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REACH

ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

EC number: 443-870-0 | CAS number: 163520-33-0

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD TG 416, oral, feed, rat:

P0 generation: NOAEL systemic toxicity and fertility: 200 ppm; actual test substance intake: males: 12.6 mg/kg bw/day, females: 18.3 mg/kg bw/day, combined sexes: 15.5 mg/kg bw/day; LOAEL systemic toxicity and fertility: 4000 ppm; actual test substance intake: males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day, combined sexes: 306.5 mg/kg bw/day

F1 generation: NOAEL systemic toxicity and fertility: 200 ppm; actual test substance intake: males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day, combined sexes: 17.3 mg/kg bw/day; LOAEL systemic toxicity and fertility: 4000 ppm; actual test substance intake: males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day, combined sexes: 353.3 mg/kg bw/day

F2 generation NOAEL systemic toxicity and fertility: 4000 ppm (highest dose tested), actual test substance intake of parental generations (P0 and F1): 329.9 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:: two-generation reproductive toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 08 Aug 1997- 27 April 1998
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:: Current version adopted in 2001
Deviations:: yes
Remarks:: Thyroid weights were not measured for F0 and F1 parental animals. Enumeration of cauda epididymal sperm reserve was not done.
Qualifier:: according to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:: Guideline in place during study conduct: adopted in 1983
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:: Adopted in 1987
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:: Adopted in 1996 and 1997
Qualifier:: according to guideline
Guideline:: other: "Reproductive Study," Japanese Ministry of Agriculture, Forestry and Fisheries, 59 NohSan No. 4200
Version / remarks:: Adopted in 1985
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Remarks:: Crl:CD(SD)BR
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, USA
- Age at receipt (P): 33 days
- Age at start of dosing (F1): 3 weeks
- Body weight at study initiation: (P) males: 161 - 230 g, females: 126 - 180 g; (F1, prior to mating) males: 392 - 607 g, females: 206 - 342 g
- Housing: individually in wire-mesh cages suspended above cage board until selection or pairing; after successful mating males stayed in wire mesh cages until scheduled necropsy, females were transferred to plastic maternity cages with nesting material (Bed-O'Cobs; The Andersons, Industrial Products Division, Maumee, USA) and stayed there until weaning on lactation Day 21. Females returned to wire mesh cages until scheduled necropsy and weaned F1 pups were housed together until PND 28 when pups were individually housed as described above.
- Diet: grounded Certified Rodent LabDiet® 5002 (PMI Nutrition International, Inc.), ad libitum
- Water: tap water in drinking water quality (reverse osmosis-treated on-site), ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Aug 1997 To: 27 Apr 1998
Route of administration:: oral: feed
Vehicle:: other: grounded diet
Remarks:: Certified Rodent LabDiet® 5002
Details on exposure:: DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: standard diet
- Storage temperature of food: room temperature, in the dark
Details on mating procedure:: - M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged: individually in plastic maternity cages with nesting material
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: On the first day of dosing, diet formulations were prepared for each dose group. Six samples were collected for each group, including the control group, from the top, middle and bottom (homogeneity sample) or middle (concentration and stability samples) of the preparations. Two sets of each were designated for homogeneity, concentration and stability samples and stored deep frozen. A formulation the portion allocated for stability sampling was stored under laboratory animal room conditions and sampled after 0, 1, 2, 4, 8 and 15 days. A 100 g sample from the middle of each formulation, including the control, was collected weekly throughout the study and stored frozen. Samples of diet from each dosage level, including controls, were analysed for test article concentration at approximately 4-week intervals for the first 3 months of the study and then at 3-month intervals thereafter. The test substance was extracted from the diet by shaking with acetonitrile for determination by high performance liquid chromatography using UV detection. The mean test substance content for the test diets sampled at the time of preparation and analysed were within the range 87.3 to 101.1% of nominal (acceptable range +10% to -15% of nominal). Homogeneity was shown to be satisfactory at all levels (i.e. mean values obtained for top, middle and bottom samples were within the range 90 - 110% of nominal and these mean % values differed by <10%). Stability was shown to be satisfactory over the time of use of the diet, i.e. % nominal values declined by <10% over 15 days storage at room temperature. Data from the analyses therefore confirmed that the diet preparations used for administration to the animals were stable for 15 days at room temperature, were homogeneous and contained the amounts of test substance specified in the protocol.
Duration of treatment / exposure:: (P) Males: 10 weeks before mating and 9 weeks during mating and thereafter until necropsy (19 weeks in total)
(P) Females: 10 weeks before mating, up to 2 weeks during mating, 3 weeks during gestation, 3 weeks during lactation till weaning of their F1 offspring on post-natal day (PND) 21 and further 2 weeks until necropsy (approx. 20 weeks in total)
(F1) Males: 18 - 19 weeks after weaning, during growth into adulthood, mating and production of F2 generation, until weaning on PND 21 of the F2 generation.
(F1) Females: 18 - 19 weeks after weaning, during growth into adulthood, mating and production of F2 generation, until weaning on PND 21 of the F2 generation.
Frequency of treatment:: daily, 7 days/week
Details on study schedule:: - F1 parental animals not mated until 10 weeks after being selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 17 weeks (F0), 14 - 15 weeks (F1)
Dose / conc.:: 20 ppm
Remarks:: actual test substance intake:
P0 generation: males: 1.2 mg/kg bw/day, females: 1.8 mg/kg bw/day
F1 generation: males: 1.5 mg/kg bw/day, females: 2.0 mg/kg bw/day
Dose / conc.:: 200 ppm
Remarks:: actual test substance intake:
P0 generation: males: 12.6 mg/kg bw/day, females: 18.3 mg/kg bw/day
F1 generation: males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day
Dose / conc.:: 4 000 ppm
Remarks:: actual test substance intake:
P0 generation: males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day
F1 generation: males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day
No. of animals per sex per dose:: 30
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: Results of a previous subchronic toxicity study showed that 4000 ppm caused a 10 - 11% reduction of body weight gain in both sexes, therefore it was expected to be a maximum tolerated dose. The 200 ppm dose level was expected to be a NOEL. The lowest dose level of 20 ppm was expected to be a certain NOEL.
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for appearance, behaviour, pharmacotoxic signs, moribundity and mortality. Females that delivered were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded weekly for all parental animals throughout the study period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual F0 and F1, male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F0 and F1, female body weights were recorded weekly until evidence of mating was observed. Once evidence of mating was observed, female body weights were recorded on gestation Days 0, 4, 7, 10, 14 and 20 and on lactation Days 1, 4, 7, 14 and 21. After weaning (lactation Day 21), weekly body weights were recorded for these females until the scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Individual F0 and F1, male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation Days 0, 4, 7, 10, 14 and 20 and lactation Days 1, 4, 7, 14 and 21.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OTHER: food efficiency
Oestrous cyclicity (parental animals):: Vaginal smears were prepared daily to determine the stage of estrus for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, smearing was continued until termination of the mating period. Vaginal smears were also performed on the day of necropsy.
Sperm parameters (parental animals):: Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 post partum: yes
- If yes, maximum of 8 pups/litter (4 sex/litter as nearly as possible); excess pups were weighed, killed and discarded on PND 4

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Detailed physical examination and weighing: PND 1, 4, 7, 15 and 21
Developmental landmarks: Balanopreputial separation from PND 40 to PND 53, vaginal patency from PND 30 to PND 53

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: All surviving animals, after the last litter of each generation was weaned
- Maternal animals: All surviving animals, after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of all euthanised or spontaneously dying animals. Examined were the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were collected and placed in 10% neutral buffered formalin.
adrenals , aorta, bone with marrow (sternebrae), brain (forebrain, midbrain, hindbrain), cervix, coagulating gland, eyes with optic nerve , gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, liver (sections of two lobes), lungs (including bronchi, fixed by inflation with, fixative), lymph node (mesenteric), ovaries and oviducts, pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary gland (submaxillary ), seminal vesicles, skeletal muscle (vastus medialis), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymides and vas deferens, thymus, thyroids (with parathyroids), trachea, urinary bladder, uterus with vagina, all gross lesions

The tissues/ organs indicated below were weighed.
adrenals, brain, epididymides (total and cauda), kidneys, liver, ovaries with oviducts, pituitary, prostate, seminal vesicles with coagulating, glands (with accessory fluids), spleen, testes, thymus, uterus with cervix

The tissues/ organs indicated below were examined microscopically in P and F1 parental animals (10/sex/group) from the control and high dose group and for all animals found dead.
adrenal gland, brain, epididymis (right): caput, corpus and cauda; cervix, coagulating gland, kidneys, liver, ovaries, oviducts, pituitary, prostate, seminal vesicles, spleen, testis (right), thymus, uterus, vagina, vas deferens, all gross (internal) lesions
Postmortem examinations (offspring):: SACRIFICE
- The F1 offspring not selected as parental animals and for examination of developmental landmarks and all F2 offspring were sacrificed at postnatal Day 21 days of age.
- These animals were subjected to postmortem examinations with emphasis on developmental morphology. One F1 and F2 pup/sex/litter was prepared for complete necropsy on PND 21

GROSS NECROPSY
- Gross necropsy with emphasis on developmental landmarks.

HISTOPATHOLOGY / ORGAN WEIGTHS
Complete necropsy was performed for one F1 and F2 pup/sex/litter as described for parental animals.
Statistics:: All analyses were conducted using two-tailed tests (except as noted below) for a minimum significance level of 5% comparing each treated group to the control group. Each mean was presented with the standard deviation and the number of animals used to calculate the mean. Data obtained from non-gravid animals were excluded from statistical analyses following the mating period. Statistical analyses were not performed when weekly food or body weight data for one or more animals were not available because the animals remained in the lactation phase. The following statistical tests were performed by a Digital® MicroVAX® 3400 computer (with appropriate programming) in this laboratory and are referenced on the report tables:
- Chi-square test with Yates' correction factor for Parental Mating and Fertility Indices
- One-way ANOVA with Dunnett's test for parental weekly body weights and weight changes, gestation and lactation body weights and body weight changes, parental food consumption, pre-coital interval, mean gestation length, number of pups born, pup body weights, absolute and relative organ weights, live litter size, sperm production rate, sperm numbers, mean day of acquisition of balanopreputial separation or vaginal perforation, mean body weight at acquisition.
- Kruskal-Wallis test with Mann-Whitney U test for sperm motility, sperm morphology, proportional postnatal survival, pup viability indices, live birth index, percent male at birth.
- Kolmogorov-Smirnov test (one-tailed test) for histopathological findings.
Reproductive indices:: Mating Index (%) for males and females = (No. of rats copulated / No. of mated pairs) x 100
Fertility Index (%) for females = (No. of females pregnant / No. of females used for mating) x 100
Fertility Index (%) for males = (No. of males siring a litter / No. of males used for mating) x 100
Gestation Index (%) = (No. of dams with live newborns / No. of pregnant dams) x 100
Offspring viability indices:: Life birth Index (%) = (No. of live offspring at Day 0 / No. of live offspring at birth) x 100
Viability Index (%) = (No. of live offspring at Day 4 / No. of live offspring at birth) x 100
Lactation Index (%) = (No. of live offspring at Day 21 / No. of live offspring at Day 4 post-selection) x 100
Clinical signs:: no effects observed
Description (incidence and severity):: There were no test substance-related signs of toxicity observed. Clinical findings in the treated groups, such as scabbing and hair loss on various body surfaces, red material around the eyes, swollen ears and soft stool, occurred similarly in the control group or infrequently.
Mortality:: mortality observed, non-treatment-related
Description (incidence):: At 4000 ppm, 2 parental (P0) males died during the study; one male died during Week 7 and another died during Week 18. The cause of death for both of these animals was determined to be urogenital tract inflammation.
At 200 ppm, 1 P0 male died during week 10; the cause of death could not be determined.
At 0 ppm, 1 control group female died during Week 4; the cause of death could not be determined.
All other parental (P0) animals survived to the scheduled necropsy.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: In the 4000 ppm group, absolute body weights of P0 males were consistently slightly below (5%) control group values from Week 6 and cumulative body weight change was 7 - 9% lower from Week 0 - 1 throughout the study period (Table 5 under "Any other information on results incl. tables"). In the 4000 ppm group, P0 males, mean body weight gains during weeks 0 - 1 through 9 - 10 were slightly lower than the control group values. The differences during weeks 0 - 1, 3 - 4, 5 - 6 and 8 - 9 were statistically significant (p <0.05 or p <0.01). Throughout the remainder of the generation (Weeks 10 - 11 through 18 - 19), mean body weight gains in the 4000 ppm group males were similar to the control group values. Cumulative body weight gains from Week 0 were reduced by 7 - 9% throughout the generation. The majority of the differences was statistically significant (p <0.05 or p <0.01). Mean body weights in the 4000 ppm group P0 males were consistently slightly lower (5%) than the control group values from Weeks 6 through 19. The difference during Week 10 was statistically significant (p <0.05).
Mean weekly body weights and body weight gains in the 20 and 200 ppm group males and females and the 4000 ppm group females were unaffected by test substance administration. The only statistically significant differences in these groups from the control values were a reduced mean body weight in the 4000 ppm group females for Week 17 (p <0.05) and a reduced mean body weight gain in the 20 ppm group males during Week 3 - 4 (p <0.05). These transient changes were not attributed to test substance administration.
During gestation, mean body weights and body weight gains in the 20, 200 and 4000 ppm groups were similar to the control group values; differences were slight and were not statistically significant.
During lactation, there was no treatment-related effect on P0 female body weight or body weight change at any dose level. In the 4000 ppm group P0 females, an increased mean body weight gain during lactation Day 1 - 4 was followed by a reduced mean body weight gain during lactation Day 4 - 7. The difference from the control group value during lactation Day 4 - 7 was statistically significant (p <0.01). No trends were apparent, and these differences during the first 6 days of lactation were attributed to biological variation. Mean body weight gain in the 4000 ppm group females was comparable to the control group value during lactation Day 7 - 14. During lactation Day 14 - 21, a mean body weight loss in the 4000 ppm group was greater than the loss observed in the control group. The difference was not statistically significant. When the entire lactation period (lactation Day 1 - 21) was evaluated, a reduced mean body weight gain in the 4000 ppm group was statistically significant at p <0.05 when compared to the control group value. Mean lactation body weights in the 4000 ppm group were similar to the control group values on lactation Days 1, 4, 7 and 14. On lactation Day 21, mean body weight in this group was 5% lower than the control group value; the difference was not statistically significant. Because the P0 female mean body weights and body weight gains were similar to the control group values throughout the remainder of the treatment period (pre-mating, gestation and post-weaning), the reduced body weights and body weight gains during the latter portion of the lactation period were attributed to biological variation. Mean lactation body weights and body weight changes in the 20 and 200 ppm group P0 females were unaffected by test substance administration. The only statistically significant differences (p <0.05 or p <0.01) from the control group were reduced mean body weight gains in the 20 and 200 ppm groups during lactation Days 4 - 7. However, mean body weight gains in these groups were greater than the control group value during lactation Days 1 - 4, and no dose-response relationship was apparent. Therefore, the differences were attributed to biological variation.
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: Food consumption, evaluated as g/animal/day and g/kg bw/day, was generally unaffected by test substance administration in all treatment groups (at 20, 200 and 4000 ppm) in both sexes.
A statistically significant (p <0.05) reduction in food consumption was observed in the 4000 ppm group males during the first week of administration (Week 0 - 1). However, because the difference was slight (4.2% when evaluated as g/animal/day), the reduction was considered not to be treatment-related. The only other statistically significant differences (p <0.05 or p <0.01) from the control group were increased g/animal/day and/or g/kg bw/day values in the 4000 ppm group females during Week 2 - 3, 7 - 8 and 17 - 18. No trends were apparent, and the differences were attributed to biological variation.
During gestation, food consumption, evaluated as g/animal/day and g/kg bw/day, was unaffected by test substance administration in the 20, 200 and 4000 ppm group P0 females. No statistically significant differences from the control group values were observed.
During lactation, food consumption was unaffected by treatment. Food consumption, assessed as g/animal/Day and g/kg bw/Day, in the 4000 ppm group P0 females was comparable to that in the control group during lactation Day 1 - 4, 4 - 7 and 7 - 14. During lactation Days 14 - 21, food consumption (g/animal/day and g/kg bw/day) in the 4000 ppm group was slightly lower than that in the control group. The differences were statistically significant (p <0.05). When the entire lactation period (lactation Day 1 - 21) was evaluated, food consumption in the 4000 ppm group females was slightly lower than that in the control group. The differences were not statistically significant. Because P0 food consumption values were similar to the control group values throughout the remainder of the treatment period (pre-mating, gestation and post-weaning), the reduction in food consumption in the latter part of the lactation period was attributed to biological variation. Food consumption in the 20 and 200 ppm group P0 females was unaffected by test substance administration throughout the lactation period. No statistically significant differences from the control group were observed.
For details on test substance intake see Table 1 - 3 under "Any other information on results incl. tables".
Food efficiency:: effects observed, treatment-related
Description (incidence and severity):: Food efficiency was reduced in the 4000 ppm group males, mainly due to the reduction in body weight gains. Food efficiency was reduced during Week 3 - 4 and between Week 5 - 6 and 9 - 10 and generally correlated to reduced body weight gain data during these weeks of treatment for the 4000 ppm group males. The reductions were statistically significant (p <0.05 or p <0.01) during Week 3 - 4, 5 - 6, 7 - 8, and 8 - 9. No treatment-related effects were observed in 20 and 200 ppm group males and females or the 4000 ppm group females. Occasionally statistically significant differences from the control group values were not observed in a dose-dependent manner.
During gestation, as for food consumption, food efficiency was unaffected by test substance administration in the 20, 200 and 4000 ppm group P0 females. No statistically significant differences from the control group values were observed.
During lactation, food efficiency was increased in the 20, 200 and 4000 ppm groups relative to the control group during lactation Day 1 - 4. During lactation Day 4 - 7, food efficiency was decreased (p <0.05) in these same groups relative to the control group. Because no trends were apparent, these differences were considered not to be test substance-related. Food efficiency in the 20 and 200 ppm groups were similar to the control groups values throughout the remainder of lactation (lactation Day 7 - 14 and 14 - 21) and when the entire lactation period (lactation Day 1 - 21) were evaluated. Food efficiency in the 4000 ppm group was similar to the control group value during lactation Day 7-14 and slightly reduced (p <0.05 or p <0.01) during lactation Day 14 - 21 and 1 - 21. Because food efficiency in the P0 females in this group was similar to the control group throughout the remainder of the treatment period (pre-mating, gestation and post-weaning), the reduction in food efficiency in the latter part of the lactation period was considered not to be test substance related.
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:: effects observed, non-treatment-related
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: At terminal necropsy of P0 animals, the incidence and/or severity of several kidney findings in the 4000 ppm group were increased compared to the control group in female animals only (Table 4 under "Any other information on results incl. tables"). These findings included nephropathy, epithelial (urothelial) hyperplasia, medullary/papillary cysts, medullary fibrosis/degeneration, papillary tubular epithelial hyperplasia and inflammation in the medulla/papilla. The increases in nephropathy and medullary/papillary cysts were statistically significant (p <0.05). The nephropathy in the 4000 ppm group females was characterized by tubular basement membrane thickening, basophilic cortical tubular epithelium and/or interstitial infiltration of mononuclear cells. The medullary/papillary cysts in these animals were lined by cuboidal tubular epithelium, often contained cellular or amorphous debris (not casts) and were located primarily in the outer medullary stripe with some extension into the inner medulla and/or papilla. Fibrosis and degenerating tubules were often present adjacent to the cysts but were also observed within the medulla and/or papilla without cyst formation. Papillary tubular epithelial hyperplasia, characterized by increased numbers of basophilic epithelial cells along the tubular basement membrane, and minimal acute inflammation of the papilla, were observed only in the 4000 ppm group females. In the 4000 ppm group females, the medullary/papillary cysts, medullary degeneration/fibrosis and inflammation were generally present in the animals with more severe nephropathy, but were also present in some animals with minimal nephropathy. In the 4000 ppm group males and females, there was an increase in the incidence and severity of hyperplasia of the epithelium lining the renal pelvis (urothelial hyperplasia). In the 4000 ppm group females, the epithelium along the papilla was most often affected, and it was usually observed in animals without evidence of pyelitis, pelvic mineralization or calculi, suggesting a test article-related effect. For the males in this group, the urothelial epithelial hyperplasia was often secondary to inflammation (pyelitis or pyelonephritis) or pelvic mineralization, and correlated with calculi observed macroscopically. Furthermore, there was no dose-response relationship. For this reason, the hyperplasia was considered not to be a test substance-related effect in the males. The incidence and severity of the kidney lesions in the 20 and 200 ppm groups were similar to those in the control group and therefore, were considered not to be test substance-related. No test substance-related microscopic findings were observed in any other tissue examined in the 4000 ppm group animals.
No test substance-related microscopic lesions were observed in the P0 animals that died during the study.
Histopathological findings: neoplastic:: no effects observed
Reproductive function: oestrous cycle:: no effects observed
Description (incidence and severity):: The regularity and duration of estrous were not affected by treatment. Individual variation in the estrous cycle occurred sporadically in all study groups without toxicological relevance.
Reproductive function: sperm measures:: no effects observed
Description (incidence and severity):: Administration of the test substance at concentrations of 20, 200 and 4000 ppm had no apparent effects on P0 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate and sperm motility and morphology). Differences between the control and treated groups were slight, without statistical significance and of none toxicological relevance.
Reproductive performance:: no effects observed
Description (incidence and severity):: Reproductive performance of P0 animals was unaffected by test substance administration at dose levels of 20, 200 and 4000 ppm (Table 6 under "Any other information on results incl. tables"). Fertility indices were 79.3%, 86.7%, 86.7% and 86.2% for the P0 males and 79.3%, 86.7%, 86.7% and 86.7% for the P0 females in the control, 20, 200 and 4000 ppm groups, respectively. Mating indices were 100%, 93.3%, 100% and 100% for both the P0 males and females in the same respective dose groups. Males, which did not sire a litter numbered 6, 4, 4 and 4 in the control, 20, 200 and 4000 ppm groups, respectively. In the same respective dose groups, 6, 2, 4 and 4 females had evidence of mating but did not deliver; all of these females were non-gravid. 2 and 4 females in the control and 20 ppm groups, respectively, had no evidence of mating; with the exception of 2 females in the 20 ppm group, all of these females were gravid. The mean numbers of days between pairing and coitus in the treated groups were comparable to the control group values.
The mean lengths of gestation were comparable between the P0 treated groups and the control group. No differences were statistically significant. The mean gestation lengths in the 20, 200 and 4000 ppm groups were 22.0, 22.0 and 21.9 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and the reproductive historical control data of the laboratory. No signs of dystocia were noted.
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic toxicity
Effect level:: 200 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: actual test substance intake (P0 generation): males: 12.6 mg/kg bw/day, females: 18.3 mg/kg bw/day, combined sexes: 15.5 mg/kg bw/day
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic toxicity
Effect level:: 4 000 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; food efficiency; histopathology: non-neoplastic
Remarks on result:: other: actual test substance intake (P0 generation): males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day, combined sexes: 306.5 mg/kg bw/day
: Key result
Dose descriptor:: NOAEL
Remarks:: reproductive toxicity
Effect level:: 4 000 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: actual test substance intake (P0 generation): males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day, combined sexes: 306.5 mg/kg bw/day
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 4 000 ppm
System:: urinary
Organ:: kidney
Treatment related:: yes
Clinical signs:: no effects observed
Description (incidence and severity):: No treatment-related clinical signs of toxicity were noted at any concentration for F1 parental animals. Clinical findings noted in the treated groups, such as hair loss and scabbing on various body surfaces and swollen ears, were noted infrequently and/or at similar frequencies in the control group.
Mortality:: no mortality observed
Description (incidence):: All F1 parental animals survived to the scheduled necropsy.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: In the 4000 ppm group, mean body weights of F1 parental animals were statistically significantly lower (10 - 11%) than the control group values at the beginning of the pre-mating phase (Table 5 under "Any other information on results incl. tables"). In males, although the difference from the control group values gradually reduced to about 5% in Week 37, values were often statistically significantly different. Cumulative body weight gain in the males was reduced throughout the generation. Mean body weight of female animals was only reduced initially.
In detail, mean body weight gains of F1 males at 4000 ppm were lower than the control group values during Week 18 - 19 and 19 - 20 ; the differences were statistically significant (p <0.01). Throughout the remainder of the generation (Week 20 - 21 through 36 - 37), mean body weight gains in the 4000 ppm group F1 males were generally slightly lower than or comparable to the control group values. The majority of the differences were not statistically significant. Cumulative body weight gain from Week 18 onwards was reduced throughout the generation. Many of the differences were statistically significant (p <0.05 or p <0.01). Mean body weights in the 4000 ppm group males were lower than the control group values, and often statistically significant (p <0.05 or p <0.01), from Week 18 through Week 37. Initially, the values were about 11% below the control group values but the difference gradually reduced to about 5% by Week 37. In the 4000 ppm group F1 females, the mean body weight for Week 18 was reduced by about 10%, and statistically significant (p <0.05), when compared to the control group value. This decrease was attributed to the significantly reduced PND 21 mean body weight in these females (p <0.01). Mean body weight gains in the 4000 ppm group females were comparable to the control group values throughout the pre-breeding period, and mean body weights for these females were within 5% of the control group value by Week 20 and were within 3% of the control group value just prior to breeding (Week 28). Mean weekly body weights and body weight gains in the 20 and 200 ppm group F1 males and females were unaffected by test article administration. Several statistically significant differences from the control group values were observed. However, the changes were transient and/or did occur without a dose-response relationship. Therefore, the changes were attributed to biological variation.
During gestation, mean body weights and body weight gains were unaffected by test substance administration in the F1 parental females in the 20, 200 and 4000 ppm groups. The only statistically significant differences from the control group included an increased mean body weight gain in the 20 ppm group during Gestation Day (GD) 14 - 20 (and consequently during GD 0 - 20), a reduced mean body weight in the 4000 ppm group on GD 14 and an increased mean body weight in the 20 ppm group on GD 20 (p <0.05). However, the differences were slight, transient and/or did not occur in a dose-dependent manner. Therefore, they were attributed to biological variation.
During lactation, mean body weights and body weight changes were unaffected by test substance administration at all dose levels. The only statistically significant differences from the control group values were reduced mean body weights in the 4000 ppm group on lactation Days 4 and 7 (p <0.05). The differences were slight (5 - 6%) and were attributed to biological variation.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: At 4000 ppm, food consumption, evaluated as g/animal/day, of F1 parental males was reduced (11%) during Week 18 - 19 through Week 21 - 22 when compared to the control group values. The differences were statistically significant (p <0.05 or p <0.01). Throughout the remainder of the generation (Week 22 - 23 through Week 36 - 37), g/animal/day food consumption values in the 4000 ppm group males was comparable to that in the control group. On a g/kg bw/day basis, food consumption values in the 4000 ppm group F1 males were often increased and statistically significant (p <0.05 or p <0.01). These increases were attributed to the differences in body weights observed between the control and 4000 ppm group males. Food consumption, evaluated as g/animal/day and g/kg bw/day, was unaffected by test substance administration in the 20 and 200 ppm group males and females and the 4000 ppm group females. Values of treated animals (g/kg bw/day) were occasionally increased and statistically significant (p <0.05 or p <0.01) when compared to the control group values. However, the corresponding g/animal/day values were comparable to the control group values, and the increases were attributed to differences in body weights between the control and treated groups.
During gestation, food consumption, evaluated as g/animal/day and g/kg bw/day, was unaffected by test substance administration at all dose levels throughout the gestation period. Statistically significant differences from the control group were increased g/animal/day values in the 20 ppm group during GD 0 - 4 and GD 14 - 20 (p <0.05). Similar increases were not observed at the higher concentrations of 200 and 4000 ppm, and no dose-response relationship was observed. Therefore, this finding was attributed to biological variation.
During lactation, food consumption (g/animal/day and g/kg bw/day) in the F1 females was unaffected by treatment at all dose levels. No statistically significant differences from the control group were observed.
For details on test substance intake see Table 1 - 3 under "Any other information on results incl. tables".
Food efficiency:: no effects observed
Description (incidence and severity):: There were no treatment-related effects on food efficiency at all dose levels in both sexes, when comapred to control.
During gestation, as for food consumption, food efficiency was unaffected by test substance administration at all dose levels throughout the gestation period.
During lactation, in the F1 females food efficiency was unaffected by treatment at all dose levels. No statistically significant differences from the control group were observed.
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:: effects observed, non-treatment-related
Description (incidence and severity):: No test substance-related effects were observed on organ weight data (absolute or relative to final body weight) for the F1 males and females at any concentration.
Mean absolute kidney weight in the 4000 ppm group males was significantly (p <0.01) lower than the control group value. However, the corresponding relative weight was similar to the control group value, and the difference was attributed to differences in final body weights between the two groups. Mean absolute and/or relative thymus gland weights in the 200 and 4000 ppm group females were higher than the control group values; the differences were statistically significant (p <0.05). However, no dose-response relationship was apparent, and similar increases were not observed in the 200 and 4000 ppm group males. Therefore, the changes were attributed to biological variation. The mean absolute right epididymis weight in the 20 ppm group males was significantly (p <0.05) higher than the control group value. However, similar increases were not observed at the higher concentrations of 200 and 4000 ppm, and no dose-response relationship in correlation to the test substance-treatment was apparent. Mean relative testes, epididymides and spleen weights were significantly increased (p <0.05 or p <0.01) in the 4000 ppm group males and/or females when compared to the control group values. However, similar increases were not observed in the corresponding absolute weights, no test substance-related microscopic findings were observed in these tissues, and the changes were attributed to differences in final body weights between the control and 4000 ppm groups. Mean relative liver weights were significantly increased (p <0.01) in the 4000 ppm group males and females when compared to the control group values. However, the corresponding absolute liver weights in the 4000 ppm group were only slightly increased compared to the control group values and there were no microscopic findings correlated with the weight increases. Therefore, the increased liver weights were considered not to be an adverse effect of test substance administration.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: At scheduled necropsy of F1 parental animals, no treatment-related internal findings were observed at any concentration. Findings in the treated groups, such as dilated renal pelves, dark red areas on the lungs, cystic oviducts, small testes, dark red contents of the ileum and reddened/enlarged lymph nodes were noted infrequently (generally in isolated animals) and/or at similar frequencies in the control group.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: In the F1 parental generation, treatment-related changes in the kidney were similar to those observed in the P0 parental generation, but also occurred in the males (Table 4 under "Any other information on results incl. tables"). Both males and females in the 4000 ppm group showed increases in the incidences of medullary cysts and medullary degeneration/fibrosis. The increases were statistically significant (p <0.05). The severity of nephropathy was very slightly increased in these males and females. Additionally, the incidence of urothelial hyperplasia that was not secondary to pelvic mineralization or pyelitis/pyelonephritis was slightly increased in the 4000 ppm group males. The cysts, degeneration/fibrosis and urothelial hyperplasia were often observed in animals with moderate to severe nephropathy in both the males and females in this group (this trend was also observed for the 4000 ppm group P0 females). In females, inflammation of the medulla/papilla was observed only in the 4000 ppm group but in males it was observed in the 20 and 4000 ppm groups in a manner that was not dose-related. Papillary tubular hyperplasia was not observed in the F1 generation animals.
Generally, test substance-related lesions were consistently observed in the kidneys in the 4000 ppm group P0 females and F1 males and females. The most consistent change was the occurrence of medullary/papillary cysts and degeneration/fibrosis in the medulla. Other changes observed (urothelial hyperplasia, papillary tubular hyperplasia, inflammation of the papilla/medulla) did not occur consistently in a dose-related manner, or were not consistent within each sex and between generations. However, these changes occurred more frequently in kidneys that contained cysts or degeneration/fibrosis, and were considered to be probable effects of the test substance. Many of the changes in the kidney were suggestive of age-related nephropathy, which is a spontaneous lesion in rats of this age and strain. The increase in the incidence and/or severity of these findings in the 4000 ppm group suggests that the test substance exacerbated the spontaneous lesions. Although cysts are often observed as part of advanced nephropathy, the increase in medullary/papillary cysts in this study is noteworthy because of their number, size, location and somewhat unusual morphologic appearance. The incidence and/or severity of these kidney lesions in the 20 and 200 ppm groups were similar to those in the control group or were not dose-responsive and were not considered to be test substance-related. No test substance-related microscopic findings were observed in any other tissue examined in the 4000 ppm group animals.
Histopathological findings: neoplastic:: no effects observed
Reproductive function: oestrous cycle:: no effects observed
Description (incidence and severity):: The regularity and duration of estrous were not affected by treatment. Individual variation in the estrous cycle occurred sporadically in all study groups without toxicological relevance.
Reproductive function: sperm measures:: no effects observed
Description (incidence and severity):: Administration of the test substance at concentrations of 20, 200 and 4000 ppm had no apparent effects on F1 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate and sperm motility and morphology). Differences between the control and treated groups were slight, without statistical significance and of none toxicological relevance.
Reproductive performance:: no effects observed
Description (incidence and severity):: Reproductive performance of F1 parental animals was unaffected by test substance administration at dose levels of 20, 200 and 4000 ppm (Table 6 under "Any other information on results incl. tables"). Fertility indices were 90.0%, 93.3%, 96.7% and 86.7% for both the F1 males and females in the control, 20, 200 and 4000 ppm groups, respectively. Mating indices were 100%, 96.7%, 96.7% and 96.7% for both males and females in the same respective dose groups. Males, which did not sire a litter numbered 3, 2, 1 and 4 in the control, 20, 200 and 4000 ppm groups, respectively. Females, which had evidence of mating but did not deliver numbered 4, 1, 0 and 3 in the same respective dose groups; with the exception of one control group female, all of these animals were non-gravid. 1, 1 and 3 females in the 20, 200 and 4000 ppm groups, respectively, had no evidence of mating; 2 of the 4000 ppm group females delivered litters, and the remaining 3 females were non-gravid. The mean numbers of days between pairing and coitus in the treated groups were similar to the control group values; differences were slight and were not statistically significant.
The mean lengths of gestation were comparable between the F1 treated groups and the control group. No differences were statistically significant. The mean gestation lengths in the 20, 200 and 4000 ppm groups were 21.9, 21.9 and 21.8 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and the reproductive historical control data of the laboratory. No signs of dystocia were noted.
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic toxicity
Effect level:: 200 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: actual test substance intake (F1 generation): males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day, combined sexes: 17.3 mg/kg bw/day
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic toxicity
Effect level:: 4 000 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; food consumption and compound intake; histopathology: non-neoplastic
Remarks on result:: other: actual test substance intake (F1 generation): males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day, combined sexes: 353.3 mg/kg bw/day
: Key result
Dose descriptor:: NOAEL
Remarks:: reproductive toxicity
Effect level:: 4 000 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: actual test substance intake (F1 generation): males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day, combined sexes: 353.3 mg/kg bw/day
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 4 000 ppm
System:: urinary
Organ:: kidney
Treatment related:: yes
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Pups, which were found dead or euthanized in extremis numbered 10, 7, 27 and 26 in the control, 20, 200 and 4000 ppm groups, respectively. In the same respective dose groups, 4, 3, 11 and 4 pups were missing and presumed to have been cannibalized. Although the numbers of pups that were found dead, euthanized in extremis and/or missing in the 200 and 4000 ppm groups were higher than in the control group, no dose-response relationship was observed. The increases were due to 1 female in each group with total litter loss (4000 ppm group) or loss of nearly the total litter (15 of 19 pups in the 200 ppm group). Therefore, no relationship to test substance administration was apparent. The general physical condition of the F1 pups during lactation was similar in all groups, including the control group.
Mortality / viability:: no mortality observed
Description (incidence and severity):: The mean live litter size, number of pups born, and percentage of males per litter at birth were unaffected by parental exposure to the test substance at all dose levels tested (Table 6 under "Any other information on results incl. tables"). No statistically significant differences from the control group values were observed. Pup survival was not affected by parental exposure in the 20, 200 and 4000 ppm groups from birth to PND 0, PND 0 - 1, PND 1 - 4, PND 4 - 7, PND 7 - 14, PND 14 - 21, birth to PND 4, and PND 4 - 21. No statistically significant differences from the control group values were observed. Pup viability in the 200 and 4000 ppm groups was slightly reduced from birth to PND 0 when compared to the control group. This was mainly attributed to one female in each group, which lost almost the whole litter or the whole litter, respectively. However, viability from birth to PND 4 pre-selection in the 4000 ppm dose group was greater than that in the 200 ppm dose group (92.3% compared to 87.9%, respectively). Since there was no evidence of a dose-response relationship or statistical significance, this decrease was considered incidental and not to be a test substance-related effect.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: In the 4000 ppm dose group, F1 pup body weight was significantly lower (11%) than the control group value on PND 21 although there was no treatment-related effect at birth (Table 6 under "Any other information on results incl. tables"). In detail, mean F1 pup body weights in the 4000 ppm group were comparable to the control group values on PND 1, 4 (before and after selection), 7 and 14. On PND 21, mean pup body weight in this group was 11% lower than the control group value. The difference was statistically significant at p <0.01. Mean F1 pup body weights were unaffected by parental treatment at concentrations of 20 and 200 ppm. No statistically significant differences from the control group were observed.
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Sexual maturation:: effects observed, treatment-related
Description (incidence and severity):: In the 4000 ppm group, there was a slight delay in the achievement of balanopreputial separation, which was due to a significant reduction in body weight/body weight gain in these prepubertal male offspring (Table 6 under "Any other information on results incl. tables"). However, this is a well-established correlation demonstrating a secondary sign of systemic toxicity, not indicating inherent reproductive toxicity potential of the test substance.
In detail, a delay in the occurrence of balanopreputial separation was evident in the 4000 ppm group. The mean days of balanopreputial separation observed for males in the control, 20, 200 and 4000 ppm groups were PND 43.3, 44.1, 44.3 and 46.2, respectively. The difference between the control and 4000 ppm group values was statistically significant (p <0.01). The mean body weights on the day of acquisition of balanopreputial separation in the 20, 200 and 4000 ppm groups were similar to the control group value. However, weekly body weights in the 4000 ppm group during the period of achievement of balanopreputial separation (Week 19 - 21) were reduced by 9 - 11% and the reduction reached statistical significance (p <0.05 or p <0.01) compared to the control group value.
In the 4000 ppm group, there was a slight delay in the achievement of vaginal patency due to a significant reduction (10 - 11%) in body weight of these peripubertal F1 female offspring (Table 6 under "Any other information on results incl. tables"). However, this is a well-established correlation demonstrating a secondary sign of systemic toxicity, not indicating inherent reproductive toxicity potential of the test substance.
In more detail, a delay in the occurrence of vaginal patency was observed in the 4000 ppm group. The mean days of vaginal patency noted for females in the control, 20, 200 and 4000 ppm groups were 33.2, 33.2, 33.7 and 34.8, respectively. The difference between the control and 4000 ppm group values was statistically significant (p <0.01). The mean body weights on the day of acquisition of vaginal patency in the 20, 200 and 4000 ppm groups were similar to the control group value. However, weekly body weights in the 4000 ppm group during the period of achievement of vaginal patency (PND 21 - Week 19) were reduced by 7 - 11% compared to the control group value. The Week 18 value was statistically significantly different (p <0.05).
The day of acquisition of both vaginal patency and balanopreputial separation has been shown to be delayed by reduced body weight. Because both of these parameters were delayed in the 4000 ppm dose group only, the delay was attributed to the clearly reduced post-weaning body weights rather than being an endocrine-mediated event.
Anogenital distance (AGD):: not examined
Nipple retention in male pups:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, non-treatment-related
Description (incidence and severity):: Organ weights (absolute and relative to final body weight) in the selected F1 weanlings were unaffected by treatment at all concentrations. Mean absolute brain weights in the 4000 ppm group males and females were lower than the control group values; the difference in the females was statistically significant (p <0.01). However, mean relative brain weights in the 4000 ppm group males and females were higher than the control group values; the differences were statistically significant (p <0.05 or p <0.01). Therefore, the differences in absolute weights were attributed to differences in body weights between the control and 4000 ppm groups. The mean absolute spleen weight in the 4000 ppm group females was reduced, and statistically significant (p <0.05), when compared to the control group value. However, mean relative spleen weight in these females was similar to the control group value, and the difference was attributed to differences in body weights between the groups.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: No internal findings that could be attributed to parental treatment with the test substance were noted at the necropsies of pups that were found dead or were euthanized in extremis. Malformations were noted in 1, 1 and 2 pups in the control, 200 and 4000 ppm groups, respectively, and included the following. One control group pup had an enlarged right kidney and agenesis of the left kidney and ureter. In the 200 ppm group, one pup had anophthalmia and exencephaly without open eyelids. At a concentration of 4000 ppm, one pup had vertebral agenesis with a filamentous tail, and another had microphthalmia. Aside from the presence or absence of milk in the stomach, the only other internal findings in pups that died or were euthanized in extremis consisted of a submeningeal hemorrhage for 1 of the control group pups and hemorrhagic bladders for 3 pups in the 200 ppm group.
At the necropsy of surplus F1 pups on PND 21, no treatment-related internal findings were noted at any concentration. Dilated renal pelves were noted for 6, 5, 2 and 4 pups in the control, 20, 200 and 4000 ppm groups, respectively. Of these, 2 pups in the 20 ppm group and 1 pup in the 200 ppm group also had distended ureters and 1 pup in the 4000 ppm group also had a cystic kidney. 1 pup in each of the 20 and 4000 ppm groups had small testes. Malformations were noted for 1 pup in each of the control and 20 ppm groups; 1 control group pup was hydrocephalic (the anomaly consisted of increased cavitation of both lateral ventricles), and 1 pup in the 20 ppm group had agenesis of the left testis and epididymis.
At the necropsy of selected F1 weanlings on PND 21, no treatment-related internal findings were observed at any concentration. 2, 2 and 1 pups in the control, 20 and 200 ppm groups, respectively, had dilated renal pelves and/or distended ureters. One pup in the 20 ppm group had a subcutaneous hemorrhage in the scapular region. No other remarkable internal findings were noted in the selected F1 weanlings.
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: Key result
Dose descriptor:: NOAEL
Remarks:: reproductive toxicity
Generation:: F1
Effect level:: 200 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: delayed sexual maturation due to reduced body weight (systemic toxicity) at 4000 ppm
Remarks on result:: other: actual test substance intake of F1 generation: males: 14.5 mg/kg bw/day, females: 20.0 mg/kg bw/day, combined sexes: 17.3 mg/kg bw/day
: Key result
Critical effects observed:: no
Clinical signs:: no effects observed
Description (incidence and severity):: There were no treatment-related findings. Pups which were found dead numbered 7, 3, 9 and 17 in the control, 20, 200 and 4000 ppm groups, respectively. In the same respective dose groups, 3, 1, 6 and 1 pups were missing and presumed to have been cannibalized. The general physical condition of the F2 pups during lactation was similar in all groups, including the control group.
Mortality / viability:: no mortality observed
Description (incidence and severity):: The mean live litter size, number of pups born, and percentage of males per litter at birth were unaffected by the F1 parental exposure in the 20, 200 and 4000 ppm groups (Table 6 under "Any other information on results incl. tables"). The only statistically significant differences from the control group were an increased mean live litter size and an increased number of pups born in the 20 ppm group (p <0.01). However, similar increases were not observed at the higher dose levels of 200 and 4000 ppm, and no dose-response relationship to the test substance was apparent. Pup survival was not affected by parental exposure in the 20, 200 and 4000 ppm groups from birth to PND 0, PND 0 - 1, PND 1 - 4, PND 4 - 7, PND 7 - 14, PND 14 - 21, birth to PND 4, and PND 4 - 21. No statistically significant differences from the control group values were observed.
Body weight and weight changes:: no effects observed
Description (incidence and severity):: Mean F2 pup body weights were unaffected by parental treatment at concentrations of 20, 200 and 4000 ppm (Table 6 under "Any other information on results incl. tables"). No statistically significant differences from the control group were observed.
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):: No adverse effects on mean organ weights (absolute and relative to final body weights) were observed in the F2 pups at any concentration. No statistically significant differences from the control group were observed.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: In addition to the absence of milk in the stomach, remarkable necropsy findings for pups found dead during the postnatal period included the following. One pup in the 4000 ppm group had a malformation (microphthalmia). In the same group, one pup of another litter had a major blood vessel variation (a retroesophageal right subclavian artery). At the necropsy of surplus F2 pups on PND 21, no treatment-related internal findings were noted at any concentration. Dilated renal pelves were noted in 3, 8, 5 and 1 pups in the control, 20, 200 and 4000 ppm groups, respectively. One of the 20 ppm group pups also had a distended ureter. Malformations were noted for two pups in one litter in the 200 ppm group. Both of these pups were hydrocephalic (the anomaly consisted of increased cavitation of both lateral ventricles and the third ventricle). At the necropsy of the selected F2 weanlings on PND 21, no treatment-related internal findings were noted. Dilated renal pelves were noted in 1 pup in each of the 20 and 200 ppm groups. No other internal findings were observed.
Histopathological findings:: not examined
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: Key result
Dose descriptor:: NOAEL
Remarks:: reproductive toxicity
Generation:: F2
Effect level:: 4 000 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: highest dose tested; actual test substance intake: P0 generation: males: 249.8 mg/kg bw/day, females: 363.2 mg/kg bw/day; F1 generation: males: 300.6 mg/kg bw/day, females: 406.0 mg/kg bw/day
: Key result
Critical effects observed:: no
: Key result
Reproductive effects observed:: yes
Lowest effective dose / conc.:: 4 000 ppm
Treatment related:: yes
Relation to other toxic effects:: reproductive effects as a secondary non-specific consequence of other toxic effects
Conclusions:: Dietary, sub-chronic administration of the test substance at a dose level of 4000 ppm (highest dose tested) to rats for 2 successive generations induced systemic toxicity (as evidenced by reductions in body weight gain and/or body weight and food intake/efficiency and an increase in microscopic kidney lesions) in the P0 and F1 generation parental males and females. Neonatal toxicity was observed as reduced F1 pup body weights in the 4000 ppm group. Delays in the achievement of vaginal patency and balanopreputial separation were observed in the F1 animals in the 4000 ppm group as secondary signs of systemic toxicity due to reduced body weight. The study provided no evidence of a reproductive toxicity potential of the test substance since the observed effects were in fact secondary to systemic toxicity.
The No Observed Adverse Effect Level (NOAEL) for both reproductive and systemic toxicity was 200 ppm, equivalent to a mean actual test substance intake of 12.6, 18.3 and 15.5 mg/kg bw/day in P0 males, females and combined sexes, respectively, and to 14.5, 20.0 and 17.3 mg/kg bw/day in F1 males, females and combined sexes, respectively. The Lowest Observed Adverse Effect Level (LOAEL) for both reproductive and systemic toxicity was 4000 ppm, equivalent to a mean actual test substance intake of about 306.5 mg/kg bw/day in P0 males and females, and to 353.3 mg/kg bw/day in F1 males and females combined. In the F2 generation, where no effects were evident within the observation period, the NOAEL for systemic and reproductive toxicity corresponded to the highest dose tested, i.e. 4000 ppm.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 15.5 mg/kg bw/day
Study duration:: subchronic
Experimental exposure time per week (hours/week):: 168
Species:: rat
Quality of whole database:: The available study was conducted according to OECD TG 416 and in compliance with GLP. As no major deviations have been noted it was given the reliability score 1.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

There is a Two-Generation Reproduction Toxicity Study with the test substance available, which was conducted in rats according to OECD TG 416 and GLP (M-185452-01-1, 1999).

In this study groups of 30 parental animals received doses of 20, 200 and 4000 ppm during the premating period, gestation and lactation corresponding to an overall mean test substance intake of 1.6 16.4 and 329.9 mg/kg bw/day. P0 and F1 animals were treated for at least 70 days prior to pairing, throughout pairing (when they were cohabitated one male with one female from the same treatment group),gestation and lactation until their pups had been weaned. Treatment commenced when the P0 animals were 6 weeks of age and, for F1 animals, at weaning on postnatal day 22 (PND 22) when at least one male and one female from each of the P0 generation litters were selected to this generation. Mated females were allowed to litter. Pups were individually identified and litter size culled to 8 pups as appropriate on PND 4. At weaning, surplus F1 and all F2 pups were euthanized and necropsied on PND 21 and an additional one male and one female per litter were selected for detailed necropsy and organ weights. Parental animals were subjected to a detailed necropsy after their pups had been weaned; selected organs were weighed. Spermatogenic endpoints (sperm motility, morphology and numbers) were recorded for all P0 and F1 males. Histopathology was conducted on the reproductive and target organs from 10 males and 10 females from the control and highest dose levels. Histopathology was also conducted on the kidneys from all P0 and F1 animals. All P0 and F1 animals were observed twice daily for clinical signs and behaviour. Body weights and food consumption were recorded weekly prior to pairing. Females were weighed on Days 0, 4, 7, 10, 14 and 20 of gestation and on Days 1, 4,7,14 and 21 of lactation. Male body weights were recorded weekly. Food consumption of females and their litters was measured during lactation. Vaginal smears were taken for 21 days prior to pairing, during pairing until mating occurred and at necropsy. Litters were examined twice daily for pup mortality, clinical signs and behaviour. Pups were weighed individually and sexed on PND 0/1, 4, 7, 14 and 21. Balanopreputial separation and vaginal opening were monitored in the F1 generation animals.

The values for overall achieved mean daily intake of the test substance (mg/kg bw/day) were:

	Compound intake [mg/kg bw/day]
Dietary level [ppm]	P0		F1		Combined generations
	♂	♀	♂	♀	♂	♀
20	1.2	1.8	1.5	2.0	1.35	1.9
200	12.6	18.3	14.5	20.0	13.55	19.15
4000	249.8	363.2	300.6	406.0	275.2	384.6

Dietary administration of 4000 ppm of the test substance to rats for two successive generations induced parental toxicity (as evidence by reductions in body weight gain and/or body weight and food intake and an increase in microscopic kidney lesions) in the P0 and F1 generation males and females. Neonatal toxicity was observed as reduced F1 pup body weight at 4000 ppm. Delays in the achievement of vaginal patency and balanopreputial separation were observed in the F1 animals in the 4000 ppm group as secondary signs of systemic toxicity due to reduced body weight. The study provided no evidence of a reproductive toxicity potential of the test substance since the observed effects were in fact secondary to systemic toxicity.

The No Observed Adverse Effect Level (NOAEL) for both reproductive and systemic toxicity was 200 ppm, equivalent to a mean actual test substance intake of 12.6, 18.3 and 15.5 mg/kg bw/day in P0 males, females and combined sexes, respectively, and to 14.5, 20.0 and 17.3 mg/kg bw/day in F1 males, females and combined sexes, respectively. The Lowest Observed Adverse Effect Level (LOAEL) for both reproductive and systemic toxicity was 4000 ppm, equivalent to a mean actual test substance intake of about 306.5 mg/kg bw/day in P0 males and females, and to 353.3 mg/kg bw/day in F1 males and females combined. In the F2 generation, where no effects were evident within the observation period, the NOAEL for systemic and reproductive toxicity corresponded to the highest dose tested, i.e. 4000 ppm.

Effects on developmental toxicity

Description of key information

OECD TG 414, oral, gavage, rat:
NOAEL maternal, systemic toxicity: 120 mg/kg bw/day
LOAEL maternal, systemic toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 120 mg/kg bw/day
LOAEL developmental toxicity: 1000 mg/kg bw/day

OECD TG 414, oral, gavage, rabbit:
NOAEL maternal, systemic toxicity: 50 mg/kg bw/day
LOAEL maternal, systemic toxicity: 500 mg/kg bw/day
NOAEL developmental toxicity: 500 mg/kg bw/day

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 23 Oct - 09 Nov 1995
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Remarks:: Several parameters according to the current version of OECD TG 414 (2018) are missing.
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: Current version adopted in 2018
Deviations:: yes
Remarks:: Animals were dosed from Gestation Day (GD) 7 - 16 (not GD 5 - 20). Measurement of thyroid weight and histopathological examination of thyroid were not done. T4, T3 and TSH blood levels were not measured. Anogenital distance was not measured.
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: Guideline in place during study conduct: adopted in 1981
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 1987
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 1984
Qualifier:: according to guideline
Guideline:: other: JMAFF (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 1985
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: Hoe: WISKf(SPF71)
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Hoechst AG, Kastengrund, Germany; SPF breeding colony
- Age at study initiation: approx. 8 - 10 weeks
- Mean body weight at Day 1: 221.5 g
- Housing: individually, in Makrolon cages (type III) on soft wood granulate
- Diet: Ssniff R-Z (V1324), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 68
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Oct 1995 To: 09 Nov 1995
Route of administration:: oral: gavage
Vehicle:: other: 1% (w/v) aqueous methylcellulose
Details on exposure:: PREPARATION OF DOSING SOLUTIONS
The test substance dosing formulation was prepared daily, immediately before dosing.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: For each concentration, samples were taken towards the start, middle and end of the dosing period (Day 7 - 16). Only the samples from the start were analysed. The archieved concentration, stability and the homogeneous distribution of the test substance in the vehicle over 4 h were acceptable.
Details on mating procedure:: - Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: detection of sperm in vaginal smears. The day of sperm detection was defined as Day 1 of gestation, and the day of mating was defined as Day 0 of pregnancy. Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.
Duration of treatment / exposure:: Day 7 - 16 of pregnancy
Frequency of treatment:: Daily
Duration of test:: 21 days
Dose / conc.:: 15 mg/kg bw/day
Dose / conc.:: 120 mg/kg bw/day
Dose / conc.:: 1 000 mg/kg bw/day
No. of animals per sex per dose:: 23 females
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: In a dose-range finding study groups of mated female Wistar rats received the test substance orally at the dose levels of 500 mg/kg bw/day (2 females) or 1000 mg/kg bw/day (4 females) from Day 7 to 16 of pregnancy and were killed on Day 21. One animal at 1000 mg/kg bw/day was found dead on Day 17 (after showing clinical signs of toxicity on Day 16). No clinical signs were observed in the other animals of the 1000 mg/kg bw/day dose group or in the animals treated with 500 mg/kg bw/day. Body weight change, food consumption and litter data did not reveal any compound-related effect. Based on the results of this study and on request of the sponsor, the dose levels of 0, 15, 120 and 1000 mg/kg bw/day were selected. 1000 mg/kg bw/day was included because it is the recommended limit dose according to guideline. The lowest dose level of 15 mg/kg bw /day was selected based on a 90-day oral repeated dose toxicity study with rats, where the NOEL was set at 15.3 mg/kg bw/day. The mid dose level, 120 mg/kg bw/day, was approximately the log mid-point between the high and low dose level.
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Several times daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 1, 4, 7, 10, 14, 17, 19 and 21 of pregnancy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Between Days 1 - 7, 7 - 10, 10 - 14, 14 - 19 and 19 - 21 of pregnancy.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes, externally and internally
- Sacrifice on Gestation Day 21
- Organs examined: thoracic and abdominal contents, Uterus
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes
- Other: The fetuses, the placentae and conceptuses undergoing resorption were removed from the uterus, weighed or measured and examined for gross external abnormalities.
Fetal examinations:: - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No
- Other: crown-rump length was recorded
Statistics:: All data were recorded on-line and compiled by a data processing system (ARTEMIS). The statistical evaluation is based on the assumption of a monotone dose-response relationship.
Statistical comparisons of the low dose groups with the simultaneous control group were only carried out if significant effects were detectable in the high dose group. Two-sided questions were generally tested as follows: a two-sided comparison with the high dose group was followed by a one-sided test for the low-dose group. In case of the caesarean section data of the multivariate statistics were first of all calculated and used in selecting relevant dose groups. For the individual parameters, sequential comparisons with the high dose group and sequential tests at the 5% level for the low dose were then conducted.

For the Wilcoxon test the exact distribution of the meaned ranks was calculated.

Food consumption: mean consumption/100 g bw was always calculated between two successive measurement times and evaluated by the rank sum test after Wilcoxon.
Body weight gain: the change in weight was determined in comparison to the initial weight. The univariate evaluation was carried out using t-tests.

The caesarean section data of the foetuses were used to calculate litter mean values. Multivariate evaluation was carried out using the test statistics of Wilks. In the univariate analysis,t-tests were used.

The number of corpora lutea, implantation sites and live foetuses, and quotas of dead embryonic primordia undergoing resorption in the animals were likewise analysed using one-sided Wilcoxon tests.

The findings obtained at autopsy, during visceral examination and skeletal examination of the foetuses were evaluated separately for the foetuses and for the litters by exact Fisher test at significance levels of 5% and 1% and compared with actual and previous control group data.
Indices:: % of pre- and post-implantation loss, % of implantation
Historical control data:: Historical data used for comparison are not given in the report.
Clinical signs:: no effects observed
Description (incidence and severity):: No clinical signs were observed in any of the animals.
Mortality:: mortality observed, treatment-related
Description (incidence):: One high dose group animal died on Day 21 of pregnancy. The mortality was considered to be treatment-related since 1000 mg/kg bw/day was about half of the acute oral LD50 value for rats and one mortality occurred on Day 17 in the dose range-finding study.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 1000 mg/kg bw/day, body weight gain was slightly but statistically significantly reduced from Day 10 until the end of the study (Table 1 under "Any other information on results incl. tables"). Body weight changes before beginning of treatment (Days 4 and 7) were not considered to be treatment-related.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: At 1000 mg/kg bw/day, food consumption was slightly but statistically significantly decreased between Days 7 - 19 of pregnancy (Table 2 under "Any other information on results incl. tables"). Decreases in food consumption before beginning of treatment (Days 1 and 7) were not considered to be treatment-related.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: Gravid uterus weights were comparable in all groups.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: At 1000 mg/kg bw/day one female exhibited whitish fluid in the renal pelvis at necropsy. The animal found dead on Day 21 showed autolysis. The uterus contained 12 well-developed foetuses.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: not examined
Histopathological findings: neoplastic:: not examined
Number of abortions:: no effects observed
Pre- and post-implantation loss:: effects observed, treatment-related
Description (incidence and severity):: No effects on relative pre-implantation loss values were observed (18.46, 13.37, 8.83, and 18.47% with ascending dose, respectively).
In the 1000 mg/kg bw/day dose group (high dose, inducing mortality of dams), the relative post-implantation loss values were increased (5.38, 4.69, 6.76, and 12.00% with ascending dose, respectively). However, as the high dose exceeds the maximum tolerable dose (MTD), this increase does not indicate an developmental toxicity potential of the test substance.
See also Table 3 under "Any other information on results incl. tables".
Total litter losses by resorption:: no effects observed
Early or late resorptions:: effects observed, treatment-related
Description (incidence and severity):: Number of early and late conceptuses undergoing resorption based on per cent of implantations ('early intrauterine deaths') was slightly but statistically significantly increased in the animals dosed at 1000 mg/kg bw/day (high dose, inducing mortality of dams) (Table 3 under "Any other information on results incl. tables").
Dead fetuses:: no effects observed
Description (incidence and severity):: Because the number of early and late conceptuses undergoing resorption based on per cent of implantations ('early intrauterine deaths') was slightly but statistically significantly increased in the animals dosed at 1000 mg/kg bw/day, the number of live foetuses based on per cent of implantations was slightly, but statistically significantly decreased (Table 3 under "Any other information on results incl. tables"). Incidence of dead foetuses ('late intrauterine deaths') was not increased.
Changes in pregnancy duration:: not examined
Changes in number of pregnant:: no effects observed
Description (incidence and severity):: With the exception of 1 female of the control group and 4 females of the 120 mg/kg bw/day mid dose group, respectively, all animals became pregnant.
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic toxicity
Effect level:: 120 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: no toxic effects observed
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic toxicity
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: maternal toxicity
: Key result
Dose descriptor:: NOAEL
Remarks:: developmental toxicity
Effect level:: 120 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: no toxic effects observed
: Key result
Dose descriptor:: LOAEL
Remarks:: developmental toxicity
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: developmental toxicity
Remarks on result:: other: Effects observed at high dose level exceeding the MTD.
: Key result
Abnormalities:: no effects observed
Fetal body weight changes:: no effects observed
Reduction in number of live offspring:: effects observed, treatment-related
Description (incidence and severity):: The number of early and late conceptuses undergoing resorption, based on per cent of implantations ('early intrauterine deaths'), was slightly but statistically significantly increased in the animals dosed at 1000 mg/kg bw/day. In direct consequence, the number of live foetuses based on per cent of implantations was slightly, but statistically significantly decreased (Table 3 under "Any other information on results incl. tables"). Incidence of dead foetuses ('late intrauterine deaths') was not increased.
One dead foetus occurred in the control group, and two dead foetuses were observed in the intermediate dose group.
Changes in sex ratio:: no effects observed
Changes in litter size and weights:: no effects observed
Changes in postnatal survival:: not examined
External malformations:: no effects observed
Description (incidence and severity):: There were no test substance-related effects. Situs inversus viscerum occurred in 1 foetus of the high dose group. In the mid dose group, 1 foetus showed diaphragmatic hernia and undescended testes (retentio testis). Statistical evaluation did not reveal differences between the groups, and the incidences were within the historical range of the rat strain used.
External examination of dead foetuses did not reveal any abnormalities.
Skeletal malformations:: no effects observed
Description (incidence and severity):: In all skeletal examinations, no test substance-related effects were observed, no statistical differences of the findings between the groups combined with dose-dependency were found, and all incidences were within the historical control data range of the strain used.
No test substance-induced variations and retardations were observed.
Visceral malformations:: no effects observed
Description (incidence and severity):: In all visceral examinations, no test substance-related effects were observed, no statistical differences of the findings between the groups combined with dose-dependency were found, and all incidences were within the historical control data range of the strain used.
No test substance-induced variations and retardations were observed.
Other effects:: no effects observed
Description (incidence and severity):: Crown-rump lengths and placental weights remained unaffected by test substance administration.
: Key result
Dose descriptor:: NOAEL
Remarks:: developmental toxicity
Effect level:: 120 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no toxic effects observed
: Key result
Dose descriptor:: LOAEL
Remarks:: developmental toxicity
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: reduction in number of live offspring
Remarks on result:: other: Effects observed at high dose level exceeding the MTD.
: Key result
Abnormalities:: no effects observed
: Key result
Developmental effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
Treatment related:: yes
Relation to maternal toxicity:: developmental effects as a secondary non-specific consequence of maternal toxicity effects
Conclusions:: In conclusion, daily administration of the test substance to rats at the regulatory limit dose level of 1000 mg/kg bw/day from Day 7 - 16 of pregnancy caused maternal, systemic toxicity and exceeded the maximum tolerable dose (as evidenced by mortality, decreased body weight gain and decreased food consumption) and a slightly increased incidence of embryonic deaths. However, there was no evidence of any test substance-induced effect on foetal morphology and no teratogenic effect. The effects reported were secondary to systemic toxicity and are not indicative for a developmental toxicity and teratogenic potential of the test substance.
Neither maternal nor embryo-foetal toxicity were observed after repeated administration of 120 mg/kg bw/day. The 'No Observed (Adverse) Effect Level' (NO(A)EL) for maternal toxicity and embryofetal effects was 120 mg/kg bw/day.

Reference 2

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 18 May - 02 Jun 1997
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study with acceptable restrictions
Remarks:: Several parameters according to the current version of OECD TG 414 (2018) are missing.
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: Current version adopted in 2018
Deviations:: yes
Remarks:: Animals were dosed from Gestation Day 6 - 18 only. Only 15 animals per group were used. Measurement of thyroid weight and histopathological examination of thyroid were not done. Anogenital distance was not measured.
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: Guideline in place during study conduct: adopted in 1981
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 1987
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 1984
Qualifier:: according to guideline
Guideline:: other: JMAFF (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 1985
GLP compliance:: yes
Limit test:: no
Species:: rabbit
Strain:: Himalayan
Remarks:: Chbb:HM(SPF) Kleinrusse
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach, Germany
- Age at study initiation: approx. 5 - 10 months
- Data on body weight not given in the study report.
- Housing: Individually housed in metal grid cages (except mating)
- Diet: commercial diet for laboratory rabbits, Ssniff K-H (V2333), ad libitum; additionally 40 - 50 g hay daily
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 25 - 75
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 May 1997 To: 02 Jun 1997
Route of administration:: oral: gavage
Vehicle:: other: 1% (w/v) aqueous methylcellulose
Details on exposure:: PREPARATION OF DOSING SOLUTIONS
Each dosing formulation at each concentration was prepared daily from a separate weighed quantity of the test substance. Formulations were stirred continuously in the animal room during dosing.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: For each concentrations, samples were taken towards the start, middle and end of the dosing period (Day 6 - 18). The archieved concentration, stability and the homogeneous distribution of the test substance in the vehicle were confirmed as acceptable in the range of 80 - 110%.
Details on mating procedure:: - Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: None; day of mating was considered as Day 0 of pregnancy. Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.
Duration of treatment / exposure:: Day 6 - 18 of pregnancy
Frequency of treatment:: Daily
Duration of test:: 29 days
Dose / conc.:: 5 mg/kg bw/day
Dose / conc.:: 50 mg/kg bw/day
Dose / conc.:: 500 mg/kg bw/day
No. of animals per sex per dose:: 15 females
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: Dose levels were based on the results of a dose-range finding study in 4 mated Himalayan rabbits, with dose levels of 250 or 500 mg/kg bw/day, respectively, from Day 6 - 18 of pregnancy (Day 0: day of mating). No mortalities occured. No compound related effects were observed at caesarian section. At 500 mg/kg bw/day, soft faeces and reduced defeacation, reduction of food consumption and body weight gain and at 250 mg/kg bw/day, no clinical signs were observed. Based on the results of this dose-range finding study the following doses were selected: 5 mg/kg bw/day - This dose level was chosen to provide consistency with the lowest no effect level in the rodent 90-day studies. 50 mg/kg bw/day - This provides a 10-fold interval between each of the other dose levels. 500 mg/kg bw/day - This caused some clinical signs, an initial reduction in body weight gain and an initial marked reduction in food consumption.
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Several times daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 0, 3, 6, 8, 11, 13, 16, 19, 23, 26 and 29 of pregnancy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Between Days 0 - 3, 3 - 6, 6 - 8, 8 - 10, 10 - 13, 13 - 16, 16 - 19, 19 - 23, 23 - 26 and 26 - 29 of pregnancy.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Gestation Day 29
- Organs examined: All animals were examined externally and internally (thoracic and abdominal) for macroscopically visible changes, with emphasis on the uterus.
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The foetuses, the placentae and conceptuses undergoing resorption were removed from the uterus, weighed or measured and examined for gross external abnormalities.
Fetal examinations:: - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No
- Other: In order to determine the viability, live foetuses were incubated for 24 h at a temperature of 32 °C and a atmospheric humidity of 60%. A record was kept of the foetuses which died during this time. Then the surviving foetuses were sacrificed and the crown-rump length recorded. All foetuses were sexed and checked for anomalies of the internal organs. Eyes, brain, heart and kidneys were cross-sectioned and examined for anomalies.
Statistics:: All data were recorded on-line and compiled by a data processing system (ARTEMIS). The statistical evaluation is based on the assumption of a monotone dose-response relationship.
Statistical comparisons of the low dose groups with the simultaneous control group were only carried out if significant effects were detectable in the high dose group. Two-sided questions were generally tested as follows: a two-sided comparison with the high dose group was followed by a one-sided test for the low-dose group. In case of the caesarean section data of the multivariate statistics were first of all calculated and used in selecting relevant dose groups. For the individual parameters, sequential comparisons with the high dose group and sequential tests at the 5% level for the low dose were then conducted.

For the Wilcoxon test the exact distribution of the meaned ranks was calculated.

Food consumption: mean consumption/100 g bw was always calculated between two successive measurement times and evaluated by the rank sum test after Wilcoxon.
Body weight gain: the change in weight was determined in comparison to the initial weight. The univariate evaluation was carried out using t-tests.

The caesarean section data of the foetuses were used to calculate litter mean values. Multivariate evaluation was carried out using the test statistics of Wilks. In the univariate analysis, t-tests were used.

The number of corpora lutea, implantation sites and live foetuses, and quotas of dead embryonic primordia undergoing resorption in the animals and the 24 h survival rate of foetuses were likewise analysed using one-sided Wilcoxon tests.

The findings obtained at autopsy, during visceral examination and skeletal examination of the foetuses were evaluated separately for the foetuses and for the litters by exact Fisher test at significance levels of 5% and 1% and compared with actual and previous control group data.
Indices:: % of pre- and post-implantation loss, % of implantation
Historical control data:: Historical control data were not included in the study report.
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: At 500 mg/kg bw/day one animal died on Day 10 of pregnancy. This animal showed reduced defecation, reduced hay intake, narrowed palpebral fissures, piloerection, reddish coloured urine and decreased activity on Day 8 and/or 9 of pregnancy. Another animal of this group exhibited reddish coloured urine on Day 20 of pregnancy and showed only two empty implantation sites at caesarean section.
At 50 mg/kg bw/day one animal showed reddish coloured urine on Day 7 and 8 of pregnancy.
No clinical signs were observed in the animals of the other study groups.
Mortality:: mortality observed, treatment-related
Description (incidence):: At 500 mg/kg bw/day one animal died on Day 10 of pregnancy. This animal showed reduced defecation, reduced hay intake, narrowed palpebral fissures, piloerection, reddish coloured urine and decreased activity on Day 8 and/or 9 of pregnancy.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 500 mg/kg bw/day there was an initial slight loss of body weight between Days 6 - 8 of pregnancy (Table 1 under "Any other information on results incl. tables"). No effects were observed in the animals of the other dose groups compared with control.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: At 500 mg/kg bw/day food consumption was markedly and statistically significantly decreased during Days 6 - 8 of pregnancy (Table 2 under "Any other information on results incl. tables"). No effects were observed in the other dose groups compared with control.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: Gravid uterus weights were comparable in all groups.
Gross pathological findings:: no effects observed
Description (incidence and severity):: No test substance-related effects were observed at necropsy of the animals. Aplasia of the right uterus was observed in 1 animal from each of the control and intermediate dose group.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: not examined
Histopathological findings: neoplastic:: not examined
Number of abortions:: effects observed, non-treatment-related
Description (incidence and severity):: At 500 mg/kg bw/day total litter loss, designated as abortion in study report, was observed in one animal. This animal had only 2 (empty) implantation sites.
Pre- and post-implantation loss:: no effects observed
Description (incidence and severity):: For details see Table 3 under "Any other information on results incl. tables".
Total litter losses by resorption:: effects observed, non-treatment-related
Description (incidence and severity):: At 500 mg/kg bw/day total litter loss, designated as abortion in study report, was observed in one animal. This animal had only 2 (empty) implantation sites.
Early or late resorptions:: no effects observed
Description (incidence and severity):: Statistical evaluation did not reveal an increase in the incidence of the numbers of early and late conceptuses undergoing resorption at caesarean section.
Dead fetuses:: no effects observed
Description (incidence and severity):: Statistical evaluation did not reveal an increase in the incidence of the numbers of dead foetuses in the animals with live foetuses at caesarean section.
3 dead foetuses were observed in the control group and 1 dead foetus occurred in each of the mid and high dose group. Statistical evaluation did not reveal differences between the groups, and the incidences were within the historical control data range of the rabbit strain used.
Changes in pregnancy duration:: not examined
Changes in number of pregnant:: no effects observed
Description (incidence and severity):: All females became pregnant.
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic toxicity
Effect level:: 50 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: no toxic effects observed
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic toxicity
Effect level:: 500 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: maternal toxicity
: Key result
Dose descriptor:: NOAEL
Remarks:: developmental toxictiy
Effect level:: 500 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: other: no toxic effects observed
: Key result
Abnormalities:: no effects observed
Fetal body weight changes:: no effects observed
Description (incidence and severity):: Foetal body weights remained unaffected by test substance administration.
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: Number of live offspring remained unaffected by test substance administration.
Changes in sex ratio:: no effects observed
Description (incidence and severity):: Sex ratios remained unaffected by test substance administration.
Changes in litter size and weights:: no effects observed
Description (incidence and severity):: Mean litter size remained unaffected by test substance administration.
Changes in postnatal survival:: no effects observed
Description (incidence and severity):: Relative survival rate 24 h after caesarean section remained unaffected by test substance administration.
External malformations:: effects observed, non-treatment-related
Description (incidence and severity):: In all external examinations, no statistically significant differences between the groups combined with dose-dependency were found. At 500 mg/kg bw/day, 3 foetuses with deflected forepaw from 2 litters were observed. The values were within the historical control data range of the rabbit strain used and were not considered treatment-related.
Skeletal malformations:: effects observed, non-treatment-related
Description (incidence and severity):: Slightly, but statistically significantly higher incidence of fused, aplastic, dislocated or fragmented caudal vertebral centres in the foetuses at 500 mg/kg bw/day were observed. However, the incidence of this finding was within the historical control data range and therefore was considered not to be treatment-related. In all other skeletal examinations, no statistically significant differences between the groups combined with dose-dependency were found. All values were within the historical control data range of the rabbit strain used.
Neither external nor skeletal defects were observed in the foetuses found dead.
Visceral malformations:: effects observed, non-treatment-related
Description (incidence and severity):: At 50 and 500 mg/kg bw/day, increased incidences of partly or completely fused lung lobes were observed. Incidences of examined foetuses accounted for 1/102, 0/74, 5/88, 10/79 with ascending dose, respectively. Incidences of examined litters accounted for 1/15, 0/15, 4/15, 5/13 with ascending dose, respectively. The difference in incidence of affected foetuses in the high dose group compared with control reached statistical significance but was within the historical control data range. Therefore, this finding was not considered treatment-related.
In all other visceral examinations, no statistically significant differences between the groups combined with dose-dependency were found. All values were within the historical control data range of the rabbit strain used.
Other effects:: no effects observed
Description (incidence and severity):: Crown-rump lengths and placental weights remained unaffected by test substance administration.
: Key result
Dose descriptor:: NOAEL
Remarks:: developmental toxicity
Effect level:: 500 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no test substance-related effects observed
: Key result
Abnormalities:: no effects observed
: Key result
Developmental effects observed:: no
Conclusions:: Under the conditions of the present study, oral administration of the test substance to the rabbit at daily doses of 500 mg/kg bw/day from Day 6 - 18 of pregnancy caused distinct maternal, systemic toxicity, as shown by mortality, body weight loss and reduced food consumption. Although this dose level exceeded the maximum tolerable dose (MTD), embryo-foetal development was unaffected.
Administration of the test substance at doses of 5 and 50 mg/kg bw/day was tolerated by the dams and their conceptuses without any signs of toxicity.
There was no evidence of any test substance-induced effect on foetal morphology. Accordingly, the test substance did not show any developmental toxicity or teratogenic potential in the rabbit.
The No Observed Adverse Effect Level (NOAEL) for maternal, systemic toxicity was 50 mg/kg bw/day and for embryo-foetal developmental toxicity and teratogenicity it was 500 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 120 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Two oral developmental studies (rat and rabbit, both RL2) conducted according to OECD TG 414 and in compliance with GLP are available. The rat was the more sensitive test species, thus the study in rat was chosen for assessment.

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

Two studies are available addressing prenatal developmental toxicity of the test substance. The studies were conducted in rats and rabbits, respectively, according to OECD test guideline 414 and GLP (M-142033-01-1, 1996; M-180231-01-1, 1998).

In the first study for prenatal developmental toxicity with rats, pregnant females were treated with the test substance at dose levels of 15, 120 and 1000 mg/kg bw/day from Gestation Day (GD) 7 until GD 16 by oral gavage.

One dam from the high dose group was found dead on Day 21 of pregnancy. This finding was considered to be treatment-related since this dose level, 1000 mg/kg bw/day, was about half of the acute oral LD50 value for rats and one mortality occurred on Day 17 in the earlier range-finding study. No test substance-related clinical signs of toxicity were observed in any of the animals. Body weight gain and food consumption were slightly decreased in the animals of the high dose group. Body weight change and food consumption were unaffected in the other groups. At necropsy, 1 female of the high dose group exhibited whitish fluid in the renal pelvis. Gravid uterus weights, foetal body weights, crown-rump lengths, litter size, sex ratios as well as placental weights remained unaffected by the administration of the test substance. Likewise, there were no differences between the numbers of dead foetuses, corpora lutea and implantations in the animals from the control and treatment groups. However, the number of early resorptions was slightly increased in the animals from the high dose group. As a consequence, the number of live foetuses was slightly decreased. Morphological examination of the foetuses obtained by caesarean section did not reveal any test substance-related effect.

The Lowest Observed Adverse Effect Level (LOAEL) for maternal toxicity (as evidenced by mortality, decreased body weight gain and decreased food consumption) and embryonic toxicity (as evidenced by a slightly increased incidence of embryonic deaths) were established at the dose level of 1000 mg/kg bw/day. However, as mortality was observed at this dose level it exceeded the maximum tolerable dose. There was no evidence of any effect on foetal morphology and no teratogenic effect at all dose levels tested. Neither maternal nor embryofoetal toxicity were observed after repeated administration of the test substance at 120 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryofoetal effects was 120 mg/kg bw/day. Thus, the test substance could not be evidenced to bear any developmental toxic and/or teratogenic potential in rat, as a rodent species.

In the second study for prenatal developmental toxicity with rabbits, test substance doses of 5, 50 and 500 mg/kg bw/day were administered to the dams from GD 6 until GD 18 by oral gavage.

One animal treated with 500 mg/kg bw/day was found dead on Day 10 of pregnancy. This animal showed reduced defecation, reduced hay intake, narrowed palpebral fissures, piloerection, reddish coloured urine and decreased activity on Day 8 and 9 of pregnancy. Another animal of this group exhibited reddish coloured urine on Day 20 of pregnancy and exhibited only two implantation sites, which were both empty, at caesarean section. One animal treated with 50 mg/kg bw/day showed reddish coloured urine on Day 7 and 8 of pregnancy. No clinical signs were observed in the animals from the other groups. At 500 mg/kg bw/day there was an initial slight loss of body weight between Days 6 - 8 of pregnancy. Food consumption was markedly decreased during this period. Body weight change and food consumption were unaffected in the mid and low dose groups. No test substance-related effects were observed at necropsy of the animals. Gravid uterus weights, foetal body weights, crown-rump lengths, sex ratios as well as placental weights remained unaffected by the administration of the test substance. There was no increase in the number of early or late conceptuses undergoing resorption. Survival rate of the foetuses over 24 h was not impaired. No compound-related defects were detected by morphological examination of the foetuses. Based on these findings a dose level of 500 mg/kg bw/day was determined to be the LOAEL for maternal systemic toxicity in rabbits under the conditions of the study. The dose level of 50 mg/kg bw/day was considered to be the NOAEL for maternal systemic toxicity. No effects were observed in the foetuses, therefore the NOAEL for developmental toxicity was considered to be 500 mg/kg bw/day in rabbits under the conditions of the study. Thus, the test substance could not be evidenced to bear any developmental toxic and/or teratogenic potential in rabbit, as a non-rodent species.

Justification for classification or non-classification

The available data on reproductive and developmental toxicity and teratogenicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

Additional information

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Parameter			Generation		Controls				20 ppm				200 ppm				4000 ppm		Dose-response
					♂		♀		♂		♀		♂		♀		♂	♀	♂	♀
Body weight gains		[g]
	Week 0-1		P0	64		27		66		24		66		26		59**		26	-	-
	Week 18-19		F1	58		39		54		41		55		37		52**		39	-	-
	Week 0-5		P0	224		97		222		90		228		93		209*		95	-	-
	Week 18-23		F1	282		135		280		137		271		128		260**		137	-	-
	Week 0-10		P0	322		134		316		125		325		129		292**		131	-	-
	Week 18-28		F1	414		186		413		189		402		182		392		185	-	-
	Week 0-19		P0	398		164		386		156		409		159		368*		158	-	-
	Week 18-37		F1	517		232		524		232		509		225		495		228	-	-

Parameter		Genera-tion	Controls		20 ppm	200 ppm	4000 ppm	Dose response
Mating index	%	P0	100.0		93.3	100.0	100.0	-
Mating index	%	F1	100.0		96.7	96.7	96.7	-
Fertility index	%	P0	79.3		86.7	86.7	86.7	-
Fertility index	%	F1	86.7		93.3	96.7	86.7	-
Gestation length	Mean [days]	P0	21.9		22.0	22.0	21.9	-
Gestation length	Mean [days]	F1	21.9		21.9	21.9	21.8	-
Gestation index	%	P0	100.0		96.2	96.2	96.2	-
Gestation index	%	F1	100.0		100.0	100.0	96.2	-
Live birth index	%	F1	98.8		99.2	95.1	94.3	-
Live birth index	%	F2	99.4		100.0	98.4	94.2	-
Litter size	Mean	F1	13.5	12.3		14.2	13.6	-
Litter size	Mean	F2	12.5	14.4**		12.9	12.4	-
Pup weight PND1	Mean [g]	F1	6.7	6.8		6.4	6.6	-
Pup weight PND1	Mean [g]	F2	6.8	6.7		6.9	6.7	-
Pup weight PND21	Mean [g]	F1	49.7	49.8		48.7	44.1**	-
Pup weight PND21	Mean [g]	F2	47.1	47.2		47.8	45.6	-
Sex ratio	% Males	F1	55.0	48.6		53.0	53.1	-
Sex ratio	% Males	F2	48.7	49.7		51.2	51.1	-
Viability index	%	F1	97.0	96.5		87.9	92.3	-
Viability index	%	F2	97.6	99.3		96.1	93.8	-
Lactation index	%	F1	97.8	94.2		99.5	100.0	-
Lactation index	%	F2	99.0	99.6		99.1	99.5	-
Sexual maturation	Males [PND]	F1	43.3	44.1		44.3	46.2**	-
Sexual maturation	Females [PND]	F1	33.2	33.2		33.7	34.8**	-
Sperm characterization	% with normal appearance	P0	99.0	99.2		99.4	99.2	-
	% with normal appearance	F1	98.9	98.8		98.8	99.2	-
	% sperm motility	P0	83.0	81.9		85.0	81.8	-
	% sperm motility	F1	80.2	79.6		77.8	80.0	-
	Sperm production rate [106/g/day]	P0	18.3	18.6		17.3	18.1	-
	Sperm production rate [106/g/day]	F1	14.5	14.3		13.5	13.9	-

Day	Dose level (mg/kg bw/day)
Day	0	15	120	1000
1	221.0	223.6	220.7	220.8
4	239.9	239.8	240.0	236.7*
7	252.9	249.8	250.4	247.4*
10	264.2	260.0	262.7	254.1*
14	280.6	275.7	280.5	269.2*
17	299.4	296.3	303.5	286.5*
19	322.3	318.8	328.0	306.5*
21	348.8	344.5	354.4	328.7*

Day	Dose level (mg/kg bw/day)
Day	0	15	120	1000
1-7	8.3	7.9	8.0	7.7*
7-10	7.8	7.5	7.7	6.9*
10-14	7.8	7.5	7.7	7.2*
14-19	8.0	7.6	8.0	7.5*
19-21	7.4	7.1	7.2	7.3

	Dose level (mg/kg bw/day)
	0	15	120	1000
Early intrauterine deaths
Total	14	14	13	30
% of implantations	5.03	4.69	5.60	12.00^#
Late intrauterine deaths
Total	1	0	2	0
% of implantations	0.35	0.00	1.16	0.00
Number of foetuses
Total	256	275	233	230
% of implantations	94.62	95.31	93.24	88.0*

Males	Compound intake [mg/kg bw/day]
dietary level [ppm]	Prior to breeding		After breeding		Overall mean
	P0	F1	P0	F1	P0	F1
20	1.4	1.7	0.9	1.0	1.2	1.5
200	14.4	17.4	9.5	9.8	12.6	14.5
4000	285.3	359.0	190.6	203.4	249.8	300.6

Females	Compound intake [mg/kg bw/day]
dietary level [ppm]	Prior to breeding		Gestation		Lactation*		Overall mean
	P0	F1	P0	F1	P0	F1	P0	F1
20	1.7	1.9	1.4	1.4	3.1	3.2	1.8	2.0
200	16.7	19.7	13.4	14.3	31.5	31.2	18.3	20.0
4000	346.5	405.2	266.8	274.2	577.4	634.6	363.2	406.0

Day	Dose level (mg/kg bw/day)
Day	0	5	50	500
0-3	80.7	43.1	62.1	102.0
3-6	-25.0	-5.9	-39.2	-31.2
6-8	-3.1	-10.4	-19.2	-60.7
8-10	-5.6	5.1	8.3	25.0
10-13	19.3	2.7	-1.1	16.1
13-16	47.4	57.5	59.0	25.4
16-19	20.4	18.9	13.4	-7.3
19-23	39.4	19.3	26.7	88.7
23-26	67.5	44.7	61.1	52.6
26-29	60.5	37.8	46.3	39.9

Day	Dose level (mg/kg bw/day)
Day	0	5	50	500
0-3	2.7	2.3	2.4	2.6
3-6	2.5A	2.6A	2.2A	2.4A
6-8	2.4	2.4	2.1	1.4*
8-10	2.6	2.6	2.4	2.5
10-13	2.6N	2.4A	2.2A	2.5A
13-16	2.1	2.3	2.0	2.0
16-19	2.7N	2.6N	2.4 N	2.1A
19-23	3.1	2.8	2.7	3.4
23-26	3.2	2.9	2.9	3.3
26-29	3.4N	3.0N	3.2A	3.3N

	Dose level (mg/kg bw/day)
	0	5	50	500
Pre-implantation loss	11.24	27.90	17.96	10.77
Post-implantation loss	5.05	10.00	8.50	10.23
Early intrauterine deaths
Total	2	10	7	8
% of implantations	2.62 N	10.00 N	7.17 N	9.37 N
Late intrauterine deaths
Total	3	0	1	1
% of implantations	2.43 N	0.00 N	1.33 N	0.85 N
Number of live fetuses
Total	102	74	88	79
% of implantations	94.95 N	90.00 N	91.50 N	89.77 N

Registration Dossier

Registration Dossier

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Diss Factsheets

ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all