch in 0.5% Tween 80). On gestational day 20, all dams were sacrificed and a laparohysterectomy was performed followed by an examination of the reproductive tract and its contents.


Severe signs of maternal toxicity were observed at the 360 mg/kg/day dose level. Consequently, the dosage was lowered to 300 mg/kg/day and the severity of the side-effects decreased. However, other signs of treatment-related effects were observed in dams of both the intermediate and high-dose groups and included decreased food consumption and decreased body weight gain.


Administration of the test substance did not have a statistically significant effect on any of the reproductive parameters examined, the incidence of foetal gross, visceral or skeletal malformations, foetal body weights or foetal sex ratios. Cleft palate was observed in three foetuses from three dams, one in an intermediate and two in high-dose group litters. However, historical data from both in-house and other laboratories show that cleft palate does occur sporadically in both untreated rats and in rats treated with non-teratogenic compounds and may be associated with maternal toxicity. The cleft palate was thought to be secondary to the maternal toxicity. Signs of foetotoxicity were observed in both the intermediate and high-dose groups as an increased incidence of minor skeletal and visceral variations. These foetotoxic effects are thought to be secondary to the maternal toxicity rather than due to any direct foetal effects. There was no evidence of any treatment-related embryotoxicity observed in this study. The no-effect level was 30 mg/kg/day in this study for both the foetus and the dam. Evidence of maternal and foetotoxicity was noted at 90 mg/kg/day (lowest effect level). The highest dose, 360 mg/kg/day, was not well tolerated by the dams; lowering the dose to 300 mg/kg/day produced what is considered as the maximum tolerated dose in the study.


The no observed adverse effect level (NOAEL) for maternal toxicity and developmental toxicity is 30 mg/kg/day based on cleft palate at 90 mg/kg bw/day and two cleft palates at 60/300 mg/kg bw/day. The NOAEL for teratogenicity is 300 mg/kg/day.


In the second key study (Raab 1986),  performed in compliance with GLP and following a method comparable to OECD TG 414 guideline, three dose levels of the test substance (100, 250, or 400 mg/kg/day) were studied for their embryo/fetal toxicity and teratogenic potential in New Zealand White rabbits. The test article was administered as a suspension in 3% cornstarch containing 0.5% Tween 80 (vehicle) once daily from day 7 through day 19 of gestation at a volume of 5.0 mL/kg/day. Control animals received an equivalent volume of vehicle.


Signs of maternal toxicity were observed in both the intermediate- and high-dose groups and were thought to be compound-related. These signs included: decreased feed consumption and decreased body weight gain in the high-dose group alone. Increased incidence of stool variations and abortions/early deliveries were also observed in the high-dose group. Administration of the test substance did not have a statistically significant effect on fetal body weights, sex ratios, the incidence of visceral or skeletal malformations, visceral variations or any of the reproductive parameters examined with the exception of a statistically significant increased incidence of resorption in the high-dose group. However, this parameter was believed to have been unduly influenced by the presence of a single litter with total resorption. Signs of fetotoxicity secondary to maternal toxicity were observed in the high-dose group only, as an increase in the incidence of the skeletal variation, fully formed 13th rib. There was no evidence of teratogenicity in this study. The no-observed-adverse-effect level was estimated to be 100 mg/kg/day for maternal toxicity and 250 mg/kg bw/day for developmental toxicity.


There are four studies included as supporting information for this endpoint. In the first study (Mallows 1987), following FIFRA 83-3 guideline and GLP compliant, the test substance was administered to a single group of rats at a dose of 300 mg/kg bw/day once daily by gavage during gestational days 6-15. At the 300 mg/kg bw/day dose level severe signs of maternal toxicity were observed. Administration of test substance produced a reduction in the mean number of viable foetuses and the mean fetal body weight, suggesting a fetotoxic response but did not have a statistically significant effect on any other reproductive parameter examined, the incidence of fetal external gross malformations, or the fetal sex ratio. Cleft palate was observed in 2 of 2064 foetuses from 2 of 158 litters in the compound-treated group. No NOAEL was estimated. In the second study (Giknis 1986), following EPA 83-3 guideline and GLP compliant, the test substance was administered to rabbits at doses of 100, 250 or 400 mg/kg bw/day once daily by gavage during gestational days 7-19 inclusive. There was no evidence of teratogenicity at the highest dose level tested of 400 mg/kg bw/day. The NOEL for maternal toxicity was >100 mg/kg/day and the NOEL for developmental toxicity was >250 mg/kg/day. In the third study (Fritz 1979a), which followed no particular guideline and was not GLP compliant, the test substance was administered orally by intubation to fertilised albino rats from Day 6 until Day 15 of pregnancy, inclusive. Animals were doses with either 0, 30, 100 or 300 mg/kg bw/day test substance. The test substance was devoid of an embryotoxic activity and did not reveal teratogenic potency in the rat. The slight delay of foetal growth noted in rats treated with 300 mg/kg bw/day was considered to be non-specific and related to maternal toxicity. In the final supporting study (Fritz 1979b), which followed no particular guideline and was not GLP compliant, the test substance was administered orally by intubation to fertilised Chinchilla rabbits from Day 6 until Day 18 of pregnancy inclusive. The dosage was selected on the basis of toxicity studies at 30, 90, and 180 mg/kg bw/day. The test substance was devoid of an embryotoxic activity and did not reveal teratogenic potency in the rabbit under the present experimental conditions.

Justification for classification or non-classification

Based on the RAC oppinion the substance is classified as Category 1B, H360D; May damage the unborn child, reproductive toxicant in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.


Comments to health hazards:


Certain Regulatory Agencies (ECHA, EU) have classified the test substance as Category 1B for developmental effects based on animal studies (ECHA RAC opinion: CLH-O-0000001412-86-139/F). This decision was based on a very low incidence of cleft palate (a malformation) in rats, the registrant considers this to be within the range of historic control incidence, non-reproducible, and occur in the presence of maternal toxicity. EFSA (European Food Safety Authority), 2017. Conclusion on the peer review of the pesticide risk assessment of the active substance propiconazole. EFSA Journal 2017;15(7):4887, 89 pp. doi:10.2903/j.efsa.2017.4887.


The position of the registrant on the global classification of this substance is that the substance should be classified as Category 2, H361D; Suspected of damaging the unborn child, based on some evidence of adverse effects on development.

Additional information