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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The results of a 2-generation-study with a structurally related compound in rats by the oral route gave no indication of a fertility impairing effect. The NOAEL for fertility was set at greater than 4000 ppm.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988 - 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
testes, epididymis weights were not taken
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH , Sulzfeld, Germany
- Weight at study initiation: (P) males: 140 - 200 g; females: 130 - 170 g
- Housing: individually in solid floor macrolone cages of type III with stainless steel lids
- Diet: powdered diet (Ssnift R 10, Ssniff Spezialdiaeten GmbH, Soest, Germany); ad libitum
- Water: Tap water was available ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Separate batches of diet were prepared at fixed concentrations for each treatment group at two week to monthly intervals.
- Mixing appropriate amounts with (Type of food): The weighed amount of test article was mixed with the final weight of the powdered diet in several steps (e. g. four steps for 30 kg formulated test article : 300 g, 5 kg, and 10 kg premixtures, 30 kg final mixture)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to three weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was removed from the males and housed individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Separate samples were taken from top, middle, and bottom of each test article formulation for groups 2 to 4 with a reference sample for the control group (untreated diet), deepfrozen, sent to Hazleton UK, and analysed for determination of test article concentrations. Stability of several formulation preparations was also examined.
Analytical method: KS-298/1
Identity: ascertained by IR spectroscopy
Duration of treatment / exposure:
P generation: 100 days premating + ca. 21 days mating, + ca. 20 days gestation with offspring F1a, + 21 days lactation up to weaning (F1a), + 14 days prior to second pairing, + ca. 21 days mating to produce a further litter (F1b) + 20 days gestation + 21 days lactation up to weaning. Σ ca. 238 days

F1a and F1b: ca. 100 days after weaning + 21 days mating, + 20 days gestation with offspring F2a, + 21 days lactation up to weaning (F2a) + 14 days prior to second pairing, + ca. 21 days mating to produce a further litter (F2b) + 20 days gestation + 21 days lactation up to weaning weaning. Σ ca. 238 days
Frequency of treatment:
The test and control diets were freely available for all animals continuously from the start of treatment until necropsy.
Details on study schedule:
- The P animals were treated for 100 days prior mating and then allowed to mate for maximally 21 days and produce one litter (F1a).

- Approximately 14 days after all the F1a offspring were weaned, the P animals were paired again, within dosage groups, for up to 21 days to produc a further litter (F1b) . Alternative pairings form the first matings were employed.

-Approximately 100 days after weaning of all F1a offspring, the selected F1 animals were allowed to mate for maximally 21 days within the same dosage group (avoiding sibling pairings) and to produce one litter (F2a).

- Approximately 14 days after all the F2a offspring were weaned, the F1 parental animals were paired again within the dosage groups, for up to 21 days to produce a further litter (F2b).

- In the second mating period of each generation, alternative pairings from the first matings were employed (avoiding sibling pairings in the Fl generation).

- The P and F1 male animals were killed and necropsied shortly after their second mating period.

- The P and F1 female animals were killed and necropsied after weaning of the F1b or F2b pups, respectively.

- All pups except those selected to produce the subsequent . generation (F1 animals) were killed and necropsied after weaning or after selection was completed.
Dose / conc.:
400 ppm
Dose / conc.:
2 000 ppm
Dose / conc.:
4 000 ppm
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of the results from the following previous studies:
a) Acute Oral Toxicity Study in Rats.
b) 90 Day Oral (Dietary Administration) Toxicity Study in Rats.
c) 90 Day Oral (Dietary Administration) Toxicity Study in Rats with Recovery Phase.
d) Dose Range-Finding Study for a Two Generation Oral (Dietary Administration) Reproduction Toxicity Study in the Rat (Ciba-Geigy Study No. 874148).
Positive control:
no
Parental animals: Observations and examinations:
MORBIDITY/MORTALITY: Yes
- Time schedule: twice daily

CLINICAL SIGNS: Yes
- Time schedule: daily
- Observations checked: signs of ill health or overt signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly during the premating and mating period. Inseminated P females were weighed on days 0, 6, 10, 15, and 20 post-coitum and lactating P females were weighed on days 1, 4, 7, 14, and 21 of lactation.

FOOD CONSUMPTION:
- Time schedule: weekly during the premating period, for days 0 to 6, 6 to 10, 10 to 15, and 15 to 20 post-coitum, and for days 1 to 4, 4 to 7, 7 to 14, and 14 to 21 during lactation.
- The food consumption was then evaluated as mean daily food consumption per measuring period.
Litter observations:
Where possible, the size of each litter was adjusted to eight pups on day 4 post-partum by eliminating extra pups by random selection to yield - as nearly as possible - four male and four female pups per litter.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the preweaning period
- Cage side observations checked: any abnormalities

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, live and dead pups, presence of external anomalies, weight, physical development (pinna unfolding, generalised hair growth, tooth eruption, eye opening), pupillary reflex, auditory response

GROSS EXAMINATION OF DEAD PUPS: Pups dying or killed during lactation were examined for external and ceral abnormalities.
Postmortem examinations (parental animals):
SACRIFICE and NECROPSY:
- All parental male animals of the P and F1 generation were killed by carbon dioxide inhalation and necropsied after termination of the respective second mating period.
- After the lactation period of 21 days after second mating or approximately on day 26 post-coitum, if an inseminated female failed to produce a viable F1b litter by that day, all P females were killed by carbon dioxide inhalation and examined macroscopically for pathological changes.
- F1 females that failed to produce a viable F2b litter after successful mating were killed and necropsied at least 26 days after insemination or after the
last day of the mating period.
- The remaining Fl females were killed by carbon dioxide inhalation and necropsied after weaning of the F2b progeny.
- Non-selected F1a weanlings and all F2b weanlings were killed and necropsied shortly after weaning.

HISTOPATHOLOGY: adrenals, cervix, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, uterus, vagina, organs which macroscopically show alterations

ORGAN WEIGHTS: adrenals, kidneys, liver, spleen
- No weights were taken from testes, epididymes
Statistics:
- Analysis of Variance was performed with one factor TREATMENT followed by the Student-Newman-Keuls test for multiple group comparisons for: body weight, body weight gain, litter weight, organ weights, and food consumption
- Analysis of Variance was performed with one factor TREATMENT - based on taking the ranks of the variables - and followed by the Student-Newman-Keuls test for multiple group comparisons for: mating performance, duration of gestation, mean pup weights, number of pups found alive, number of pups found dead, number of pups (days 1, 4, 7, 14, and 21 post-partum), live birth index, viability indices, weaning index, pinna unfolding, hair growth, incisor eruption and eye opening
Reproductive indices:
- mating performance (in %): sum of days until successful insemination x 100 / number of inseminated animals
- Insemination index (in %): number of inseminated animals x 100 / number of paired animals
- fecundity index (in %): number of pregnant animals x 100 / number of inseminated animals
- fertility index (in %): number of pregnant animals x 100 / number of paired animals
- gestation index (in %): number of females with live pups x 100 / number of pregnant animals
- sex ratio (in %): number of males x 100 /number of pups; number of females x 100 / number of pups
Offspring viability indices:
- Live birth index (in %): number of pups alive on day 1 x 100 / number of pups born
- viability indices (in %): number of pups alive on day 4/7/14/21 x 100 / number of pups alive on day 1/4/7/14
- weaning index (in %): number of pups alive on day 21 x 100 / number of pups alive on day 4
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES: Two males of group 3 (2000 ppm) showed clinical signs during the treatment period. In one male animal, the left hindlimb was swollen from day 164 to 205 of treatment. In the second male, the ankle of the right hindlimb was blue-red in color and swollen from day 92 to 126 of treatment and the ankles of both hindlimbs were severely swollen from day 127 to 159 of treatment. From day 160 to 205 of treatment, the ankles of the hindlimbs and of the left forelimb were severely swollen. Similar clinical changes were also observed in four group 4 (4000 ppm) animals. One male showed the left hindlimb slightly swollen on day 153 of treatment. The second animal showed a swollen left forelimb from day 178 to 184 of treatment and the left hindlimb was blue-red in color and severely swollen from day 190 to 203 of treatment. In the third animal, hindlimbs were swollen from day 136 to 183 of treatment and from day 184 to 205 of treatment, hindlimbs and left forelimb were swollen. The fourth animal showed swollen hindlimbs from day 114 to 125 of treatment. In consequence of the described clinical signs observed in groups 3 and 4 (2000 and 4000 ppm), the locomotion of these animals was limited. All these clinical changes are considered to be related to treatment.

FEMALES: Four control animals showed clinical signs as inflammation of the left eye or eyes incrusted with blood and/or injury on the tail or bloody chest and fur loss on chest or forelimbs on several days during the course of the study. Two females of group 2 (400 ppm) showed eye effects. One showed blindness of the right eye first on day 4 post-partum of the second lactation period and then a swollen right eye from day 14 post-partum until necropsy. Autopsy of this animal revealed a microphthalmia of the right eye. In the other animal, an inflammation of the left eye was observed starting from day 14 of dosing and lasting to the 7th day prior to start of second mating. Nine animals of group 3 (2000 ppm) showed clinical signs on various days of treatment. Two of these only had loss of fur on either chest and neck or abdomen. One only had an eye inflammation. Two only had a hindlimb swollen while one only had ears reddened and swollen. One animal had reddened and swollen ears in addition to swollen forelimbs and hindlimbs. One animal was found dead while one had ankle and hindlimb swelling in addition to an eye opacity. Three animals of group 4 (4000 ppm) showed clinical changes. In one of them, an injury and fur loss on head and neck were observed from day 80 to 70 prior start of first mating, and the other two animals showed reddened and/or swollen hindlimbs on some days during the second post-partum treatment period. The observed clinical changes in forelimbs and/or hindlimbs and/or reddened and swollen ears in group 3 (2000 ppm) and group 4 (4000 ppm) are considered to be related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES: The mean body weights of group 3 (2000 ppm) were statistically significantly reduced from week 10 of treatment through the second week of first mating, except for week 13, and prior to and during the second mating. The mean body weights of group 2 (400 ppm) and group 3 (2000 ppm) were comparable with the control group. In group 4 (4000 ppm), mean body weight gain was statistically significantly reduced over treatment weeks 4 to 6, 9 to 10, 13 to 14, between the first and second matings and over all weeks prior to and during the second mating. This finding is considered to be related to treatment. Group mean body weight gain of group 3 (2000 ppm) was reduced during weeks 4 to 5 of treatment prior to first mating while group 2 (400 ppm) mean body weight gain was comparable with the control group.

FEMALES: The mean body weights of group 2 (400 ppm) and group 4 (4000 ppm) were generally statistically significantly reduced from week 6 of treatment through week 15. Since group 3 (2000 ppm) was not affected and since there was no difference between groups 2 and 4, this effect is considered questionable. This is supported by the absence of body weight changes during gestation and lactation compared to the control group. All test groups had a decreased mean body weight compared to control during week 21 of treatment but this was not dose-related. Group mean body weight gain prior to first mating was sporadically lower in all dose groups and is considered a questionable effect because there were no consistent dose responses. Mean body weight gain was generally comparable for all groups at the other measurement periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, mean daily food consumption was comparable for all groups. In females, Group 4 (4000 ppm) mean food consumption was increased compared to control during days 10 to 20 of first gestation and days 1 to 4 of first lactation. Sporadic increases were also seen with group 3 (2000 ppm) animals. Group 2 (400 ppm) findings were similar to the control group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Necrotizing changes and inflammatory response - partly with accommodating responses of the parenchyma - observed in the female genital tract and in the adrenal gland, liver, and lymph node of male and female animals result from treatment with the test article. Just as acute purulent nephritis or pyelitis and diffuse polymorphonuclear cell infiltration in the spleen from male and female animals of group 4 - without signs of necrotizing changes - suggest an effect of treatment with the test article. A relationship to treatment with the test article cannot definitively be excluded in the case of an endometrial polyp and of a squamous cell carcinoma in the uterus, each observed in one animal. The other pathological findings are regularly observed in rats and therefore considered as "background pathology".
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test substance on mating performance and fertility.
Dose descriptor:
NOAEL
Effect level:
400 ppm (nominal)
Sex:
male/female
Basis for effect level:
gross pathology
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES: Three control animals showed clinical signs as bloody urine or left ear slightly swollen or inflammation on the left eye on a few days during the treatment period. One animal of group 2 (400 ppm) showed a bloody incrusted right eye on some days during the treatment period. Eleven animals of group 3 (2000 ppm) showed clinical changes in various days of treatment. Three of these only had hindlimbs swollen while one only had ears swollen. Four animals showed a shaggy coat in addition to swollen forelimbs and/or hindlimbs. Two further animals showed a bloody incrusted nose or blood in the sawdust and red-colored urine on single days. One animal of group 3 (2000 ppm) was found dead on day 196 of treatment. This animal showed clinical changes as swollen forelimbs and hindlimbs and shaggy coat from day 120 to 195 of treatment. At necropsy, a swelling of both hindlimbs and left forelimb, bilateral hydronephrosis, an enlarged right lymph node of the right kidney, and a partly reddened and thickened mucosa of the urinary bladder were found. In nine animals of group 4 (4000 ppm), similar clinical changes were observed during the treatment period. Four animals only showed swollen hindlimbs. Three showed a shaggy coat in addition to swollen hindlimbs while one only showed a shaggy coat. One animal only showed shaggy coat. One animal of group 4 (4000 ppm) was killed moribund on day 148 of treatment. It showed clinical signs as shaggy coat and swollen forelimbs and hindlimbs, and therefore, the locomotion of this animal was limited. The observed clinical changes in group 3 (2000 ppm) and group 4 (4000 ppm) are considered to be related to treatment.

FEMALES: 21 control animals showed clinical signs as shaggy coat on some days during the course of the study, mainly during the first post-partum treatment period. One of these additionally showed an inflammation of the left eye on some days. 24 animals of group 2 (400 ppm) showed clinical changes on various days. 19 animals only showed a shaggy coat. Three additional animals showed shaggy coat in addition to hair loss at limbs and/or abdomen and/or a wound on the neck and/or an inflammation of the eye. One animal showed shaggy coat and swellings on the right side of abdomen, neck, and forepaws, partly bloody incrusted, mainly during the second postpartum treatment period. One animal of group 2 (400 ppm) was found dead on day 2 post-partum after first mating. This animal showed shaggy coat, pale eyes, and a bad physical condition prior death. Necropsy revealed colored kidneys and liver and consistency of cervical marrow softer than normal. 24 animals of group 3 (2000 ppm) showed clinical changes on various days of treatment. 16 animals only showed a shaggy coat. Three animals had swollen hindlimbs in addition to a shaggy coat. One of these additionally showed a swollen ear. One animal only showed swollen hindlimbs. Three animals showed an inflammation of the eye or an abnormal position of the head in addition to shaggy coat, and one additional animal had a wound on the neck. All 25 animals of group 4 (4000 ppm) showed clinical signs. 17 only showed a shaggy coat on several days during the first post-partum treatment period. Four animals had a shaggy coat in addition to front teeth cut and/or an injury on the neck and/or ear or right eye reddish encrusted. Two animals showed a shaggy coat and swollen forelimbs and hindlimbs and pale eyes or abnormal position of the head, and dragging of the left hindlimb and pale eyes. One animal was killed moribund on day 20 after first post-partum treatment period. This animal showed a shaggy coat, swollen forelimbs and hindlimbs, and locomotion was limited. Necropsy revealed spleen with granulated surface and cut surface and permeated with follicles, liver showing white inclusions, and talocalcanean joints of hindlimbs and forelimbs not movable, and metatarsal swollen. A second animal that showed a shaggy coat and an inflammation of the right eye was found dead on day 19 post-coitum of the second gestation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES: The mean body weights of group 4 (4000 ppm) were statistically significantly reduced from week 10 to 15 and 24 to 26 of treatment. The mean body weights of group 2 (400 ppm) and group 3 (2000 ppm) were comparable with the control group. In group 4 (4000 ppm), mean body weight gain was statistically significantly reduced over treatment weeks 4 to 5, 9 to 10, 11 to 12, and 25 to 26. Therefore, overall body weight gain was also reduced from weeks 1 to 15 and 24 to 26 of treatment. This finding is considered to be related to treatment. In group 2 (400 ppm) and group 3 (2000 ppm), body weight gains were slightly reduced during weeks 2 to 3 and 11 to 12 of treatment. The differences were small and although values were statistically significant, these findings are considered not to be related to treatment.

FEMALES: The mean body weights of all three dose groups were generally comparable with the control group prior to first and second mating as well as during gestation and lactation. Group body weight gain did not show any dose-related effect, although statistically significant differences were occasionally observed in all dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean daily food consumption was comparable for all groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
MALES: At 2000 ppm (group 3) and 4000 ppm (group 4), the mean weights of adrenals, kidneys, and spleen were higher than in the control group. The mean liver weight of group 4 (4000 ppm) was lower than in the control group. The observed changes in organ weights are considered to be related to treatment and similar changes were also observed in the P male animals.

FEMALES: In group 3 (2000 ppm) and group 4 (4000 ppm), the mean weights of the kidneys and spleen were higher than in the control group. Similar changes were also observed in the P female animals and therefore, these findings are considered to be related to treatment. In addition, the mean weights of the liver and adrenals were increased in group 4 (4000 ppm). These findings are also considered to be treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MALES: At necropsy, six control animals showed minor findings in the kidneys and one additional animal a reduced left testis and epididymis. In five animals of group 2 (400 ppm), findings as testes and epididymis reduced in size or dilatation of the renal pelvis or kidneys yellowish in color were detected at necropsy. At necropsy, 18 surviving animals of group 3 (2000 ppm) showed findings. Five of these only had enlarged mesenteric lymph nodes and one only had a grossly granulated cut surface of the spleen. Seven animals had either renal pelvis dilatation and/or discolored kidneys or testis reduced in size or grossly granulated cut surface of the spleen in addition to enlarged mesenteric lymph nodes. Five animals showed swollen hindlimb(s) at necropsy. Three of these had in addition enlarged mesenteric lymph nodes and/or grossly granulated cut surface of the spleen and/or spleen with yellowish thickened tissue. 20 surviving animals of group 4 (4000 ppm) showed findings at necropsy. Two of these only had enlarged mesenteric lymph nodes. 15 animals has either grossly granulated cut surface of the spleen and/or kidneys yellowish in color and/or spotted and/or inclusions in the spleen and/or liver and/or right seminal vesicle reduced in size and/or dilatation of the renal pelvis in addition to enlarged mesenteric lymph nodes. Two additional animals showed left testis reduced in size, right testis enlarged, a tissue formation close to epididymis and kidneys yellowish in color or spleen adhered to abdominal wall and abnormally shaped in addition to enlarged mesenteric lymph nodes. One additional animal with swollen hindlimb had left hindfoot with thickened talocalcanean joint, kidneys yellowish in color, inclusions in the liver, and also enlarged mesenteric lymph nodes. One additional animal that was killed moribund showed kidneys and liver swollen, white follicles in the spleen, spleen swollen, urinary bladder extremely filled, both forelimbs and right hindlimb swollen, right hindfoot callously thickened from talocalcanean joint dorsally to metatarsal, tibia bent cranially, right and left carpals in total cartilage - like thickened. The described necropsy findings in group 3 (2000 ppm) and group 4 (4000 ppm) are considered to be related to treatment.

FEMALES: Necropsy revealed right adrenal reduced in size, left adrenal and liver of firm consistency, left kidney containing yellowish, subcapsular, and cortical areas, swelling of mesenteric lymph nodes, and thymus dark red. The observed clinical changes as swollen limbs in group 3 (2000 ppm) and group 4 (4000 ppm) are considered to be related to treatment. At necropsy, one control animal only showed an ovary covered with vesicle. Four surviving animals of group 2 (400 ppm) showed findings at necropsy. Two animals had a unilateral dilatation of the renal pelvis, and in one animal, the uterus was filled with liquid. The fourth animal which externally showed a swelling of the right part of the body, displayed at necropsy an ulcer filled with yellowish liquid under the neck and an ulcer at one nipple on the right side. At necropsy, twelve animals of group 3 (2000 ppm) showed findings. Eight of these only had enlarged mesenteric lymph nodes, and one only had a unilateral dilatation of the renal pelvis. Three animals showed a thickening of the talocalcanean joint, two of these in addition to enlarged mesenteric lymph nodes. At necropsy, 23 surviving animals of group 4 (4000 ppm) showed findings. Five animals only had enlarged mesenteric lymph nodes. 16 animals showed enlarged mesenteric lymph nodes in addition to either changes in the kidneys and/or adrenals, and/or liver, and/or spleen. Two animals only showed findings as unilateral dilatation of the renal pelvis or left adrenal spotted at necropsy. The described necropsy findings in group 3 (2000 ppm) and group 4 (4000 ppm) are considered to be related to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrotizing changes and inflammatory response - partly with accommodating responses of the parenchyma - observed in the female genital tract, in the pituitary gland and kidney of female animals, in the adrenal gland, and liver, in the spleen, and lymph node of male and females animals result from treatment with the test article. Just as the purulent pyelitis of three male animals - without signs of necrotizing changes - suggest an effect of treatment with the test article. The other pathological findings are regularly observed in rats and therefore considered as "background pathology".
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test article on mating performance and fertility.
Dose descriptor:
NOAEL
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
F1a LITTER DATA - P GENERATION
The mean duration of gestation was similar and normal in all groups. Mean litter findings with respect to rearing and viability data as well as litter and pup weights were similar in all groups. There was no effect of treatment with the test substance on the physical parameters examined (pinna unfolding, hair growth, incisor eruption, and eye opening). The employed functional tests on day 21 post-partum (pupillary reflex and auditory response) revealed no effect of treatment. Malformations were observed in two pups of the control group only. One pup showed an unilateral hydronephrosis and one further an aplasia of the tail and atresia ani. The incidence of variations was comparable in all groups.

F1b LITTER DATA - P GENERATION
The mean duration of gestation was similar and normal in all groups. On days 1 and 4 post-partum, the mean number of live pups was slightly decreased in group 4 (4000 ppm), however, the live birth index was comparable with the control group. During lactation, pup losses were high in all groups, including the control group and therefore, the weaning index was low in all groups. In group 3 (2000 ppm), pup losses were higher than in the control group but due to the fact that values of the high dose group (4000 ppm) were comparable with the control group, this finding is considered to be incidental. Sex distribution of Fib pups was similar in all groups. The mean pup weight was generally comparable for all groups from day 1 to 7 post-partum. On days 14 and 21 post-partum, mean pup weights for group 3 (2000 ppm) and group 4 (4000 ppm) were slightly lower than in the control group. This finding is considered to be incidental because mean pup weight of group 4 (4000 ppm) was higher than in group 3 (2000 ppm) and in addition, the mean pup weight of group 4 (4000 ppm) was comparable with the F2a pup weight of the control group. In group 2 (400 ppm), mean pup weights were comparable to the control group. There was no effect of treatment on the physical parameters examined (pinna unfolding, hair growth, incisor eruption, and eye opening). The employed functional tests on day 21 post-partum (pupillary reflex, auditory response) did not reveal any treatment-related effect. Malformations were observed in two pups each of group 2 (400 ppm) and group 3 (2000 ppm). In group 2 (400 ppm), one pup showed a unilateral anophthalmia and one a bilateral hydronephrosis. In one pup of group 3 (2000 ppm), the tail was rudimentarily developed and in the second, a unilateral hydronephrosis was found. In one female of group 3 (2000 ppm) which was found dead on day 22 postcoitum, spina bifida and exencephalocele were observed in one pup at necropsy. The nature and frequency of these malformations are considered to be incidental. The nature and incidence of variations did not reveal any treatment-related effect.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: enlarged mesenteric lymph nodes, and/or findings in the kidneys, spleen, and/or liver in the 2000 and 4000 ppm dose groups. 400 ppm corresponds approximately to 25 to 31 mg/kg bw/day.
F2a LITTER DATA – F1 GENERATION
The mean duration of gestation was similar and normal in all groups. During lactation, pup losses were high in all groups including the control group and therefore, the weaning index was low in all groups. As the highest weaning index was observed in the high dose group (4000 ppm), these findings are considered to be incidental. Sex distribution of F2a pups was similar in all groups. The mean pup weight was generally comparable for all groups from day 1 to 14 post-partum. On day 21 post-partum, mean pup weights for all dose groups were lower than in the control group. This finding is considered to be incidental and not to be related to treatment because mean weights of the F2b pups on day 21 postpartum were generally higher. There was no effect of treatment with test material on the physical parameters examined (pinna unfolding, hair growth, incisor eruption, and eye opening). The employed functional tests on day 21 post-partum (pupillary reflex and auditory response) revealed no effect of treatment. No malformations were observed in any of the groups. The nature and incidence of variations revealed no treatment-related effects.

F2b LITTER DATA – F1 GENERATION
The mean duration of gestation was similar and normal in all groups. During lactation, pup losses were observed in all groups including the control group. Weaning index of group 2 (400 ppm) and group 3 (2000 ppm) was lower than in the control group. This finding is considered to be incidental because weaning index of group 4 (4000 ppm) was higher than in the control group. The sex distribution of F2b pups was similar in all groups. The mean pup weight was generally comparable for all groups from day 1 to 14 post-partum. On day 21 post-partum, mean pup weight for group 4 (4000 ppm) was slightly lower than in the control group. This finding is considered to be incidental. In group 2 (400 ppm) and group 3 (2000 ppm), mean pup weights were comparable with the control group on day 21 post-partum. There was no effect of treatment on the physical parameters examined (pinna unfolding, hair growth, incisor eruption, and eye opening). The employed functional tests on day 21 post-partum (pupillary reflex and auditory response) revealed no treatment-related effect. Malformations were observed in one pup each of group 2 (400 ppm) and group 3 (2000 ppm). One pup in group 2 (400 ppm) showed a bilateral hydronephrosis and one in group 3 (2000 ppm) an anophthalmia. The single occurrence of these malformations is considered to be incidental. The nature and incidence of variations did not reveal any treatment-related effect.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 4 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Reproductive effects observed:
no

There was no effect on mating performance and fertility in P and F1 animals treated with the test substance admixed to the diet at concentrations of 400, 2000, and 4000 ppm.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No reproductive toxicity studies are available. From a structurally analogue substance a 2-generation-study is available.

(See attached document, chapter 13, for read across justification)

Data from the structurally related compound:

 

In a two-generation study according to OECD TG 416 the test substance was administered orally in feed to groups of 30 male and 30 female rats (in the P generation) and of 25 male and 25 female rats (in the F1 generation) of the Sprague Dawley strain at doses of 0, 400, 2000, 4000 ppm. After 100 days of premating treatment, the P parental animals were mated for up to 21 days and the females were allowed to litter and to rear their offspring (F1a generation) to weaning. Approximately 14 days after all the F1a offspring were weaned, the P parental animals were paired again, for up to 21 days (alternative pairs). The P females were allowed to litter and to rear their offspring (F1b generation) to weaning. After a 100 day maturation period after weaning, 25 male and 25 female animals per group of the F1a parental animals were mated for up to 21 days and the females were allowed to litter and to rear their offspring (F2a generation) to weaning. Approximately 14 days after all the F2a offspring were weaned, the F1 parental animals were paired again, within the dose groups, for up to 21 days with alternative pairing. The F1 females were allowed to litter and to rear their offspring (F2b generation) to weaning.

 

Among the F1 animals one male from the 2'000 ppm group and one male and two females from the 4000 ppm group died due to toxicity. At 2'000 and 4'000 ppm clinical changes such as reddened and/or swollen ears and/or extremities were observed.

 

At 4000 ppm, mean body weight gain of P and F1 male animals was reduced during the treatment period. Administration of 2000 and 4000 ppm induced changes in organ weights of P and F1 animals (increased mean weights of adrenals, kidneys, and spleen as well as decreased mean liver weight in P and F1 male animals). At 2000 and 4000 ppm, necropsy findings in the kidneys, spleen, and/or liver, and enlarged mesenteric lymph nodes were detected in P and F1 animals. Histopathological examination of the control and high dose group revealed necrotizing changes and inflammatory response - partly with accommodating response of the parenchyma - observed in the genital tract of the P and F1 females, in the adrenal gland, liver and lymph node of P and F1 male and female animals, in the pituitary gland and kidney of F1 females and in the spleen of F1 males and females.

 

There was no effect of treatment in either generation on mating performance and fertility. The duration of gestation was similar and normal in the P and F1 females of all groups. Viability and post natal growth of the F1a, F1b, F2a, and F2b pups was not affected by treatment. There were no effects of treatment on the physical development of the F1a, F1b, F2a, and F2b pups and on the nature and incidence of malformations observed in the F1a, F1b, F2a, and F2b pups.

 

In conclusion, administration of the test substance by admixture to the basic powdered diet at concentrations of 2000 and 4000 ppm revealed toxic effects in the P and F1 animals. Administration of 400 ppm did not elicit any toxic effects on males and females of the P and F1 generation or on their offspring. This dose level corresponds approximately to a 25 to 31 mg/kg/day dose level and is considered to represent the NOEL (no observed effect level) of the test substance. There was no effect on mating performance and fertility in P and F1 animals treated with the test substance admixed to the diet at concentrations of 400, 2000, and 4000 ppm. Therefore, the NOAEL for fertility was set at greater than 4000 ppm.

Effects on developmental toxicity

Description of key information

The results of a teratogenicity study with a structurally related compound in rats by the oral route gave no indication of a developmental toxic or teratogenic effect. The NOAEL for teratogenicity was set at greater than 1200 mg/kg body weight.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1983-1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study predating GLP. QAU statement included
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Remarks:
but QAU statement included
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-Geigy, WST
- Age at study initiation: 2 months
- Weight at study initiation: 180 - 200 g
- Housing: 4 per cage in Makrolon cages
- Diet: NAFAG No. 890, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % aqueous solution of CMC (sodium carboxymethylcellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The suspension of the test material was freshly prepared daily by homogenization and stirring (magnetic stirrer), and administered at a rate of 10 ml/kg of body weight.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male / 3 females
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 until day 15 of pregnancy
Frequency of treatment:
once daily
Duration of test:
15 days
Remarks:
Doses / Concentrations:
0, 200, 600, 1200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: On the basis of the preliminary experiment where 12 fertilized rats each were administered orally by intubation with the vehicle and 1000 mg/kg bw of the test substance per day in a 0.5% aqueous solution of sodium carboxymethylcellulose (CMC) from day 6 until day 15 of pregnancy. Treatment at these dose levels caused some depression in both bodyweight gain and food consumption of the mother animals. No adverse effects were recorded for the progeny at sacrifice shortly before term in comparison with the vehicle control. On the basis of the foregoing results the doses for the main study were selected at 0, 200, 600 and 1200 mg/kg of body weight.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily during the period of treatment

FOOD CONSUMPTION: Yes
- Time schedule: on days 6, 11, 16 and 21 of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: especially ovaries and uterus (mucosa and contents, including amniotic fluid), and placentae as well as abortions and resorption sites
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
Fetal examinations:
- External examinations: Yes: in one third per litter
- Skeletal examinations: Yes: in two third per litter
Statistics:
Chi square-test
Details on maternal toxic effects:
The dams of the high-dose (1200 mg/kg d) and intermediate dose (600 mg/kg d) groups reacted to the treatment in a dose-related fashion by a reduced body-weight gain and food consumption in comparison with the vehicle control. Female No. 49 of the intermediate dose group showed some vaginal bleeding on day 15 p.c.). In the high-dose- group diarrhea was noted for female No. 83 on day 8 p.c. Female No. 85 was found moribund and killed for humane reasons on day 14 p.c.; severe inflammation of caecum (distended by a large amount of purulent material) was observed at autopsy. Females Nos. 84 and 94 died spontaneously on days 15 and 13, respectively; abscess of the large intestine was also found in these animals.

The pregnancy rates expressed as average number of implantation sites per female were comparable for all groups in the present experiment. The numbers of embryonic and/or fetal deaths (resorptions) were not increased at either dose. The male to female ratios of the fetuses were also comparable for all groups.
Dose descriptor:
NOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
In comparison with the vehicle control, the average body-weight of the live fetuses was not diminished at the intermediate and high-dose groups; a slight but significant increase was noted for the live fetuses of the low-dose group. This finding, however, is not assumed to be of a biological relevance. The gross inspection of the live fetuses revealed one fetus each with brachymelia of hind-limb (unilateral) and omphalocele, respectively, in the intermediate dose group. Omphalocele also occurred in one fetus of the high-dose group. One instance of brachycaudia was recorded for the vehicle control. Carrying out the slicing technique for "visceral" examination, agenesis of testis (unilateral) was found in one fetus of the vehicle control. The skeletal assessment revealed instances of irregular sternebral ossification in the dose groups and in the vehicle control; abnormal "wide suture" associated with incomplete ossification of the basioccipital bone was found in one fetus of the high-dose group, in addition. Concerning the status of skeletal maturation of the fetuses shortly before term, some delay, as indicated by a slightly enhanced number of still incompletely ossified calcanei and 5th sternebra, was recorded for the high-dose group in comparison with the vehicle control.
Dose descriptor:
NOAEL
Effect level:
>= 1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: highest dose tested
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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

No developmental toxicity studies are available. From a structurally analogue substance a teratogenicity study is available.

(See attached document, chapter 13, for read across justification)

Data from the structurally related compound:

 

In a teratogenicity study according to OECD TG 414 the test substance was administered in 0.5 % CMC to pregnant Sprague Dawley rats at dose levels of 0, 200, 600 and 1200 mg/kg of body weight from day 6 until day 15.

 

The dams of the intermediate and high-dose group reacted to the treatment by a dose - related reduction in body-weight gain and food consumption. In a close relation to these findings, the progeny of the high-dose group displayed a slight delay of skeletal maturation; calcanei and 5th sternebrae were affected in this respect. The progeny of the intermediate and low-dose groups remained unaffected by the treatment. Occasional anomalies and/or malformations were found in all dose groups and in the controls (vehicle and historical).

 

One instance each of brachymelia (hind-limb, unilateral) and omphalocele, respectively, occurred in the intermediate-dose group; omphalocele was also recorded for one fetus of the high-dose group. Brachycaudia and unilateral agenesis of testis, respectively, were recorded for one fetus each of the vehicle control. These occasional anomalies and malformations are considered to be of a spontaneous origin and not related to the treatment, the incidence of omphalocele being 0.04% in the historical control population of the breed of rats used for the present experiment. Likewise, the occurrence of sternebral anomalies in all groups as well as the one fetus of the high-dose group showing "wide suture" (associated with incompletely ossified basioccipital bone) were not assumed to be of an experimental significance. The overall incidence of sternebral anomalies was recorded to be 0.41% in the historical control.

 

To summarize, the test substance was devoid of an embryotoxic activity and did not reveal teratogenic potency in the rat under the present experimental conditions. A slight delay of physiological growth was noted for the foetuses of the high-dose group but considered to be non-specific and related to maternal toxicity.

Justification for classification or non-classification

No classification is required according to Annex VI of Directive 67/548/EEC and according to the Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008.