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Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No information available.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD and Japanese testing guidelines
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).
Deviations:
no
GLP compliance:
not specified
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals: Seventy males and 70 females of Crj:CD(SD) strain rats (SPF) were purchased from Japan Charles River Co., Ltd., (Atsugi-shi, Kanagawa) at 8-weeks of age on September 20, 1993, quarantined and acclimatized to the test environment for 7 days. 48 males and 48 females which showed no abnormalities in general condition during the acclimatization period were allocated to each group after 8 days of preliminary housing period at 10-weeks of age.
At the end of grouping, the males weighed 339-400 g and the females weighed 226-265 g.

Animal husbandry: The animals were housed in an animal room (W 5.7 x D 10.0 x H 2.5 m, 142.5m3) kept by barrier system under the target environmental conditions at 22-26°C of temperature, 45-65 % of relative humidity with 15 times per hour of ventilation frequency, and 12 hour lighting of 150-300 lux (lighting at 7: 00 am and turn off at 7:00 pm).
Animals were housed individually in a cage with aluminum front and stainless steel mesh-floor (W 15.8 x D 23.8 x H 16.0 cm, Space: 6017 cm3). Cages were set on a water-flush breeding instrument (W 691.0 x D 79.0 x H 195.0 cm) supplied by Tokyo Giken Service Co. Ltd. (Fuchu-shi, Tokyo). However, during the mating period, males were housed in cages with aluminum front and stainless steel mesh-floor (W 36.8 x D 25.0 x H 16.0 cm, Space: 14720 cm3). Dam after 18 days of gestation was housed in a cage with aluminum front and stainless steel mesh-floor (W 36.8 x D 25.0 x H 16.0 cm) with a lactation tray and nesting materials (Alpha-dry) until day 4 of lactation. The cages were exchanged once every other week, and food suppliers were exchanged once a week.
The food used was NMF solid food (treated with radiation sterilization) supplied by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo). The food was available ad libitum. The analysis of contaminant in the food used in this study was conducted in Japan Food Research Laboratories (Shibuya-ku, Tokyo) on the request by Oriental Yeast Co., Ltd.
The animals were allowed free access to drinking water of tap water. The analyses of tap water were performed 4 times in a year in Examination Center of Life Science of Shizuoka (Hamamatsu-shi, Shizuoka). The food, water and nesting material supplied did not contain any contaminants which were considered to have affected the results of the study.
There were no environmental deviations which might have affected the reliability of the study data during the duration of housing period.

Test groups: Animals were stratified by the body weights and allocated to each test group by randomization method on October 5, 1993.
Animals were identified by ear-punch and attaching animal identification card with animal identification number (animal ID-No.) to cages. Before grouping, animals were identified by tentative animal numbers.
The remained animals were euthanized by carbon dioxide.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Exact amount of test substance was dissolved into corn oil (Nacalai Tesque, Kyoto-Shi, Kyoto) to make the specified concentrations of 1.2, 6 and 30 mg/ml. After the preparation, the preparations were stored in a refrigerator until use. As shown in Reference data 5, the test substance preparation was conducted at the frequency of once a week or more, and the preparations were used within 7 days after preparation. In the case of 1.2 mg/ml, the preparation was confirmed to be stable at least for 8 days after preparation.
Details on mating procedure:
After 14 days of observation period for estrus cycles before mating, female animals were placed in cages together with male animal of the same group in one-to-one base for 2 weeks as the longest period. In the next morning, the presence of sperms in the vaginal smears was determined as the established copulation and as 0 day of gestation. The observation of estrus cycles was conducted until the day of copulation. The number of days between each estrus stage were estimated to be the number of days of estrus cycle, and mean estrus cycle durations were calculated.
From the results of mating, the copulation index [(number of animals of copulation/number of animals placed together) x 100] was calculated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of homogeneity and concentration of the dosing preparation was conducted on the sample taken randomly from batches of each group prepared at the start of administration. The mean values of the concentration in each preparation were within 99.3-104 %, and the coefficient of variation was lower than 0.8 % indicating that the preparations were properly adjusted.
Duration of treatment / exposure:
Male animals were administered for 45 days consecutively. Female animals were administered for 41-48 days. Infertile female animal was administered for 44 days until the day before necropsy.
Frequency of treatment:
The test substance was administered by gavage once a day (7 days/week) using a stomach tube.
Details on study schedule:
Male animals were administered for 45 days consecutively from 14 days before mating, during 14 days of mating period and up to 17 days after the end of mating period. Female animals were administered from 14 days before mating, during mating period (until the day of copulation confirmation, 14 days at the longest), gestation period after copulation and up to day 3 of lactation after delivery (41-48 days). Infertile female animal was administered for 44 days until the day before necropsy (day 25 of gestation).
Remarks:
Doses / Concentrations:
0, 6, 30, 150 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals of each sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Reason for selection of the species: The use of rat is indicated by OECD Guideline. The species was selected considering the stability in estrus cycle, reproduction and genetic.
Reasons for the selection of dose levels: Dose levels were selected by considering the results of “Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Preliminary Study in Rats” (study number 2265) conducted previously. In this preliminary study, dose levels of 0, 75, 150, 300 and 600 mg/kg were administered to males and females for 14 consecutive days, and all animals treated with 600 mg/kg died. Suppression in body weight gain was seen in male groups treated with 300 mg/kg or more and in females treated with 600 mg/kg. Decrease in food intake was seen in male groups treated with 150 mg/kg or more, and females treated with 300 mg/kg or more.
Among the organ weight at necropsy, both absolute and relative weights of liver increased in male groups treated with 75 mg/kg or more, and in female groups treated with 150 mg/kg or more.
At necropsy, enlargement of liver was seen in males treated with 150 or 300 mg/kg and females treated with 300 mg/kg.
From the above results and taking into account of the longer administration period in the main study, 150 mg/kg was selected as the highest dose level. The lower dose levels of 30 and 6 mg/kg were selected by dividing the highest dose level by common geometric ratio of 5.
Positive control:
Positive control not utilised in this study.
Parental animals: Observations and examinations:
Mating: After 14 days of observation period for estrus cycles before mating, female animals were placed in cages together with male animal of the same group in one-to-one base for 2 weeks as the longest period. In the next morning, the presence of sperms in the vaginal smears was determined as the established copulation and as 0 day of gestation. The observation of estrus cycles was conducted until the day of copulation.
From the results of mating, the copulation index [(number of animals of copulation/number of animals placed together) x 100] was calculated.

Observation on the natural delivery and lactation period: The observations of delivery were done from 9:00 am to 10:00 am during days 20-25 of gestation. By confirmation of the completion of delivery during this observation time, the day was recorded as day 0 of lactation. In the case of delivery after 10:00 am, the next day was recorded as day 0 of lactation.
In addition, gestation period (number of days between day 0 of gestation and day 0 of lactation), fertility index [(number of pregnant animals/number of copulated animals) x 100], gestation index [(number of females with live birth/number of pregnant female) x 100], implantation index [(number of implantation sites/number of corpora lutea of pregnancy) x 100], delivery index [(number of birth/number of implantation sites) x 100], live birth index [(number of live birth/number of birth) x 100] were calculated.
Female which showed no delivery until 9:00 am on day 25 of gestation was necropsied, and female without implantation site was judged to be infertile.
At the natural delivery, the status of delivery was observed, and the condition of lactation was observed until day 4 of lactation. At the necropsy on day 4 of lactation, numbers of corpora lutea of pregnancy and implantation sites were counted.
Oestrous cyclicity (parental animals):
The number of days between each estrus stage were estimated to be the number of days of estrus cycle, and mean estrus cycle durations were calculated.
Sperm parameters (parental animals):
Not specified
Litter observations:
After parturition, number of birth (live birth + still birth) were examined, and sex was identified and sex ratio (males/females) was calculated. Presence of external anomaly was also examined. In addition, body weight of each litter was weighed separately by sex on day 0 and 4 of lactation and mean body weight of each sex was calculated.
Furthermore, the viability index on day 4 [(number of live neonates on day 4 of lactation/number of live birth) x 100] were calculated.
Postmortem examinations (parental animals):
Male animals
Necropsy and organ weight: Macroscopic observations of organs and tissues were performed on the animals euthanized by exsanguinations after blood sampling. Thymus, liver, kidney, testis and epididymides were weighed, and the ratio of organ weight to body weight (relative weight) was calculated. In addition, thymus, liver, kidney, brain, heart, spleen, adrenals, seminal vesicles, prostate, pituitary and the tissues with abnormal findings (lung, mass in the abdominal cavity) of all animals were fixed in 10% neutral buffered formaldehyde solution. The testis and the epididymides were fixed in Bouin solution.
Histopathology: Paraffin sections from the fixed organs and tissues indicated below were prepared by Histo Science Laboratory Co., Ltd., (Oume-shi, Tokyo) and routinely stained with hematoxylin-eosin. In addition, kidney was stained with PAS reaction. Microscopic observations were conducted in BioSafety Research Center.
Fertile male: Brain, thymus, heart, liver, kidney, spleen, adrenals, testis of all animals of the control group and 150 mg/kg group and the tissues with abnormal findings in all groups. Thymus, liver, kidney and adrenals of all animals of 6 and 30 mg/kg groups.
Infertile male: Brain, thymus, heart, liver, kidney, spleen, adrenals, testis, epididymides, seminal vesicles, prostate and pituitary.

Female animals
Necropsy and organ weight: The animals necropsied are shown below and the presence of abnormalities in the organs and tissues was observed.
Dead animal: Animals were necropsied immediately after found dead. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution.
Female with natural delivery: Animals were euthanized by exsanguination under ether anesthesia on day 4 of lactation, and main organs were observed macroscopically. And then, thymus, liver, kidney and ovaries were weighed, and the ratio of organ weight to body weight (relative weight) was calculated. In addition, thymus, liver, kidney, ovaries, brain, heart, spleen, adrenals, pituitary and the tissues with abnormal findings of all animals were fixed in 10% neutral buffered formaldehyde solution.
Female without natural delivery: On day 25 of gestation, animals were euthanized by exsanguinations under ether anesthesia, and macroscopic observations of main organs were performed. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution. Animal with no implantation sites was judged to be infertile.
Female whose pups all died: On the day or the next day when the death or cannibalism of pups were found, parental females were euthanized by exsanguinations under ether anesthesia, and macroscopic observations of main organs were performed. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution. The numbers of corpora lutea of pregnancy and implantation sites were examined at the necropsy.
Histopathology: Paraffin sections from the fixed organs and tissues indicated below were prepared by Histo Science Laboratory Co., Ltd., (Oume-shi, Tokyo) and routinely stained with hematoxylin-eosin. In addition, kidney were stained with PAS reaction. Microscopic observations were conducted in BioSafety Research Center.
Dead animal: Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord.
Female with natural delivery: Brain, thymus, heart, liver, kidney, spleen, adrenals, ovaries of all animals of the control group and 150 mg/kg group, and the tissues with abnormal findings of all animals of all groups. Thymus, liver, kidney and adrenals of all animals of 6 mg/kg group and 30 mg/kg group.
Infertile female: Brain, thymus, heart, liver, kidney, spleen, adrenals, vagina, ovaries and pituitary.
Female whose pups all died: Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord.
Postmortem examinations (offspring):
On day 4 of lactation, all neonates were sacrificed and main organs were macroscopically observed. Neonates died during lactation period were also necropsied and main organs were macroscopically observed.
Statistics:
Data of this study were recorded using a computer system, and the results of the study were statistically analyzed using the following methods.
The mean value per dam was used as one sample for the results of neonates during lactation period. Levels of significance were two steps of * : P < 0.05 and ** : P < 0.01.

Multiple comparison analysis was applied for body weight, food intake, number of corpora lutea of pregnancy, implantation site, number of birth, number of still birth, sex ratio, mean value of estrus cycle duration, gestation period, implantation index, delivery index, live birth index, incidence of external anomaly, viability index on day 4 of neonates, organ weight, ratio of organ weight to body weight (relative organ weight), hematology and blood chemistry.
First, the homogeneity was assessed by Bartlett test. If homogeneity was obtained, one way analysis of variance was applied. If variance was significant and the numbers of samples were equal among groups, Dunnett multiple comparison was applied. In the case of unequal numbers of samples, the significances of difference between the control group and each treated group were assessed by Scheffé multiple comparison.
When the data showed non-homogeneity by Bartlett test, the data were analyzed by Kruskal-Wallis rank sum test. Dunnett rank test was applied if significance was obtained and numbers of sample were equal, and in the case of unequal numbers of samples, Scheffé rank test was applied for the analysis of significant difference between the control group and each treated group.
X2 test was applied for gestation index, copulation index and fertility index.
Fisher’s direct probability method was applied for the enlargement of liver in macroscopic findings and the centrilobular hypertrophy of hepatocytes in microscopic examination.
Reproductive indices:
Multiple comparison analysis was applied for number of corpora lutea of pregnancy, implantation site, number of birth, number of still birth, sex ratio, mean value of estrus cycle duration, gestation period, implantation index, delivery index, live birth index, incidence of external anomaly.
Offspring viability indices:
Multiple comparison analysis was applied for viability index on day 4 of neonates
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Effect on parental animals
Copulation and fertility: Copulation was established in all animals in all groups.
Fertilization was successful in all of test substance administered groups, but was unsuccessful in one pair of the control group. In the estrus cycle observation, pseudopregnancy (continuous diestrus smear image) was not observed, and estrus cycles of 4-5 day were seen in all groups and no inter-group differences were seen in the mean duration of estrus cycles.

Delivery and lactation: The mean durations of gestation were within the range of 22.5-22.9 days in each group, and no prolongation of gestation was observed. One animal (animal No. 2305) in 150 mg/kg group delivered 9 neonates and died during delivery. At necropsy, uterus was examined and 6 residual fetuses were found in the uterus. In addition, the number of implantation sites in 150 mg/kg group decreased compared to the control group. The number of females whose pups all died during lactation period was 4 (animal No. 2204, 2303, 2309, 2312). Viability index on day 4 were slightly lower both in male and female of 150 mg/kg group. However, no statistically significant differences were noted. No abnormal delivery was seen in the control group, 6 mg/kg group or 30 mg/kg group. No inter-group differences were observed in the number of corpora lutea, number of implantation sites, sex ratio, gestation index, implantation index, delivery index, live birth index and viability index on day 4 of neonate.
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Detailed in results
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Detailed in results
Effect on neonates
External examination: No external abnormalities were seen in all groups.
Body weight: No inter group differences were seen both in male and female in the body weights until day 4 of lactation.
Necropsy: In the necropsy of dead neonates until day 4 of lactation, thymic remnant in the neck was sporadically seen in test substance administered group.
In the necropsy of live neonates sacrificed on 4 day of lactation, thymic remnant in the neck, white patch/zone in liver, pale and pelvic dilatation in kidney were sporadically seen in the control group, 6 mg/kg group and 30 mg/kg group.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects gross pathology; organ weights; histopathology; birth index; live birth index; litter size; litter weight; pup weight; sex ratio; survival index; viability index;
Reproductive effects observed:
not specified

This report was translated from the original Japanese report. The translated copy is appended below for reference.

Conclusions:
From the results, no effect in male fertility by the administration of 6-tert-butyl-2, 4-xylenol was seen at the dose level of 150 mg/kg/day. Therefore, no observed effect level is estimated to be 150 mg/kg/day.
Concerning to the effect in female reproduction and development and growth of neonate, numbers of dams whose pups all died increased in 150 mg/kg group. Therefore, no observed effect level is estimated to be 30 mg/kg/day.
Executive summary:

In order to evaluate the toxicological nature of existing chemical substances, the repeated dose toxicity as well as the effect on reproduction and development were studied by the administration of 6-tert-butyl-2, 4-xylenol at the dose levels of 0 (vehicle control), 6, 30 and 150 mg/kg/day to rats for the period from 14 days before mating, during mating period, gestation period and to day 3 of lactation.

 

The general toxicological effect as well as the effect on reproduction and development were studied following the OECD Guidelines for “Testing of Chemicals; Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test, Extended Steering Group Document No. 3” (March 22, 1990).

The conduct of the study was designed to fulfill the standard described in “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).

 

No effect due to the test substance administration was observed in the copulation capability, fertility or estrus cycle observation. In the observation at parturition, one female of 150 mg/kg group died during delivery. In addition, there were 3 females whose pups all died in this group. The viability index on day 4 was inclined to show low value, and it is suggested that the test substance has the possibility to induce disorder in parturition or lactation function. However, no effect due to the test substance administration was seen during gestation period or delivery duration. In the external examination of neonates, no external anomalies were observed, and the body weight of neonates increased normally until day 4 of lactation. Abnormal findings considered to be due to the test substance administration were not seen in the rate of stillbirth and death rate of pups, or on the necropsy on day 4 of lactation.

From the above results, no effect on male fertility were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 150 mg/kg/day. The effect on fertility of female and development and growth of neonates were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 30 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In order to evaluate the toxicological nature of existing chemical substances, the repeated dose toxicity as well as the effect on reproduction and development were studied by the administration of 6-tert-butyl-2, 4-xylenol at the dose levels of 0 (vehicle control), 6, 30 and 150 mg/kg/day to rats for the period from 14 days before mating, during mating period, gestation period and to day 3 of lactation.

 

The general toxicological effect as well as the effect on reproduction and development were studied following the OECD Guidelines for “Testing of Chemicals; Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test, Extended Steering Group Document No. 3” (March 22, 1990).

The conduct of the study was designed to fulfill the standard described in “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).

 

No effect due to the test substance administration was observed in the copulation capability, fertility or estrus cycle observation. In the observation at parturition, one female of 150 mg/kg group died during delivery. In addition, there were 3 females whose pups all died in this group. The viability index on day 4 was inclined to show low value, and it is suggested that the test substance has the possibility to induce disorder in parturition or lactation function. However, no effect due to the test substance administration was seen during gestation period or delivery duration. In the external examination of neonates, no external anomalies were observed, and the body weight of neonates increased normally until day 4 of lactation. Abnormal findings considered to be due to the test substance administration were not seen in the rate of stillbirth and death rate of pups, or on the necropsy on day 4 of lactation.

From the above results, no effect on male fertility were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 150 mg/kg/day. The effect on fertility of female and development and growth of neonates were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 30 mg/kg/day.

Short description of key information:

NOAEL Maternal toxicity 30 mg/kg bw/day (actual dose received)

Justification for selection of Effect on fertility via oral route:

Main study on the substance itself.

Effects on developmental toxicity

Description of key information

NOAEL Devleopmental toxicity 40 mg/kg bw/day (actual dose received)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
April to October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP. Read across to structural analogue; justification for read across is attached below.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), 2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany, Niederlassung Kisslegg, Germany
- Age at study initiation: 25 - 36 weeks
- Weight at study initiation: 2265 to 3375 g
- Housing: individually in stainless steel cages equipped with an automatic cleaning system; a piece of wood and a haystick were also provided for environmental enrichment.
- Diet (e.g. ad libitum): pelleted standard Kliba Nafag 3418 (batch no. 70/07) rabbit maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days, under test conditions and after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 - 70%
- Temperature and humidity values outside of the ranges given above occasionally occurred, usually following room cleaning; they were considered not to have any influence on the study.
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs
Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC and 0.1% Tween 80 in highly purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The dose formulations were prepared weekly. The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle (0.5% CMC and highly purified water) were added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle (0.5% CMC and highly purified water) was added. Separate formulations were prepared for each concentration. The correct amount of 0.1% Tween 80 was added each morning to the daily portions of the dose formulations. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. Dose formulations in glass beakers were stored in the refrigerator (2 - 8 °C) where they were stable for 8 days.

VEHICLE
- Identification: 0.5% CMC, 0.1% Tween 80, highly purified water
- Source: Fluka Chemie GmbH, 9471 Buchs / Switzerland
- Batch Number: 1367388 (CMC), 34707017 (Tween 80)
- Storage Conditions: Room temperature (20 ± 5 °C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical monitoring:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken again as above. The aliquots for analysis of dose formulations were frozen and stored at -20 ± 5 °C until analysis.
The samples were analyzed by gas chromatography coupled to a flame ionization detector following an analytical procedure provided by the Sponsor and adapted at the testing facilities. The test item was used as the analytical standard.

Results of the analytical monitoring:
The identity of the test item was confirmed by its retention time. The test item content of 13 out of 18 samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test item in 0.5% CMC/0.1% Tween 80 was demonstrated for 4 out of 6 groups. It is indicated that homogeneity was easier to achieve for the lower content levels of dose groups 2 and 3 and that correct sampling was difficult due to formulation properties. This had an impact on results of storage stability as well. 2 out of 6 results met the limit of ±10% of nominal only. Deviating results were predominantly above nominal content which is not a sign of test item degradation. Therefore, the application formulations were considered to be stable for at least 8 days when kept at 2-8 °C. In conclusion, the results obtained within this part confirm the correct preparation of application formulations during the conduct of this study.
Details on mating procedure:
After acclimatization, females were placed in cages with sexually mature males (1:1) until copulation had been observed. After mating, the females were removed and placed in individual cages. The day of mating was designated day 0 post coitum.
The male rabbits were from the same source and strain as the females and were used for the mating only; their fertility had been proven and was continuously monitored.
Duration of treatment / exposure:
22 days (from day 6 to 27 post coitum)
Frequency of treatment:
once daily
Duration of test:
27 days (sacrifice was done on day 28)
Remarks:
Doses / Concentrations:
20, 40, 80 mg/kg bw/day
Basis:
actual ingested
(the application volume was 4 mL/kg bw)
No. of animals per sex per dose:
20 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous non-GLP dose range finding toxicity study (RCC B55170, 2008)
Maternal examinations:
MORTALITY AND CLINICAL SIGNS
The dams were examined for mortality twice daily. Cage-side clinical observations were made once daily up to the day of necropsy for assessment of clinical symptoms.

BODY WEIGHTS
Body weights were recorded daily for day 0 to day 28 post coitum.

FOOD CONSUMPTION
Food Consumption was recorded for the following periods: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24 and 24-28 post-coitum.

BLOOD SAMPLING
Blood samples (approximately 1 mL) were collected from the ear vein from 10 females each in groups 0, 20, 40 and 80 mg/kg bw/day. The samples were taken 24 h before the first administration (day 5 post coitum) and 24 h after the last administration (day 28 post coitum, directly before the necropsy). Plasma samples were prepared from the blood and frozen (-80 °C) for possible examination of the level of T3, T4 and TSH. However, based on the results of this study, these further investigations were not considered necessary.

NECROPSY
At the scheduled necropsy on day 28 post coitum, females were sacrificed by an intravenous injection of sodium pentobarbital and the fetuses removed by Caesarean section. Any female sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and its contents.
Gross macroscopic examination of all internal organs was performed, and particular attention was given to the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 28 post coitum to enable the calculation of the corrected body weight gain.
The following organs from all sacrificed females were weighed: liver, adrenal glands, thyroid gland, pituitary gland.
The above organs (but only half of the liver) were preserved in neutral phosphate buffered 4% formaldehyde solution for possible histopathological examination. The other half of the liver was placed immediately in a bag in pieces of 4 - 5 g and put in liquid nitrogen for storage at -80 °C for possible examination of necessary parameters (for example enzyme induction). However, based on the results of this study, these further investigations were not considered necessary.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. The fetuses were dissected, the organs examined and any abnormal findings were recorded. The sex of each fetus was noted.
2. After the skin had been removed, the cranium was examined for the degree of ossification.
3. From half of the fetuses the heads were separated from the trunks and fixed in Bouin's fixative. They were serially sectioned and examined (evaluation of the internal structures of the heads, including the eyes, brain, nasal passages and tongue) according to the method of Wilson JG (In: Teratology: Principles and Techniques. Eds., J.G. Wilson and J. Warkany, University of Chicago Press, 265-277,1965). Descriptions of any abnormal findings were recorded. After examination, the sections were preserved in a solution of ethyl alcohol and glycerin (one head per container). From the other half of the fetuses the heads were not separated but processed and stained as described in the next paragraph.
4. From all fetuses the skin with the exception of over the paws and the dorsal-cervical fat pads were removed and discarded. The trunks of the fetuses without heads and the fetuses with heads were processed through solutions of ethanol, glacial acetic with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage; the procedure were based on the method of Inouye M (Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and Alizarin red-S. Congenital Anomalies 16, 171-173, 1976). The skeletons were examined and all abnormal findings and variations were recorded
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated
- The Dunnett-test [Dunnett CW, J. Amer. Statist. Assoc. 50, 1096-1121, 1955] (many to one t-test) based on a pooled variance estimate was applied if the variables could not be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test [Miller RG, Simultaneous Statistical Inference, Springer Verlag, New York,1981] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
-Fisher's exact-test [Fisher RA, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh, 1950] was applied for the macroscopical findings.
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study.
One dam of the high dose group (80 mg/kg bw/day) aborted on day 27 post coitum. This female was necropsied on day 28 post coitum, as scheduled. No other clinical signs or symptoms were noted for any dam in any group.

BODY WEIGHTS
In the high dose group (80 mg/kg bw/day), mean percentage body weight gain was generally statistically significantly reduced from day 9 post coitum onwards. Over days 6-28, mean body weight increased by 3.3% compared to 6.3% in the control group. Although mean body weight also became reduced in comparison to the control group as the study progressed, it was at no time statistically significantly reduced. Mean corrected body weight gain was slightly reduced (-5.9% compared to -4.7% in the control group). This was considered to be a sign of maternal toxicity.
In the mid and low dose groups (40 and 20 mg/kg bw/day) mean body weight, body weight gain and corrected body weight gain were not affected by treatment with the test item.

FOOD CONSUMPTION
In the high dose group (80 mg/kg bw/day), mean food consumption was reduced, at times statistically significantly, throughout the treatment period (-24.9% over days 6-28 post coitum, compared to the control group). This was considered to be a result of treatment with the test item. In the 40 mg/kg bw/day group, there was a slight transient reduction in food consumption over days 9-12 (-9.2%) and 21-24 post coitum (-21.6%) but this was not statistically significant and did not have an effect on the mean body weight in this dose group. In the low dose group treated with 20 mg/kg bw/day, mean food consumption was not affected by treatment.

REPRODUCTION PARAMETERS (for details, see table below)
All females in all groups were mated.
One female each in the control and the mid dose groups (0 and 40 mg/kg bw/day) were not pregnant as well as 2 in the high dose group (80 mg/kg bw/day). These occurrences were considered to be incidental. Post-implantation loss was statistically significantly increased in the high dose group (27.5% in 12 out of 17 dams compared to 10.0% in 9 out of 19 dams in the control group). As a result, the total number of fetuses was statistically significantly reduced (4.6 fetuses per dam compared to 6.2 in the control group). This was considered to be a test item-related effect. One female in the high dose group aborted on day 27 post coitum. This was considered likely to be a further post-implantation loss and therefore may also be test item-related. In the low and mid dose groups, no test item-related effects were noted in the reproduction data.

NECROPSY
Gross examination of the dams after necropsy revealed several abnormalities in the high dose group (80 mg/kg bw/day) when compared to the remaining groups; the findings consisted of raised crateriform area in the stomach containing hair, pale kidneys, and enlarged gall bladder. Due to the increase in the incidence of these findings in this dose group, they were considered likely to be related to treatment with the test item.
Findings in the stomach were noted in one dam each in the low (20 mg/kg bw/day) and mid (40 mg/kg bw/day) dose groups. The low dose dam had a raised area on the pylorus and the mid dose dam had a red area in the stomach. Since these were the only findings in the stomach in these dose groups, they were considered likely to be incidental.
Referring to the organ weights, the weights of the left and right thyroid and adrenal glands were increased in the high dose group. These increases were considered to be probably test item-related but since they were not statistically significant, they were not considered to be adverse. In the low and mid dose groups, the organ weights were similar to those of the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at the same dose as maternal toxicity

Details on embryotoxic / teratogenic effects:
SEX RATIO
No test item-related effects on the sex ratio of the fetuses were noted in any group.

FETAL WEIGHTS
In the high dose group (80 mg/kg bw/day), mean fetal weights were statistically significantly reduced (30.5 g versus 32.7 g in the control group; p<0.01). This was considered to be an effect of the observed maternal toxicity in this dose group.
Mean fetal weights in the low and mid dose groups (20 and 40 mg/kg bw/day) were within control range (31.7 and 32.4 g, respectively) and thus were not affected by treatment with the test item. The statistically significant reduction of body weight of the males on an individual basis in the low dose group (31.7 g versus 33.4 g in the control group; p>0.05; see table below) was within the range of the historical control data for litters of this size and therefore was incidental.

EXAMINATION OF SECTIONED HEADS
Examination of fixed sectioned fetal heads did not reveal any test item-related effects. In fact, in the high dose, one fetuse of 41 (i.e. 2% of the fetuses) examined showed dilation of the lateral brain ventricles. The finding was incidental.

EXTERNAL AND VISCERAL EXAMINATIONS (for details, see the attached tables)
The main findings were summarized in a table (see below). During visceral examination of the fetuses, findings were noted in:
63% examined fetuses (in 100% litters) in the control group;
58% examined fetuses (in 100% litters) in the low dose group (20 mg/kg bw/day);
59% examined fetuses (in 94% litters) in the mid dose group (40 mg/kg bw/day);
66% examined fetuses (in 94% litters) in the high dose group (80 mg/kg bw/day).
Abnormalities were noted in 2% of fetuses in the control group, 3% in the low and mid dose groups, and 9% in the high dose group. Whereas the findings in the low and mid dose groups were considered to be incidental, in the high dose group, the higher incidence of abnormalities was considered to be test item-related, since there was also a higher incidence of post-implantation loss at the same dose level. Since maternal toxicity was noted in this dose group and there was no clear pattern in the nature of the findings, a direct effect of the test item on the fetuses is not suspected. None of the variations noted were considered to be test item-related. Although there was an increased incidence of an additional artery arising from the aortic arch, this was within the range of the historical control data.

SKELETAL EXAMINATIONS (for details, see the attached tables)
In the high dose group 4, the number of supernumerary ribs was marginally increased on a fetus basis compared to the concurrent control, but was not statistically significant on a litter basis. There was a statistically significant increase in incompletely ossified limbs/digits in all 3 treated groups (20, 40 and 80 mg/kg bw/day). The level in the low dose group was within the range of the historical control data and was therefore considered unlikely to be of toxicological relevance. The level in the mid dose group was within the historical control data on a litter level but outside on a fetus level. This was considered to be a transient and non-adverse effect of the test item. The level in the high dose group was outside the historical control range and was considered to be related to the maternal toxicity at this dose level.
During cartilage examination of the fetuses, abnormal findings were noted in:
6% examined fetuses (in 16% litters) in group 1
4% examined fetuses (in 20% litters) in group 2
7% examined fetuses (in 33% litters) in group 3
10% examined fetuses (in 41% litters) in group 4
In the high dose group, there was a decreased incidence of short costal cartilage 10 as well as a decreased incidence of costal cartilage 7 not reaching the sternum. These findings, together with the increased incidence of supernumerary costal cartilages, suggest a possible minor disturbance in the development of the axial skeleton. This would not be unexpected in the presence of the observed maternal toxicity at this dose level. In the mid dose group, these effects only attained statistical significance on a fetal level and therefore the toxicological relevance is unclear.
Key result
Dose descriptor:
NOAEC
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See remarks
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Summary of reproduction data:

 

Reproduction Parameters

 

Dose groups (mg/kg bw/day)

0

20

40

80

NUMBER OF DAMS

19

20

18

17

CORPORA LUTEA

 

 

 

 

 

Total

154

148

135

130

 

Mean/Dam

8.1

7.4

7.5

7.6

PRE-IMPLANTATION LOSS

24

24

30

21

 

% OF CORP. LUTEA

15.6

16.2

22.2

16.2

 

MEAN

1.3

1.2

1.7

1.2

 

NUMBER OF DAMS AFFECTED

11

12

11

10

IMPLANTATION SITES

130

124

105

109

 

% OF CORP. LUTEA

84.4

83.8

77.8

83.8

 

MEAN

6.8

6.2

5.8

6.4

POST-IMPLANTATION LOSS

13

5

11

30

 

% OF IMPLANT. SITES

10

4

10.5

27.5**

 

MEAN

0.7

0.3

0.6

1.8

 

NUMBER OF DAMS AFFECTED

9

4

6

12

EMBRYONIC/FETAL DEATHS TOTAL

13

5

11

30

EMBRYONIC RESORPTIONS

12

5

10

23

 

% OF IMPLANT. SITES

9.2

4

9.5

21.1**

 

MEAN

0.6

0.3

0.6

1.4

 

NUMBER OF DAMS AFFECTED

9

4

6

8

FETAL RESORPTIONS

1

0

1

7

 

% OF IMPLANT. SITES

0.8

 

1

6.4*

 

MEAN

0.1

 

0.1

0.4

 

NUMBER OF DAMS AFFECTED

1

 

1

4

FETUSES

 

 

 

 

 

TOTAL

117

119

94

79

 

% OF IMPLANT. SITES

90

96

89.5

72.5**

 

MEAN

6.2

6

5.2

4.6

 

LIVE FETUSES

117

119

94

79

 

DEAD FETUSES

0

0

0

0

 

ABNORMAL FETUSES

0

0

0

0

SEX OF THE FETUSES

 

 

 

 

TOTAL MALES

57

53

46

33

 

% OF FETUSES

48.7

44.5

48.9

41.8

 

MEAN

3

2.7

2.6

1.9

TOTAL FEMALES

60

66

48

46

 

% OF FETUSES

51.3

55.5

51.1

58.2

 

MEAN

3.2

3.3

2.7

2.7

LIVE MALES

57

53

46

33

LIVE FEMALES

60

66

48

46

WEIGHTS OF LIVE FETUSES (g)

(LITTER BASIS)

 

 

 

 

TOTAL FETUSES

 

 

 

 

 

N (LITTERS)

19

20

18

17

 

MEAN

33.3

32.4

33.3

30.4*

MALES

 

 

 

 

 

N (LITTERS)

19

19

17

16

 

MEAN

34

32.5

33.6

31.2*

FEMALES

 

 

 

 

 

N (LITTERS)

17

20

16

16

 

MEAN

32.6

32.3

32.1

30.8

WEIGHTS OF LIVE FETUSES (g)

(INDIV. BASIS)

 

 

 

 

TOTAL FETUSES

 

 

 

 

 

N (LITTERS)

117

119

94

79

 

MEAN

32.7

31.7

32.4

30.5**

MALES

 

 

 

 

 

N (LITTERS)

57

53

46

33

 

MEAN

33.4

31.7*

33.3

30.9**

FEMALES

 

 

 

 

 

N (LITTERS)

60

66

48

46

 

MEAN

32

31.7

31.6

30.2*

*, p<0.05; **, p<0.01

Summary of fetal data: see attached document.

Conclusions:
Some development toxicity related to the test substance was seen, however at a dose level which clearly was toxic to the dams. Based on the results of this study, the maternal NOAEL was set at 40 mg/kg body weight/day. The NOAEL for developmental toxicity also was set at 40 mg/kg body weight/day.
Executive summary:

This study is read across to the structural analogue CAS125643-61-0.

The purpose of the present study was to detect effects on the pregnant rabbit and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 to 27 post coitum (i.e., from implantation to the day prior to Caesarean section) at doses of 0, 20, 40 or 80 mg/kg body weight/day. Mortality, clinical signs, changes in body weights and body weight gain, and changes in food consumption were recorded. The dams were sacrificed on day 28 post coitum, examined for gross lesions, and the organ weights of the liver, adrenal glands, thyroid gland and pituitary gland were recorded. The fetuses were removed by Caesarean section, examined and allocated to visceral or skeletal examination.

Whereas no indication for maternal toxicity was seen at 20 and 40 mg/kg bw/day, treatment with 80 mg/kg body weight/day, resulted in following test item related findings: The mean food consumption was reduced, at times statistically significantly, throughout the treatment period; the mean body weight gain and body weight were also reduced; post-implantation loss was statistically significantly increased and as a result, the total number of fetuses was statistically significantly reduced; one case of abortion was reported; necropsy revealed several lesions, especially in the stomach, kidneys and gall bladder.

Referring to developmental toxicity, no indication for developmental toxicity was seen at 20 and 40 mg/kg bw/day. At 80 mg/kg body weight/day, the mean fetal weights were statistically significantly reduced. There were more visceral abnormalities in the fetuses than in the control group. There was reduced cranial ossification, as well as reduced digit/limb ossification. The findings from the skeletal and cartilage examinations suggest a possible minor disturbance in the development of the axial skeletons of the fetuses. Nevertheless, since the fetal findings were seen at the highest tested dose of 80 mg/kg bw/day which clearly was toxic to the dams, they were considered to be attributable to maternal toxicity.

In conclusion, development toxicity related to the test substance was seen, however at a dose level which clearly was toxic to the dams.

Based on the results of this study, the maternal NOAEL was set at 40 mg/kg body weight/day. The NOAEL for developmental toxicity also was set at 40 mg/kg body weight/day.

 

No classification and labelling is applicable.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit
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

This study is read across to the structural analogue CAS125643-61-0.

The purpose of the present study was to detect effects on the pregnant rabbit and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 to 27 post coitum (i.e., from implantation to the day prior to Caesarean section) at doses of 0, 20, 40 or 80 mg/kg body weight/day. Mortality, clinical signs, changes in body weights and body weight gain, and changes in food consumption were recorded. The dams were sacrificed on day 28 post coitum, examined for gross lesions, and the organ weights of the liver, adrenal glands, thyroid gland and pituitary gland were recorded. The fetuses were removed by Caesarean section, examined and allocated to visceral or skeletal examination.

Whereas no indication for maternal toxicity was seen at 20 and 40 mg/kg bw/day, treatment with 80 mg/kg body weight/day, resulted in following test item related findings: The mean food consumption was reduced, at times statistically significantly, throughout the treatment period; the mean body weight gain and body weight were also reduced; post-implantation loss was statistically significantly increased and as a result, the total number of fetuses was statistically significantly reduced; one case of abortion was reported; necropsy revealed several lesions, especially in the stomach, kidneys and gall bladder.

Referring to developmental toxicity, no indication for developmental toxicity was seen at 20 and 40 mg/kg bw/day. At 80 mg/kg body weight/day, the mean fetal weights were statistically significantly reduced. There were more visceral abnormalities in the fetuses than in the control group. There was reduced cranial ossification, as well as reduced digit/limb ossification. The findings from the skeletal and cartilage examinations suggest a possible minor disturbance in the development of the axial skeletons of the fetuses. Nevertheless, since the fetal findings were seen at the highest tested dose of 80 mg/kg bw/day which clearly was toxic to the dams, they were considered to be attributable to maternal toxicity.

In conclusion, some development toxicity related to the test substance was seen, however at a dose level which clearly was toxic to the dams.

Based on the results of this study, the maternal NOAEL was set at 40 mg/kg body weight/day. The NOAEL for developmental toxicity also was set at 40 mg/kg body weight/day.

Justification for selection of Effect on developmental toxicity: via oral route:

GLP compliant study; read across to structural analogue.

Justification for classification or non-classification

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for reproductive effects is therefore required.

Additional information