Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Repeated dose toxicity study

In this study performed according to OECD 408, no gross or histopathological changes in any of the reproductive organs (M/F rats) were observed following 90 days of treatment with the read across analouge, at doses up to 500 mg/kg bw/day.

Multi-generation reproductive toxicity study

The study-derived NOAEL for the read across analogue was 1000 mg/kg bw/day for reproductive and developmental toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Remarks:
Repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across data
Qualifier:
according to guideline
Guideline:
other: OECD 408
Version / remarks:
1998
Principles of method if other than guideline:
According to OECD 408
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD IGS BR strain
Details on species / strain selection:
Male and female Sprague-Dawley Crl:CD IGS BR strain rats were obtained from Charles River (UK) Ltd. (Margate, Kent)
Sex:
male/female
Details on test animals or test system and environmental conditions:
After 6 days of acclimatization, the animals were randomly allocated to treatment groups using a total randomization method and the group mean bodyweights were thereafter determined to ensure similarity between the test groups. Animals were housed according to sex in two groups of three and one group of four. At the initiation of treatment males weighed 131 to 172 g, females weighed 122 to 155 g, and were 6 to 8 weeks old. The study room was supplied with at least 15 air changes per hour. Temperature ranged from 19 to 23C and humidity from 40 to 70%. A 12:12-hour light-dark cycle period was used. Food was available ad lib, as was water. Wooden chew blocks and cardboard fun tunnels were provided for environmental enrichment.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The dose volume was 4 mL/kg/d.
Details on mating procedure:
No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were stored in darkness at 4C and were tested for stability, homogeneity, and concentration. The formulations were observed to be stable for at least two weeks and to be within acceptable concentration limits (mean nominal concentrations of +/- 3%).
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once per day
Details on study schedule:
No Data Available
Remarks:
0 (vehicle), 5, 30 and 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected based on the results of a 14-day dose range finding (DRF) study. In the DRF, 3 rats per sex per dose were treated at 0 and 1000 mg/kg bw/day. No deaths were observed, however increased salivation, some slightly reduced body weights, and some pale kidneys were observed at 1000 mg/kg. Therefore, 500 mg/kg bw/day was selected as top dose in the definitive study.
Positive control:
Not included
Parental animals: Observations and examinations:
The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters included hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration), total leukocyte count, differential leukocyte count, platelet count, reticulocyte count, prothrombin time, and activated partial thromboplastin time. Blood chemistry parameters included urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorous, aspartate aminotransferase (AST), alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, and total bilirubin.
Oestrous cyclicity (parental animals):
No Data Available
Sperm parameters (parental animals):
No Data Available
Litter observations:
No Data Available
Postmortem examinations (parental animals):
All rats were sacrificed on day 91. The following organs were weighed at all dose levels: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg: adrenals, thoracic aorta, bone and bone marrow (femur including stifle joint and sternum), brain (cerebrum, cerebellum, and pons), cecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum (including Peyer patches), jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), esophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, midthoracic, and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, and uterus.
Postmortem examinations (offspring):
No Data Available
Statistics:
Homogeneity of means: ANOVA or ANCOVA and Bartlett test.
Transformed data: Williams test (parametric) or the Shirley test (nonparametric).
Data showing no dose-dependency, but homogeneity of the mean: Dunnett (parametric) or Steel (non-parametric).
Pairwise tests (student t test parametric or Mann-Whitney U test nonparametric) were used on a case-by-case basis.
Histopathology data: Chi-square analysis and Kruskal-Wallis one-way nonparametric analysis.
Reproductive indices:
No Data Available
Offspring viability indices:
No Data Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity.One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
All animals survived to planned death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects on behaviour, functional performance, or sensory reactivity were 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 microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Dose descriptor:
LOAEL
Effect level:
<= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Critical effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Conclusions:
In this study performed according to OECD 408, no gross or histopathological changes in any of the reproductive organs (M/F rats) were observed following 90 days of treatment with the test chemical, at doses up to 500 mg/kg bw/day.
Executive summary:

The chemical was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 5, 30 and 500 mg/kg/day for a total of 90 days. Dose levels were selected based on the results of a dose range-finding (DRF) study. In the DRF, three rats per sex per dose level were treated at 0 and 1000 mg/kg/day for 14 days. No unscheduled deaths were observed in the DRF, and clinical signs were limited to increased salivation at 1000 mg/kg. Male rats treated at 1000 mg/kg weighed slightly less than the controls on the 4th day of study. At necropsy, 2 males and 1 female treated at 1000 mg/kg showed pale kidneys. Based on these data, doses of 0, 5, 30 and 500 mg/kg/day were selected in the definitive 90-day study. The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters includedhemoglobin, erythrocytecount, hematocrit, erythrocyte indices (meancorpuscular hemoglobin, mean corpuscular volume, and meancorpuscular hemoglobinconcentration), total leukocyte count,differential leukocyte count, platelet count, reticulocytecount, prothrombin time, and activated partial thromboplastintime. Blood chemistry parameters included urea,glucose, total protein, albumin, albumin/globulin ratio, sodium,potassium, chloride, calcium, inorganic phosphorous, aspartateaminotransferase (AST), alanine aminotransferase, alkalinephosphatase, creatinine, total cholesterol, and total bilirubin.All rats were sacrificed on day 91.Thefollowing organs were weighedat all dose levels:adrenals, brain, epididymides, heart, kidneys, liver, ovaries,spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg:adrenals, thoracic aorta, bone and bonemarrow (femur including stifle joint and sternum), brain (cerebrum,cerebellum, and pons), cecum, colon, duodenum,epididymides, eyes, gross lesions, heart, ileum (including Peyerpatches), jejunum, kidneys, liver, lungs (with bronchi), lymphnodes (cervical and mesenteric), mammary glands, muscle (skeletal),esophagus, ovaries, pancreas, pituitary, prostate, rectum,salivary glands (submaxillary), sciatic nerve, seminal vesicles,skin (hind limb), spinal cord (cervical, midthoracic, and lumbar),spleen, stomach, testes, thymus, thyroid/parathyroid, tongue,trachea, urinary bladder, and uterus.All animals survived to planned death. Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity. One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical. No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed. Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed. No adverse effects on food intake, food efficiency, or water intake were observed during the study period. The ophthalmoscopic examination revealed no treatment-related effects at 500 mg/kg. No treatment-related effects on haematology were observed. Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study. No gross pathological effects were observed at any dose level. Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders). The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group. Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Read across data
Qualifier:
no guideline followed
Principles of method if other than guideline:
The toxicological effects of the test chemical on reproductive performance and development (if any) were investigated across several generations of rats that were exposed to the chemical by diet.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Details on mating procedure:
Two litters were obtained from each generation. The first litters from each parental generation were assigned to a long-term toxicity study. The second litters from the F0 generation (F1b) were used to generate the F2a and F2b generations. The F2b generation was then used to generate the F3a and F3b generations.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Animals were treated for 63 days before mating. They were mated for a period of 20 days. From day 12 post-partum and throughout the remainder of lactation, the dosing of the nursing dams at 500 and 1000 mg/kg was reduced to avoid marked overdosing of the pups. After weaning, the dams were returned to their respective treatment levels.
Frequency of treatment:
Daily
Details on study schedule:
No further details.
Remarks:
0 (plain diet), 0 (plain diet containing placebo beadlets), 250, 500 and 1000 mg/kg bw/day.
No. of animals per sex per dose:
The F0 generation included 210 male and 420 female rats. These animals were assigned to six different treatment groups.
Control animals:
yes, plain diet
other: Control group 2: plain diet with placebo beadlets
Details on study design:
Two litters were obtained from each generation. The first litters from each parental generation were assigned to a long-term toxicity study. The second litters from the F0 generation (F1b) were used to generate the F2a and F2b generations. The F2b generation was then used to generate the F3a and F3b generations. Animals were treated for 63 days before mating. They were mated for a period of 20 days. From day 12 post-partum and throughout the remainder of lactation, the dosing of the nursing dams at 500 and 1000 mg/kg was reduced to avoid marked overdosing of the pups. After weaning, the dams were returned to their respective treatment levels.
Positive control:
Not included.
Parental animals: Observations and examinations:
Clinical signs, body weights, food intake, and food efficiency were recorded throughout the study. Reproductive parameters included pregnancy rates, pregnancy performance, and gestation lengths.
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Not examined.
Litter observations:
Viability, litter size, litter weight, and pup weight.
Postmortem examinations (offspring):
Organ weight analyses and histopathological examinations were performed on selected F3 pups.
Statistics:
Not specified.
Clinical signs:
no effects observed
Mortality:
not specified
Description (incidence):
Some rats died, mostly male rats of the F0 generation. Deaths were attributed to hemorrhagic events based on circumstantial evidence.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gains were generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in body weight gains were observed between dosed animals and the control group that received placebo beadlets.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food intakes were generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in food intakes were observed between dosed animals and the control group that received placebo beadlets.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food efficiency was generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in food efficiency was observed between dosed animals and the control group that received placebo beadlets.
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
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant effects were observed on mating performance, pregnancy rate or gestation length.
The above results are for all parental generations of the study.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant effects on pup viability were observed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gains were generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in body weight gains were observed between dosed animals and the control group that received placebo beadlets.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food intakes were generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in food intakes were observed between dosed animals and the control group that received placebo beadlets.
Food efficiency:
no effects observed
Description (incidence and severity):
Food efficiency was generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in food iefficiency was observed between dosed animals and the control group that received placebo beadlets.
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
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weight data obtained from selected F3b pups revealed no treatment-related effects.
Gross pathological findings:
not specified
Histopathological findings:
no effects observed
Description (incidence and severity):
The histopathological examination of selected F3b pups revealed no treatment-related effects.
The above results are for all offspring generations.
Dose descriptor:
NOAEL
Generation:
other: F1, F2, F3
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
food consumption and compound intake
food efficiency
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Reproductive effects observed:
no
Conclusions:
The study-derived NOAEL for reproductive and developmental toxicity was 1000 mg/kg bw/day in this multi-generation toxicity study.
Executive summary:

The chemical was given by diet at dose levels corresponding to 0, 100, 250, 500 and 1000 mg/kg bw/day. The F0 generation included 210 male and 420 female rats. These animals were assigned to six different treatment groups. The two control groups received treatment at 0 mg/kg bw/day, either by plain diet or diet containing placebo beadlets. The other groups received the test chemical by diet at dosages of 100, 250, 500 and 1000 mg/kg bw/day. Two litters were obtained from each generation. The first litters from each parental generation were assigned to a long-term toxicity study. The second litters from the F0 generation (F1b) were used to generate the F2a and F2b generations. The F2b generation was then used to generate the F3a and F3b generations. Animals were treated for 63 days before mating. They were mated for a period of 20 days. From day 12 post-partum and throughout the remainder of lactation, the dosing of the nursing dams at 500 and 1000 mg/kg was reduced to avoid marked overdosing of the pups. After weaning, the dams were returned to their respective treatment levels. Clinical signs, body weights, food intake, and food efficiency were recorded throughout the study. Reproductive parameters included pregnancy rates, pregnancy performance, and gestation lengths. Litter data were recorded, and additional organ weight analyses and histopathological examinations were performed on selected F3 pups. No clinical signs of toxicity were observed at any dose level. Some rats died, mostly male rats of the F0 generation. Deaths were attributed to hemorrhagic events based on circumstantial evidence. Body weight gain, food intake and food efficiency were generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in body weight gain, food intake or food efficiency were observed between dosed animals and the control group that received placebo beadlets. At necropsy, no treatment-related effects were observed apart from expected discoloration. No significant effects were observed on mating performance, pregnancy rate or gestation length. The averages for litter size, pup mortality, litter weight, and pup weight were unaffected by treatment. The organ weight analysis and histopathological examination of selected F3b pups revealed no treatment-related effects. The study-derived NOAEL for reproductive and developmental toxicity was 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1 and 2 sources.
Additional information

Study 1

The read across analogue was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 5, 30 and 500 mg/kg/day for a total of 90 days. Dose levels were selected based on the results of a dose range-finding (DRF) study. In the DRF, three rats per sex per dose level were treated at 0 and 1000 mg/kg/day for 14 days. No unscheduled deaths were observed in the DRF, and clinical signs were limited to increased salivation at 1000 mg/kg. Male rats treated at 1000 mg/kg weighed slightly less than the controls on the 4th day of study. At necropsy, 2 males and 1 female treated at 1000 mg/kg showed pale kidneys. Based on these data, doses of 0, 5, 30 and 500 mg/kg/day were selected in the definitive 90-day study. The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters includedhemoglobin, erythrocytecount, hematocrit, erythrocyte indices (meancorpuscular hemoglobin, mean corpuscular volume, and meancorpuscular hemoglobinconcentration), total leukocyte count,differential leukocyte count, platelet count, reticulocytecount, prothrombin time, and activated partial thromboplastintime. Blood chemistry parameters included urea,glucose, total protein, albumin, albumin/globulin ratio, sodium,potassium, chloride, calcium, inorganic phosphorous, aspartateaminotransferase (AST), alanine aminotransferase, alkalinephosphatase, creatinine, total cholesterol, and total bilirubin.All rats were sacrificed on day 91.Thefollowing organs were weighedat all dose levels:adrenals, brain, epididymides, heart, kidneys, liver, ovaries,spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg:adrenals, thoracic aorta, bone and bonemarrow (femur including stifle joint and sternum), brain (cerebrum,cerebellum, and pons), cecum, colon, duodenum,epididymides, eyes, gross lesions, heart, ileum (including Peyerpatches), jejunum, kidneys, liver, lungs (with bronchi), lymphnodes (cervical and mesenteric), mammary glands, muscle (skeletal),esophagus, ovaries, pancreas, pituitary, prostate, rectum,salivary glands (submaxillary), sciatic nerve, seminal vesicles,skin (hind limb), spinal cord (cervical, midthoracic, and lumbar),spleen, stomach, testes, thymus, thyroid/parathyroid, tongue,trachea, urinary bladder, and uterus.All animals survived to planned death. Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity. One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical. No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed. Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed. No adverse effects on food intake, food efficiency, or water intake were observed during the study period. The ophthalmoscopic examination revealed no treatment-related effects at 500 mg/kg. No treatment-related effects on haematology were observed. Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study. No gross pathological effects were observed at any dose level. Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders). The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group. Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.

Study 2

The read across analogue was given by diet at dose levels corresponding to 0, 100, 250, 500 and 1000 mg/kg bw/day. The F0 generation included 210 male and 420 female rats. These animals were assigned to six different treatment groups. The two control groups received treatment at 0 mg/kg bw/day, either by plain diet or diet containing placebo beadlets. The other groups received the test chemical by diet at dosages of 100, 250, 500 and 1000 mg/kg bw/day. Two litters were obtained from each generation. The first litters from each parental generation were assigned to a long-term toxicity study. The second litters from the F0 generation (F1b) were used to generate the F2a and F2b generations. The F2b generation was then used to generate the F3a and F3b generations. Animals were treated for 63 days before mating. They were mated for a period of 20 days. From day 12 post-partum and throughout the remainder of lactation, the dosing of the nursing dams at 500 and 1000 mg/kg was reduced to avoid marked overdosing of the pups. After weaning, the dams were returned to their respective treatment levels. Clinical signs, body weights, food intake, and food efficiency were recorded throughout the study. Reproductive parameters included pregnancy rates, pregnancy performance, and gestation lengths. Litter data were recorded, and additional organ weight analyses and histopathological examinations were performed on selected F3 pups. No clinical signs of toxicity were observed at any dose level. Some rats died, mostly male rats of the F0 generation. Deaths were attributed to hemorrhagic events based on circumstantial evidence. Body weight gain, food intake and food efficiency were generally greater in the control group that received plain diet versus the control group that received placebo beadlets. No marked differences in body weight gain, food intake or food efficiency were observed between dosed animals and the control group that received placebo beadlets. At necropsy, no treatment-related effects were observed apart from expected discoloration. No significant effects were observed on mating performance, pregnancy rate or gestation length. The averages for litter size, pup mortality, litter weight, and pup weight were unaffected by treatment. The organ weight analysis and histopathological examination of selected F3b pups revealed no treatment-related effects. The study-derived NOAEL for reproductive and developmental toxicity was 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Study 1

NOAEL for maternal systemic toxicity was concluded at 100 mg/kg bw/day. This effect level was based on observed effects on body weight gain and food intake at 400 mg/kg bw/day. NOAEL for developmental toxicity was concluded at 400 mg/kg bw/day. The study was performed according to OECD 414.

Study 2

The study-derived NOAEL for maternal and developmental toxicity was at 500 mg/kg bw/day. Increased incidence of gestation losses at 1000 mg/kg was the only manifestation of developmental toxicity attributed to the test chemical. The authors of the study considered this effect to be secondary to alterations in maternal homeostasis at 1000 mg/kg leading to disturbed physiological support needed for embryo survival. The study was comparable to OECD 414.

Study 3

The study-derived NOAEL for embryotoxicity and teratogenicity was 1000 mg/kg bw/day. The study was limited in detail with regard to examinations and presentation of data.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Read across data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Germany)
- Age at study initiation: about 70-84 days
- Weight at study initiation: 148.7 - 183.6 g
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Olive Oil
- Test substance preparation: At the beginning of the administration period and thereafter at intervals which took into account the analytical results of the stability verification. For the preparation of the solutions an appropriate amount of the test substance was weighed depending on the dose group, in a graduated beaker, topped up with olive oil, and subsequently thoroughly mixed using a magnetic stirrer.
- Concentration in vehicle: 500, 2000 and 8000 mg/100 ml
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): 10R0449/02050
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Check of stability and concentration control was performed by HPLC. Since the test substance was a true solutions, investigations concerning homogeneity were not necessary.
Details on mating procedure:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (= detection of vaginal plug / sperm). The animals arrived on the same day (i.e. day 0 p.c.) at the experimental laboratory. The following day was designed "day 1" post coitum (p.c.). Animals were assigned to the test groups by taken random selection.
Duration of treatment / exposure:
Day 6 through Day 19 post coitum (p.c.)
Frequency of treatment:
Once Daily
Duration of test:
Up to GD 20
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control Group
Dose / conc.:
25 mg/kg bw/day
Remarks:
Low Dose Group
Dose / conc.:
100 mg/kg bw/day
Remarks:
Mid Dose Group
Dose / conc.:
400 mg/kg bw/day
Remarks:
High Dose Group
No. of animals per sex per dose:
25 animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
No Data Available
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No Data Available

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No Data Available

BODY WEIGHT: Yes
- Time schedule for examinations: No Data Available

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day # 19
- Organs examined: All visceral and reproductive organs.

OTHER: No Data Available
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: Dead fetuses, Calculation of conception rate and pre- and post implantation losseswere carried out.
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:
Statistical analyses were performed according to following
schedule:
- DUNNETT-test (two-sided): Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- FISHER'S EXACT test (one-sided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- WILCOXON-test (one-sided): Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
- KRUSKAL-WALLIS-test (two-sided): Liver weights
Indices:
Implantation Index, Resorption Index, Viability Index.
Historical control data:
The historical control data used for interpretation of findings refer to the same test facility, the same rat strain and supplier of the animals and cover a period of about 24 months (June 2001 - June 2003, 15 studies).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high dose and the majority (22 out of 25) of the mid dose animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed salivation persisted in the respective females only for a few minutes after the actual gavaging had taken place. After cessation of treatment on day 19 p.c., salivation did not occur any longer.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the high dose dams was statistically significantly reduced (9% below the concurrent control value) at initiation of treatment (days 6 - 8 p.c.). On the following days of the treatment period, however, food consumption of the high dose rats reached or even exceeded control values. The food consumption of the females of low and mid dose dams was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls. The transient reductions in food consumption at 400 mg/kg bw were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
21 high dose dams showed dark-yellow discolored urine on gestation days 12 - 20 p.c. which is probably related to a chemical reaction of the test substance or its metabolites with the bedding or with components of the air and does not represent a toxicologically relevant finding.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No indications for disturbances of the general behavior.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Uterus weight: The mean gravid uterus weights of the animals of all test groups were not influenced by the administration of the test substance.

Liver weight: Absolute and relative mean liver weights were statistically significantly increased at the mid and high dose groups and were about 9 or 29% (absolute) and 8 or 29% (relative) above control values. These weight increases, which are considered to be substance-induced, are indicative of hepatic changes primarily caused by microsomal enzyme induction. Absolute and relative liver weights of the low dose dams, however, were similar to the control values and did not show any toxicologically significant changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There occurred no substance-related observations at necropsy in any of the dams of all test groups. Only very few spontaneous findings were recorded for single low and mid dose rats (one hydrometra in low dose female which consequently did not become pregnant, hemorrhagic thymus in one mid dose female). No association to the test compound was assumed for these findings due to their scattered occurrence without any relation to dosing.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
All high dose and the majority (22 out of 25) of the mid dose animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed salivation persisted in the respective females only for a few minutes after the actual gavaging had taken place. After cessation of treatment on day 19 p.c., salivation did not occur any longer. There were no substance-related or spontaneous mortalities in any of the groups. Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20). The mean food consumption of the high dose dams was statistically significantly reduced (9% below the concurrent control value) at initiation of treatment (days 6 - 8 p.c.). On the following days of the treatment period, however, food consumption of the high dose rats reached or even exceeded control values. The food consumption of the females of low and mid dose dams was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls. The transient reductions in food consumption at 400 mg/kg bw were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing. 21 high dose dams showed dark-yellow discolored urine on gestation days 12 - 20 p.c. which is probably related to a chemical reaction of the test substance or its metabolites with the bedding or with components of the air and does not represent a toxicologically relevant finding. The mean gravid uterus weights of the animals of all test groups were not influenced by the administration of the test substance. Absolute and relative mean liver weights were statistically significantly increased at the mid and high dose groups and were about 9 or 29% (absolute) and 8 or 29% (relative) above control values. These weight increases, which are considered to be substance-induced, are indicative of hepatic changes primarily caused by microsomal enzyme induction. Absolute and relative liver weights of the low dose dams, however, were similar to the control values and did not show any toxicologically significant changes. There occurred no substance-related observations at necropsy in any of the dams of all test groups. Only very few spontaneous findings were recorded for single low and mid dose rats (one hydrometra in low dose female which consequently did not become pregnant, hemorrhagic thymus in one mid dose female). No association to the test compound was assumed for these findings due to their scattered occurrence without any relation to dosing.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
The conception rate reached 96% in the controls, 92% in the low and the high dose groups, and 100% in the mid dose. There were no substance-related and/or biologically relevant differences between the test groups in the conception rate, in the mean number of corpora lutea and implantation sites.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the test groups in the pre- and the postimplantation losses. The pre- and the postimplantation loss values in the 25 and 100 mg/kg groups, however, were above the upper ranges of the historical control values and the mean number of live fetuses/low dose dam was statistically significantly below the concurrent and the historical control value. These differences appeared without any dose-response relationship and thus are not considered to reflect any substance-induced effect.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the test groups in the number of resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the test groups in the number of early or late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between viable and dead fetus.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The conception rate reached 96% in the controls, 92% in the low and the high dose groups, and 100% in the mid dose. There were no substance-related and/or biologically relevant differences between the test groups in the conception rate, in the mean number of corpora lutea and implantation sites, the pre- and the postimplantation losses, and the number of resorptions and viable fetuses. The pre- and the postimplantation loss values in the 25 and 100 mg/kg groups, however, were above the upper ranges of the historical control values and the mean number of live fetuses/low dose dam was statistically significantly below the concurrent and the historical control value. These differences appeared without any dose-response relationship and thus are not considered to reflect any substance-induced effect. They can well be explained by the fact that one low dose dam and two mid dose dams resorbed all of their implants and thus had
no viable fetuses (the same was also observed for one control dam). Moreover, one low dose, which had 7 implantation sites, resorbed 6 implants and had only one live fetus at terminal sacrifice. If these 5 rats are excluded from the calculation of the means, pre- and postimplantation loss values as well as the mean number of live fetuses/dam fit well into the historical control ranges with one unimportant exception (preimplantation loss value at 25 mg/kg bw/day).
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
dermal irritation
early or late resorptions
food consumption and compound intake
gross pathology
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
urinalysis
Dose descriptor:
LOAEL
Effect level:
<= 400 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal body weights in all test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in all test groups was comparable with that of the control fetuses.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean fetal body weights in all test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Most of the observed malformations were limited to one multiply malformed mid dose fetus. The external examination of this fetus revealed gastroschisis, anal atresia, malrotated left hindlimb, and a thread-like tail.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During its skeletal evaluation additional malformations like severely malformed sternum and absent vertebrae were recorded. The other malformations that occurred were anasarca (in one control fetus), situs inversus (in one low dose fetus), and cleft sternum (in another control fetus).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue variations, exclusively in the form of dilated renal pelvis and ureters were observed to be sporadic, spontaneous and non treatment related.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The mean fetal body weights in all test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values. The sex distribution of the fetuses in all test groups was comparable with that of the control fetuses. Most of the observed malformations were limited to one multiply malformed mid dose fetus. The external examination of this fetus revealed gastroschisis, anal atresia, malrotated left hindlimb, and a thread-like tail. During its skeletal evaluation additional malformations like severely malformed sternum and absent vertebrae were recorded. The other malformations that occurred were anasarca (in one control fetus), situs inversus (in one low dose fetus), and cleft sternum (in another control fetus). Soft tissue variations, exclusively in the form of dilated renal pelvis and ureters were observed to be sporadic, spontaneous and non treatment related.
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Developmental effects observed:
no
Treatment related:
no
Conclusions:
NOAEL for maternal systemic toxicity was concluded at 100 mg/kg bw/day. This effect level was based on observed effects on body weight gain and food intake at 400 mg/kg bw/day. NOAEL for developmental toxicity was concluded at 400 mg/kg bw/day.
Executive summary:

The chemical was given by oral gavage to 25 presumed pregnant rats per dose levelat 0 (vehicle), 25, 100 and 400 mg/kg bw/day from GD 6 to 19. Dose levels were selected based on the results of dose range-finding study. All animals survived to planned death (scheduled on GD 20). Clinical signs were limited to transient salivation at ≥100 mg/kg and discolored urine at 400 mg/kg. None of these effects were considered to be of toxicological significance. No significant effects on maternal body weight were observed. Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20). Collectively, the observed effects on body weight gain and food intake at 400 mg/kg were taken as signs of maternal systemic toxicity. No significant effects were observed on mean gravid uterus weight. Absolute and relative liver weights were significantly increased at ≥100 mg/kg. Necropsy findings were unremarkable. The conception rates were 96, 92, 100 and 92% at 0, 25, 100 and 400 mg/kg, respectively. Pre- and post-implantation losses were significantly increased at 25 and 100 mg/kg compared to the control data. This effect was not attributed to the test chemical as no such effect was observed at 400 mg/kg. The mean number of live fetuses was significantly lower at 25 mg/kg (mean, 7.4) compared to the control data (mean, 8.7). This effect was not attributed to the test chemical as no such effect was observed at 100 or 400 mg/kg. No significant effects were observed in the mean numbers of corpora lutea, implantation sites, or resorptions. The number of abortions were 1, 1, 2 and 0 at 0, 25, 100 and 400 mg/kg, respectively. Mean placental weight was significantly increased at 400 mg/kg (mean, 0.45 g) compared to the control group (mean, 0.41 g) but was well within the historical control range (0.32 to 0.58 g). No significant effects on mean fetal body weight were observed among the groups. Sex ratio was unaffected by treatment. No treatment-related gross, visceral or skeletal malformations were observed. The combined number of malformations expressed as % of fetuses affected were 1.0, 0.6, 0.5 and 0.0% at 0, 25, 100 and 400 mg/kg, respectively. No external variations were observed. No treatment-related gross, visceral or skeletal variations were observed. The combined number of variations expressed as % of fetuses affected were 53, 54, 52 and 49% at 0, 25, 100 and 400 mg/kg, respectively. The combined number of variations expressed as % of litters affected was 100% at all dose levels. NOAEL for maternal systemic toxicity was concluded at 100 mg/kg bw/day. This effect level was based on observed effects on body weight gain and food intake at 400 mg/kg bw/day. NOAEL for developmental toxicity was concluded at 400 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Read across data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Oswaldo Cruz Foundation Central Animal House breeding stock,
- Age at study initiation: Approx 9 weeks
- Weight at study initiation: Approximately 200gm
- Fasting period before study:
- Housing: individually in standard plastic cages with stainless-steel coverlids and white pinewood shavings as bedding.
- Diet (e.g. ad libitum): ad libitum, A pelleted diet (Nuvital® for laboratory rats and mice, Nuvilab Ltd, Curitiba, PR, Brazil).
- Water (e.g. ad libitum): ad libitum, Filtered tap water.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C
- Humidity (%): approx. 70%
- Photoperiod (hrs dark / hrs light): 12-h light:12-h dark photoperiod (lights on from 8:00–20:00 h).
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No Data Available

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test chemical was more soluble in corn oil than in water.
- Concentration in vehicle: No data available
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data Available
Details on mating procedure:
Mating was carried out by placing two females into the cage of one male for 2 h (6:00–8:00 a.m.) and confirmed by the presence of sperm in the vaginal smear. The day on which spermatozoa were found in the smear was designated as day 0 of pregnancy.
Duration of treatment / exposure:
From GD 6 to GD 15
Frequency of treatment:
Daily
Duration of test:
Up to GD 21
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control Group
Dose / conc.:
125 mg/kg bw/day
Remarks:
Low Dose Group
Dose / conc.:
250 mg/kg bw/day
Remarks:
Mid Dose Group
Dose / conc.:
500 mg/kg bw/day
Remarks:
High Dose Group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Top dose group
No. of animals per sex per dose:
0 mg/kg: 17 animals
125 mg/kg: 17 animals
250 mg/kg: 17 animals
500 mg/kg: 18 animals
1000 mg/kg: 20 animals
Control animals:
yes, concurrent vehicle
Details on study design:
No Data Available
Maternal examinations:
Clinical signs, Maternal weight, Maternal weight gain, Gestation length.
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:
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
Data were evaluated by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Values are the mean ± standard deviation (SD), or the median and range (minimum-maximum values). Proportions were analyzed by the Chi-square test or by the Fisher exact test if any expected frequency was smaller than 5. Calculations were performed using a GraphPad Prism 5® statistical software.
Indices:
Resorption Index, Viability Index, Implantation Index
Historical control data:
No Data Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity were observed at 500 and 1000 mg/kg from GD 7. At 500 mg/kg, the clinical signs were restricted to hypoactivity (one animal) andchromodacryorrhea(four animals). At 1000 mg/kg, the clinical signs were comprised of piloerection (10 animals), vocalization (4 animals), hyperactivity (one animal), chromodacryorrhea(7 animals, hypoactivity (one animal), hair loss (one animal), lacrimation (one animal) and lethargy (one animal). No clinical signs of toxicity were observed after cessation of treatment
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
All animals survived to planned death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No significant effects on maternal weight were observed at GD 0 or GD 21. A significant decrease in maternal weight gain from GD 6 to 15 was observed at 1000 mg/kg (mean, 21.2 g) compared to the control group (mean, 35.8 g). No other significant effects on maternal weight gain were observed. Maternal body weight gain corrected for gravid uterus weight was unaffected by treatment.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No alteration in maternal organ weights. The test chemical did not decrease gravid uterus weight at any dose level, nor did it reduce the net maternal weight gain in pregnancy (i.e., the maternal weight gain minus the gravid uterus weight).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathology abnormalities were observed at any dose level.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
All adult rats survived to planned death. Clinical signs of toxicity were observed at 500 and 1000 mg/kg from GD 7. At 500 mg/kg, the clinical signs were restricted to hypoactivity (one animal) and chromodacryorrhea (four animals). At 1000 mg/kg, the clinical signs were comprised of piloerection (10 animals), vocalization (4 animals), hyperactivity (one animal), chromodacryorrhea (7 animals, hypoactivity (one animal), hair loss (one animal), lacrimation (one animal) and lethargy (one animal). No clinical signs of toxicity were observed after cessation of treatment. No significant effects on maternal weight were observed at GD 0 or GD 21. A significant decrease in maternal weight gain from GD 6 to 15 was observed at 1000 mg/kg (mean, 21.2 g) compared to the control group (mean, 35.8 g). No other significant effects on maternal weight gain were observed. Maternal body weight gain corrected for gravid uterus weight was unaffected by treatment.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One animal treated at 1000 mg/kg aborted. No other abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant effects on the number of implantation sites per pregnant female were observed.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Increased incidence of gestation losses at 1000 mg/kg was the only manifestation of maternal developmental toxicity attributed to the test chemical. The authors of the study considered this effect to be secondary to alterations in maternal homeostasis at 1000 mg/kg leading to disturbed physiological support needed for embryo survival.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant effects on the total number of resorptions (early or late) were observed.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was observed at 1000 mg/kg. No other dead foetuses were observed in the study.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
The pregnancy rates were 94, 94, 82, 94 and 60% at 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The effect at 1000 mg/kg was of unclear toxicological significance according to the authors based on in-huse data.
Details on maternal toxic effects:
A significant decrease in maternal weight gain from GD 6 to 15 was observed at 1000 mg/kg (mean, 21.2 g) compared to the control group (mean, 35.8 g). No other significant effects on maternal weight gain were observed. Maternal body weight gain corrected for gravid uterus weight was unaffected by treatment. The pregnancy rates were 94, 94, 82, 94 and 60% at 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The mean numbers of corpora lutea and implantation sites were similar among all groups. One animal treated at 1000 mg/kg aborted. No significant effects on the total number of resorptions were observed. Increased incidence of gestation losses at 1000 mg/kg was the only manifestation of maternal developmental toxicity attributed to the test chemical. The authors of the study considered this effect to be secondary to alterations in maternal homeostasis at 1000 mg/kg leading to disturbed physiological support needed for embryo survival.
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
gross pathology
mortality
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
Dose descriptor:
LOAEL
Effect level:
<= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
total litter losses by resorption
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant effects on mean fetal body weight were observed.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio (F/M) was somewhat lower at 1000 mg/kg (ratio, 0.7) compared to the control group (ratio, 1.3). This effect was not considered to be of toxicological significance in the study.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related gross abnormalities were observed.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related skeletal abnormalities were observed.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related visceral abnormalities were observed.
Details on embryotoxic / teratogenic effects:
The mean numbers of live fetuses per litter were 10.8, 11.4, 11.4, 11.2 and 10.9 at 0, 125, 250, 500 and 1000 mg/kg, respectively, with no statistically significant differences observed. No significant effects on mean fetal body weight were observed. Sex ratio (F/M) was somewhat lower at 1000 mg/kg (ratio, 0.7) compared to the control group (ratio, 1.3). No treatment-related gross, visceral or skeletal malformations/variations were observed.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Conclusions:
The study-derived NOAEL for maternal and developmental toxicity was at 500 mg/kg bw/day. Increased incidence of gestation losses at 1000 mg/kg was the only manifestation of developmental toxicity attributed to the test chemical. The authors of the study considered this effect to be secondary to alterations in maternal homeostasis at 1000 mg/kg leading to disturbed physiological support needed for embryo survival.
Executive summary:

The chemical was given to by oral gavage to 17 to 20 (mean, 17.8) presumed pregnant rats per dose level at 0 (vehicle), 125, 250, 500 and 1000 mg/kg bw/day from gestation day (GD) 6 to 15. The animals were examined for mortality and clinical signs of toxicity before being sacrificed on GD 21. Body weights were recorded on GD 0, 6, 11, 15 and 21. The gravid uterus from each animal was removed and weighed with its content. Ovaries were removed and the number of corpora lutea was determined. The numbers of live and dead fetuses were counted, as was the number of resorptions classified as either early resorptions; intermediate resorptions; or late resorptions. All living fetuses were weighed and examined for external abnormalities. One-third of the fetuses from each litter were examined for visceral malformations and variations. The remaining fetuses were examined for skeletal malformations and variations. All adult rats survived to planned death. Clinical signs of toxicity were observed at 500 and 1000 mg/kg from GD 7. At 500 mg/kg, the clinical signs were restricted to hypoactivity (one animal) andchromodacryorrhea(four animals). At 1000 mg/kg, the clinical signs were comprised of piloerection (10 animals), vocalization (4 animals), hyperactivity (one animal), chromodacryorrhea(7 animals, hypoactivity (one animal), hair loss (one animal), lacrimation (one animal) and lethargy (one animal). No clinical signs of toxicity were observed after cessation of treatment. No significant effects on maternal weight were observed at GD 0 or GD 21. A significant decrease in maternal weight gain from GD 6 to 15 was observed at 1000 mg/kg (mean, 21.2 g) compared to the control group (mean, 35.8 g). No other significant effects on maternal weight gain were observed. Maternal body weight gain corrected for gravid uterus weight was unaffected by treatment.The pregnancy rates were 94, 94, 82, 94 and 60% at 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The mean numbers of corpora lutea and implantation sites were similar among all groups. One animal treated at 1000 mg/kg aborted. No significant effects on the total number of resorptions were observed. One dead fetus was observed at 1000 mg/kg. The mean numbers of live fetuses per litter were 10.8, 11.4, 11.4, 11.2 and 10.9 at 0, 125, 250, 500 and 1000 mg/kg, respectively, with no statistically significant differences observed. No significant effects on mean fetal body weight were observed. Sex ratio (F/M) was somewhat lower at 1000 mg/kg (ratio, 0.7) compared to the control group (ratio, 1.3). No treatment-related gross, visceral or skeletal malformations or variations were observed. The study-derived NOAEL for maternal and developmental toxicity was at 500 mg/kg bw/day. Increased incidence of gestation losses at 1000 mg/kg was the only manifestation of developmental toxicity attributed to the test chemical. The authors of the study considered this effect to be secondary to alterations in maternal homeostasis at 1000 mg/kg leading to disturbed physiological support needed for embryo survival.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Read across data
Qualifier:
no guideline followed
Principles of method if other than guideline:
The chemical was given by diet to 160 presumed pregnant rats at doses equivalent to 0, 250, 500 and 1000 mg/kg bw/day. Doses were given from gestation day 7 to 16. Effects on development were investigated.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Ftillinsdorf albino rats.
Details on test animals or test system and environmental conditions:
Not specified.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test substance was given in beadlets containing 11.5% active ingredient.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Not specified.
Duration of treatment / exposure:
Doses were given as a feed admixture from day 7 through day 16 of gestation.
Frequency of treatment:
Once daily.
Duration of test:
Up to GD 21
Remarks:
0 (place beadlets), 250, 500 and 1000 mg/kg bw/day
No. of animals per sex per dose:
Not specified, however 160 mated female albino rats were divided into the 4 different groups.
Control animals:
yes, plain diet
Details on study design:
No further details provided.
Maternal examinations:
The animals were observed daily. Body weights were recorded before treatment, during treatment, and before sacrifice. On gestation day 21, the dams were divided into two groups. Dams in the first group were sacrificed on GD 21 and the uteri and fetuses were examined. Dams of the second group were allowed to deliver and raise their young. On day 23 of lactation, dams of the second group and their young were sacrificed for post-mortem examination.
Ovaries and uterine content:
Dams in the first group were sacrificed on GD 21 and the uteri and fetuses were examined.
Fetal examinations:
Dams in the first group were sacrificed on GD 21 and the uteri and fetuses were examined. On day 23 of lactation, dams of the second group and their young were sacrificed for post-mortem examination.
Statistics:
Not specified
Indices:
Not specified
Historical control data:
Not specified
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the highest level of 1000 mg/kg/day there was a slight reduction in body weight gain. This effect was of unclear toxicological significance.
Details on results:
No treatment-related maternal death, and no signs of maternal toxicity were noted in any dosage groups.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
mortality
Details on embryotoxic / teratogenic effects:
As reported in the article, there was neither embryotoxicity nor teratogenic effect in rats dosed up to 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was neither embryotoxicity nor teratogenic effect in rats dosed up to 1000 mg/kg/day.
Key result
Developmental effects observed:
no
Conclusions:
The study-derived NOAEL for developmental toxicity was 1000 mg/kg bw/day.
Executive summary:

The chemical was given by diet to 160 presumed pregnant rats at doses equivalent to 0, 250, 500 and 1000 mg/kg bw/day. Doses were given from gestation day 7 to 16. The animals were observed daily. Body weights were recorded before treatment, during treatment, and before sacrifice. On gestation day 21, the dams were divided into two groups. Dams in the first group were sacrificed on GD 21 and the uteri and fetuses were examined. Dams of the second group were allowed to deliver and raise their young. On day 23 of lactation, dams of the second group and their young were sacrificed for post-mortem examination. No treatment-related mortality or clinical signs were observed. At the highest level of 1000 mg/kg/day there was a slight reduction in body weight gain. This effect was of unclear toxicological significance. No indications of embryotoxicity or teratogenicity were observed up to 1000 mg/kg bw/day. The study-derived NOAEL for developmental toxicity was 1000 mg/kg bw/day. The study was limited in detail with regard to examinations and the presentation of data.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 2 and 4 sources.
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

Study 1

The read across analogue was given by oral gavage to 25 presumed pregnant rats per dose levelat 0 (vehicle), 25, 100 and 400 mg/kg bw/day from GD 6 to 19. Dose levels were selected based on the results of dose range-finding study. All animals survived to planned death (scheduled on GD 20). Clinical signs were limited to transient salivation at ≥100 mg/kg and discolored urine at 400 mg/kg. None of these effects were considered to be of toxicological significance. No significant effects on maternal body weight were observed. Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20). Collectively, the observed effects on body weight gain and food intake at 400 mg/kg were taken as signs of maternal systemic toxicity. No significant effects were observed on mean gravid uterus weight. Absolute and relative liver weights were significantly increased at ≥100 mg/kg. Necropsy findings were unremarkable. The conception rates were 96, 92, 100 and 92% at 0, 25, 100 and 400 mg/kg, respectively. Pre- and post-implantation losses were significantly increased at 25 and 100 mg/kg compared to the control data. This effect was not attributed to the test chemical as no such effect was observed at 400 mg/kg. The mean number of live fetuses was significantly lower at 25 mg/kg (mean, 7.4) compared to the control data (mean, 8.7). This effect was not attributed to the test chemical as no such effect was observed at 100 or 400 mg/kg. No significant effects were observed in the mean numbers of corpora lutea, implantation sites, or resorptions. The number of abortions were 1, 1, 2 and 0 at 0, 25, 100 and 400 mg/kg, respectively. Mean placental weight was significantly increased at 400 mg/kg (mean, 0.45 g) compared to the control group (mean, 0.41 g) but was well within the historical control range (0.32 to 0.58 g). No significant effects on mean fetal body weight were observed among the groups. Sex ratio was unaffected by treatment. No treatment-related gross, visceral or skeletal malformations were observed. The combined number of malformations expressed as % of fetuses affected were 1.0, 0.6, 0.5 and 0.0% at 0, 25, 100 and 400 mg/kg, respectively. No external variations were observed. No treatment-related gross, visceral or skeletal variations were observed. The combined number of variations expressed as % of fetuses affected were 53, 54, 52 and 49% at 0, 25, 100 and 400 mg/kg, respectively.The combined number of variations expressed as % of litters affected was 100% at all dose levels. NOAEL for maternal systemic toxicity was concluded at 100 mg/kg bw/day. This effect level was based on observed effects on body weight gain and food intake at 400 mg/kg bw/day. NOAEL for developmental toxicity was concluded at 400 mg/kg bw/day.

Study2

The read across analogue was given to by oral gavage to 17 to 20 (mean, 17.8) presumed pregnant rats per dose level at 0 (vehicle), 125, 250, 500 and 1000 mg/kg bw/day from gestation day (GD) 6 to 15. The animals were examined for mortality and clinical signs of toxicity before being sacrificed on GD 21. Body weights were recorded on GD 0, 6, 11, 15 and 21. The gravid uterus from each animal was removed and weighed with its content. Ovaries were removed and the number of corpora lutea was determined. The numbers of live and dead fetuses were counted, as was the number of resorptions classified as either early resorptions; intermediate resorptions; or late resorptions. All living fetuses were weighed and examined for external abnormalities. One-third of the fetuses from each litter were examined for visceral malformations and variations. The remaining fetuses were examined for skeletal malformations and variations. All adult rats survived to planned death. Clinical signs of toxicity were observed at 500 and 1000 mg/kg from GD 7. At 500 mg/kg, the clinical signs were restricted to hypoactivity (one animal) andchromodacryorrhea(four animals). At 1000 mg/kg, the clinical signs were comprised of piloerection (10 animals), vocalization (4 animals), hyperactivity (one animal), chromodacryorrhea(7 animals, hypoactivity (one animal), hair loss (one animal), lacrimation (one animal) and lethargy (one animal). No clinical signs of toxicity were observed after cessation of treatment. No significant effects on maternal weight were observed at GD 0 or GD 21. A significant decrease in maternal weight gain from GD 6 to 15 was observed at 1000 mg/kg (mean, 21.2 g) compared to the control group (mean, 35.8 g). No other significant effects on maternal weight gain were observed. Maternal body weight gain corrected for gravid uterus weight was unaffected by treatment.The pregnancy rates were 94, 94, 82, 94 and 60% at 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The mean numbers of corpora lutea and implantation sites were similar among all groups. One animal treated at 1000 mg/kg aborted. No significant effects on the total number of resorptions were observed. One dead fetus was observed at 1000 mg/kg. The mean numbers of live fetuses per litter were 10.8, 11.4, 11.4, 11.2 and 10.9 at 0, 125, 250, 500 and 1000 mg/kg, respectively, with no statistically significant differences observed. No significant effects on mean fetal body weight were observed. Sex ratio (F/M) was somewhat lower at 1000 mg/kg (ratio, 0.7) compared to the control group (ratio, 1.3). No treatment-related gross, visceral or skeletal malformations/variations were observed.The study-derived NOAEL for maternal and developmental toxicity was at 500 mg/kg bw/day. Increased incidence of gestation losses at 1000 mg/kg was the only manifestation of developmental toxicity attributed to the test chemical. The authors of the study considered this effect to be secondary to alterations in maternal homeostasis at 1000 mg/kg leading to disturbed physiological support needed for embryo survival.

Study 3

The read across analogue was given by diet to 160 presumed pregnant rats at doses equivalent to 0, 250, 500 and 1000 mg/kg bw/day. Doses were given from gestation day 7 to 16. The animals were observed daily. Body weights were recorded before treatment, during treatment, and before sacrifice. On gestation day 21, the dams were divided into two groups. Dams in the first group were sacrificed on GD 21 and the uteri and fetuses were examined. Dams of the second group were allowed to deliver and raise their young. On day 23 of lactation, dams of the second group and their young were sacrificed for post-mortem examination. No treatment-related mortality or clinical signs were observed. At the highest level of 1000 mg/kg/day there was a slight reduction in body weight gain. This effect was of unclear toxicological significance. No indications of embryotoxicity or teratogenicity were observed up to 1000 mg/kg bw/day. The study-derived NOAEL for developmental toxicity was 1000 mg/kg bw/day.

Justification for classification or non-classification

In the OECD 414 study conducted with read across analogue No. 1, no adverse effects on development were reported up to the highest dose tested (i.e. 400 mg/kg/day). In the second developmental toxicity study, conducted with read across analogue No. 2, observed effects on development were limited to increased incidence of gestation losses at the top dose level (i.e. 1000 mg/kg/day). The authors of the study, which included nearly all relevant aspects of prenatal developmental toxicity as foreseen to be investigated in OECD 414, attributed this effect to maternal systemic toxicity. In the limited developmental toxicity study conducted with read across analogue No. 3, no embryotoxicity or teratogenicity were reported upon maternal exposure from gestation day 7 to 16 at doses up to 1000 mg/kg bw/day. By weight of evidence, treatment with the registered substance in rats as per OECD 414 can be assumed to produce no adverse effects on development. In the study performed according to OECD 408, treatment with the read across analogue No. 1 in rats, by oral gavage up to 500 mg/kg bw/day, did not produce any gross or histopathological effects in the testes, epididymides, seminal vesicles, prostate, ovaries, uterus. In a multi-generation reproductive toxicity study, treatment with the read across analogue No. 2 in rats, at dietary concentrations corresponding to 0, 100, 250, 500 and 1000 mg/kg bw/day, did not produce any adverse effects on reproductive performance or development. By weight of evidence, the registered substance is regarded to be classified as Not Classified for Toxicity to reproduction according to Regulation EC No 1272/2008. The read-across justification is provided in Section 13 of the dossier.

Additional information