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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the SOP of the International Research and Development Corporation (IRDC) and followed GLP.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1987
Reference Type:
other: Report
Title:
Unnamed
Year:
1985

Materials and methods

Principles of method if other than guideline:
The study was conducted according to the SOP of the International Research and Development Corporation (IRDC)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Purity 99.9 %

Test animals

Species:
rat
Strain:
other: Charles River COBS CD
Sex:
male/female
Details on test animals and environmental conditions:
At test initiation, the males were 40 days old and weighed between 159 and 194 g; the females also were 40 days old and weighed between 121 and 147 g.

Administration / exposure

Route of administration:
oral: drinking water
Details on mating procedure:
Mating was conducted after 80 days of treatment; for this purpose, males and females of the same group were put together at a ratio of one male and two females; the mating period was 15 days.
The F0 females were mated twice. The first mating resulted in the F1a generation. After a resting period of 10 days following the lactation of the F1a, the females were mated a second time to produce the F1b generation. The males selected for the second mating procedure were different from those used for the first mating of the F0 females.
After weaning, 13 F1b males and 26 F1b females per group were randomly selected to form the parental F1 generation.
After a period of at least 87 days of treatment, the F1b parental animals were mated as described above to produce the F2a generation. After a period of at least 10 days following completion of the lactation period, the F1b females were subjected to a further mating with males different from those used for the first mating, and produced the F2b generation.
As mentioned above, the duration of the mating period was 15 days. Vaginal smears of the females were examined daily for the appearance of a copulation plug or for the presence of sperm; the day when copulation could be evidenced was defined as day 0 of gestation. If mating could not be evidenced during the first 10 days of the mating period, then the concerned female was placed together with a further male, which had been mated successfully before with another female of the same group; the remating period did not exceed 5 days.
Details on analytical verification of doses or concentrations:
Analytical monitoring of the test concentrations:
The test substance was checked for purity prior test initiation and at the end of the test. Samples of the test solutions were taken for verification of the test substance concentration prior to test initiation and on study weeks 1, 4, 8 and 13. The test material analysis revealed mean bronopol concentrations ranging between 95 and 107% of the target levels.
Stability of bronopol in aqueous solutions (Lock JI (1985) Report No: DTM 85003):
The stability of aqueous bronopol solutions (0.005 to 0.6%) was tested at different pH (4 – 7.9) over a period of 8 days. The results showed, that bronopol in aqueous solution was stable at pH 4.
Duration of treatment / exposure:
Duration of exposure before mating: 80 days for both, males and females
Duration of exposure in general: The treatment was started 80 days prior mating, and mating duration was about 15 days. Treatment was continued thereafter until the end of the weaning period of the F1 young, which corresponded to the time point at which the F0 parents were sacrifice for necropsy.
Treatment of the parental F1 rats was started at weaning and was continued until weaning of the F2 young and thereafter for 33 to 47 days.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 70, 200 mg/kg bw/day
Basis:

No. of animals per sex per dose:
For both, the F0 and the F1 parental generation, the test comprised 3 treatment groups and one control group; each group consisted of 13 male and 26 female rats.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical signs: All animals were checked twice daily for mortality and clinical symptoms of toxicity. They furthermore were subjected to additional detailed observation once a week.
Body weight:
Males: Body weights of the male animals were recorded at test initiation and weekly thereafter until sacrifice.
Females: Body weights of the F0 females were recorded weekly until successful mating, and thereafter between the F1a and F1b matings. For the F1 females, recording of body weight between F2a and F2b matings was omitted by inadvertence.
Females during gestation: The pregnant females were weighed on day 0, 6, 15 and 20 of gestation.
Females during lactation: Females that had delivered were weighed on day 0, 7, 14 and 21 of lactation.
Food consumption: Food consumption for both, the F0 and the F1 generation, was measured weekly from test initiation until sacrifice, except for the mating period.
Females during gestation: The food consumption of the pregnant females was measured for the 0-6, 6-15, 15-20 and 0-20 day intervals during gestation.
Females during lactation: The food consumption of the females during lactation was measured for the 0-7, 7-14, 14-21 and 0-21 day intervals.
Water consumption:
Water consumption for both, the F0 and the F1 generation, was measured weekly from test initiation until sacrifice, except for the mating period.
Females during gestation: The water consumption of the pregnant females was measured for the 0-6, 6-15, 15-20 and 0-20 day intervals during gestation.
Females during lactation: The water consumption of the females during lactation was measured for the 0-7, 7-14, 14-21 and 0-21 day intervals.
For the F0 females, recording of water consumption between F1a and F1b matings was omitted by inadvertence.
The determination of the test substance intake was calculated on the basis of the mean water consumption and was expressed in mg test substance/kg bw/day.
Litter observations:
Reproduction, delivery and litter data: Parental F0 and F1b animals: Mating performance, fertility indices for both, males and females, and gestation length were considered. Difficulties in parturition were recorded. All pregnant females were allowed to deliver and the day of delivery was defined as day 0 of lactation.
All litters were examined for litter size, number of stillbirths, number of live births and gross abnormalities. On lactation day 4, all the litters were reduced to 10 pups of equal sex distribution (if possible) to provided a homogeneous group size for the evaluation of nursing, survival and growth. The dams were kept together with their litters for a period of 3 weeks and were observed for survival and for nesting and nursing behaviour. The removed pups were examined for external abnormalities and were then sacrificed. Dead pups were recorded and subjected to necropsy.
F1a pups:
Body weights of the pups were recorded on day 0, 4, 7, 14 and 21 of lactation. The number of pups per sex was determined on day 4 of lactation (i.e. prior to reduce the litters) and on day 21.
F2a pups:
The F2a pups were subjected to similar examinations as described above.
F2b pups: The F2a pups were subjected to similar examinations as described above.
Postmortem examinations (parental animals):
Non pregnant females: On the 25th day following separation from the males, non pregnant females were sacrificed and their uteri were examined for implantations.
F0 parents: After selection of the F1 parental animals (F1b), ten F0 parental rats per sex and group were sacrificed and selected tissues were preserved in fixative. The remaining F0 rats were examined for external abnormalities and were then sacrificed and discarded.
F1b parents: About 33 to 47 days following weaning of the F2b generation, ten F1b parental rats per sex and group were sacrificed and subjected to complete gross and histo-pathology. The remaining F1b rats were examined for external abnormalities and were then sacrificed and discarded.

Macroscopic examination: Following sacrifice, the 10 selected parental F0 and F1 rats/sex/group were subjected to a complete external examination. This was followed by the in situ examination of the contents of the abdominal, thoracic and cranial cavities. The contents of the different cavities were then removed and dissected for further examination. Following organs and tissues were collected and placed in phosphate-buffered neutral: Adrenal, aorta, brain, colon, duodenum, epididymis, esophagus, eye, heart, ileum, jejunum, kidney, liver, lung, lymph node, mammary gland, ovary, pancreas, pituitary, peripheral nerve, prostate, salivary gland, seminal vesicle, skeletal muscle, skin, spleen, stomach, testis, thymus, thyroid and parathyroid, trachea, urinary bladder, uterus and cervix, all gross lesions.
Organ weight: The body weights of the selected animals (see above) were recorded post mortem and following organs were weighed: Adrenal, heart, kidney, liver, ovary, testis, thyroid/parathyroid.
Microscopic examination: A series of organs/tissues removed from the selected animals (see above) were further prepared for histopathological examination. Samples of following organs/tissues were taken for paraffin embedding: Adrenal, colon, epididymis, esophagus, heart, ileum, jejunum, kidney, liver, lung, ovary, prostate, spleen, stomach, testis, thyroid, trachea, urinary bladder, uterus and cervix, and all gross lesions. Paraffin sections were then HE-stained for microscopic examination.
Postmortem examinations (offspring):
F1b and F2b pups: Five weaned pups per sex and group were sacrificed and selected tissues were subjected to histopathological examination.
Remaining F2b pups,: the remaining F2b pups were examined for external abnormalities, and were sacrificed and discarded.
F1a and F2a pups: After weaning, the F1a and F2a pups were examined for external abnormalities and were then sacrificed and discarded.
Dead pups: Dead pups were recorded and subjected to necropsy. The pups found dead before and on day 4 of lactation were subjected to heart examination; for this purpose, the hearts were dissected according to a method described by Staples RE (Teratology 9: A37-A38, 1974).

Macroscopic examination: Following sacrifice the 5 selected F1b and F2b pups/sex/group were subjected to a complete external examination. This was followed by the in situ examination of the contents of the abdominal, thoracic and cranial cavities. The contents of the different cavities were then removed and dissected for further examination. Following organs and tissues were collected and placed in phosphate-buffered neutral: Adrenal, aorta, brain, colon, duodenum, epididymis, esophagus, eye, heart, ileum, jejunum, kidney, liver, lung, lymph node, mammary gland, ovary, pancreas, pituitary, peripheral nerve, prostate, salivary gland, seminal vesicle, skeletal muscle, skin, spleen, stomach, testis, thymus, thyroid and parathyroid, trachea, urinary bladder, uterus and cervix, all gross lesions.
Organ weight: The body weights of the selected animals (see above) were recorded post mortem and following organs were weighed: Adrenal, heart, kidney, liver, ovary, testis, thyroid/parathyroid.
Microscopic examination: A series of organs/tissues removed from the selected animals (see above) were further prepared for histopathological examination. Samples of following organs/tissues were taken for paraffin embedding: Adrenal, colon, epididymis, esophagus, heart, ileum, jejunum, kidney, liver, lung, ovary, prostate, spleen, stomach, testis, thyroid, trachea, urinary bladder, uterus and cervix, and all gross lesions. Paraffin sections were then HE-stained for microscopic examination.
Statistics:
Parental body weights and organ weights: These parameters were statistically assessed y means of the one-way analysis of variance, Bartlett´s test for homogeneity of variance and the t-test.
Male and female fertility: Fertility and copulatory indices were assessed by means of the Chi-square test and/or Fisher´s exact probability test.
Pup survival indices: The pup survival indices were compares using the Mann-Whitney U test.
Litter size and pup body weights and organ weights: These parameters were statistically assessed y means of the one-way analysis of variance, Bartlett´s test for homogeneity of variance and the t-test.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

One control female died during study week 11 and one female of the 25 mg/kg bw/day group died during week 25, on gestation day 22 of the F1b mating. In the 200 mg/kg bw/day group, one male and one female died during study week 1; a further female died during week 7 and another one was sacrificed in extremis during week 18, on lactation day 21 of the F1a mating. One female died during week 25, on gestation day 22 of the F1b mating and a further one was sacrificed in extremis during study week 26, on lactation day 3 of the F1b mating. Clinical signs of toxicity only were reported for these 8 animals that either died or were sacrificed in extremis and were summarized in the table below. Necropsy of the animals that died or were sacrificed in extremis revealed abnormalities and/or lesions, Necropsy of the F0 parents, which survived the treatment and were sacrificed, revealed no treatment-related abnormalities. Mortalities were increased in the 200 mg/kg bw/day group, but neither the clinical symptoms nor the findings at necropsy showed consistent trends indicating a relationship to the treatment. Therefore the increased mortality seen at 200 mg/kg bw/day group was considered to rather be incidental than treatment-related.

Body weight:
Body weight for the males and females of the 200 mg/kg bw/day group were below control values starting from the first study week, throughout the whole experimental phase. The differences in mean body weight for the 200 mg/kg bw females versus control were significant at week 20, 21, 30 and 31. The decrease in body weight gain was seen as treatment-related effect. The remaining test groups showed no treatment-related effects
The body weights and body weight gain for the 200 mg/kg bw females during gestation were below control values for both, the F1a and the F1b mating. Body weights and body weight change in the remaining test groups (25 and 70 mg/kg bw/day) were inconspicuous.
The body weights and the body weight gain for the 200 mg/kg bw females during the F1a lactation period were below control values; body weights and body weight change in the remaining test groups (25 and 70 mg/kg bw) were inconspicuous. However during the F1b lactation, losses in body weights and body weight gain were reported for the control, the 25 and the 70 mg/kg bw groups; for the females of the 200 mg/kg bw group, a slight increased weight gain was seen.

Food consumption:
Food consumption of all treated males was slightly below control values. For the females, a decrease in food consumption was seen on week 1 and 2 particularly in the 200 mg/kg bw group. This decrease in food consumption was considered to be treatment-related.
For both matings (F1a & F1b), the treated F0 females showed food consumption similar to that of the control females.
For both matings (F1a & F1b), food consumption of the 200 mg/kg bw F0 females during lactation was slightly reduced compared to that of the control females. This effect was considered to be treatment-related.

Water consumption:
F0 males and females:
The mean water consumption for the control F0 males over the complete study period of 31 weeks ranged between ca. 29 and 38 g/animal/day. For the F0 males of the 25 mg/kg bw group, water consumption was less than in control and ranged between ca. 27 and 36 g/animal/day. For the 70 mg/kg bw group, water consumption over the whole study period also was reduced and ranged between 24 and 35 g/animal/day. In the highest test group, the decrease in water consumption of the F0 males was more pronounced that in the remaining groups and ranged between 20 and 29 g/animal/day. Water consumption of the control F0 females during the first 12 study weeks ranged between 26 and 35 g/animal/day. For the treated groups, water consumption was decreased and ranged between 24 and 33 g/animal/day for the 25 mg/kg bw group, 21 and 27 g/animal/day for the 70 mg/kg bw group, and 18 and 24 g/animal/day for the 200 mg/kg bw group. For both, the F0 males and females, the decrease in water consumption seen in the treated groups was a dose-dependent, treatment-related effect.
F0 females over the gestation period, mating F1a and F1b:
The mean daily water consumption for the control F0 females from day 0 to day 20 of gestation was about 41 g/animal/day for mating F1a and about 46 g/animal/day for mating F1b. Considering the treated groups, for mating F1a, mean water consumption was about 39, 34 and 27 g/animal/day respectively for the 25, the 70 and the 200 mg/kg bw group; for mating F1b, the water consumption respectively was 43, 37 and 32 g/animal/day. In all treated groups and for both, the F1a and the F1b gestation periods, water consumption for the F0 females was decreased when compared to control. The decrease in water consumption was seen as a dose-dependent, treatment-related effect.
F0 females over the lactation period, mating F1a and F1b:
The mean daily water consumption for the control F0 females from day 0 to day 21 of gestation was about 90 g/animal/day for mating F1a and 79 g/animal/day for mating F1b. Considering the treated groups, for mating F1a, mean water consumption was about 90, 81 and 63 g/animal/day respectively for the 25, the 70 and the 200 mg/kg bw group; for mating F1b, the water consumption respectively was about 82, 61 and 61 g/animal/day. Especially in the 70 and the 200 mg/kg bw groups, water consumption of the F0 females was decreased when compared to control. This was true for both, the F1a and the F1b lactation periods. Water consumption in the 25 mg/kg bw group was similar to control. The decrease in water consumption seen at the mid and the high dose was a dose-dependent, treatment-related effect.

F1 parents, mortality, clinical signs of toxicity, necropsy:
Three cases of mortality were reported, which however rather were incidental than treatment-related. In fact, one female of each treatment group (25, 70 and 200 mg/kg bw); the females of the low and the high doses died, died at the beginning of the F2b mating period whereas the female of the mid-dose group died on gestation day 14 of the F2a mating.
Excepted for hair loss and brown staining of the forelimbs reported for the mid-dose female that died, no clinical signs of toxicity were seen. Necropsy of the animals that died revealed congested lungs in the low dose female, and discoloured liver, black foci on the stomach and enlarged kidneys, lymph nodes, spleen and thymus in the mid dose female; no gross lesions were seen in the high dose female. Necropsy of the F1 parents which survived the treatment and were sacrificed, revealed no treatment-related abnormalities.

F1 parents, body weight:
Body weight for the males and females of the 200 mg/kg bw/day group were moderately to significantly below control values; this was particularly true for the first 22 weeks for males and for the 14 first weeks for the females. The decrease in body weight gain was seen as treatment-related effect. The remaining test groups showed no treatment-related effects.
F1 females, body weights during gestation:
The body weights and body weight gain for the 200 mg/kg bw females during gestation were below control values for both, the F2a and the F2b mating. This effect was treatment-related. Body weights and body weight change in the remaining test groups (25 and 70 mg/kg bw/day) were inconspicuous.
F1 females during lactation, body weights:
The body weights and the body weight gain for all treated females during the F2a and F2b lactation periods were within control range and therefore inconspicuous.
F1 parents, food consumption:
Food consumption in the 200 mg/kg bw group was below control values over the F1 generation period, as evidenced by the amounts calculated in g per animal and day; the fact that food consumption expressed as g/kg bw/day was within control range was related to the reduced body weights of the 200 mg/kg bw animals.
F1 female during gestation, food consumption:
For both, mating F2a and mating F2b, the treated F1 females of the 200 mg/kg bw group showed reduced food consumption when compared to the control females; the effect was treatment-related. The remaining treated groups were inconspicuous.
F1 females during lactation, food consumption:
For both, mating F2a and mating F2b, the treated F1 females of the 200 mg/kg bw group showed reduced food consumption when compared to the control females; the effect was treatment-related. The remaining treated groups were inconspicuous.

F1 parents and F1 females during gestation and lactation, water consumption:
F1 males and females:
The mean water consumption for the control males over the complete F1 generation period (4 weeks to 41 weeks old) ranged between ca. 23 and 45 g/animal/day. For the F1 males of the 25 mg/kg bw group, water consumption was less than in control and ranged between ca. 21 and 38 g/animal/day. For the 70 mg/kg bw group, water consumption over the whole study period also was reduced and ranged between ca. 18 and 38.6 g/animal/day. In the highest test group, the decrease in water consumption of the F1 males was more pronounced that in the remaining groups and ranged between 14 and 30 g/animal/day. Water consumption of the control F1 females ranged between 20 and 50 g/animal/day. For the treated groups, water consumption was decreased and ranged between ca. 18 and 42 g/animal/day for the 25 mg/kg bw group, 16 and 34 g/animal/day for the 70 mg/kg bw group and between 13 and 30 g/animal/day for the 200 mg/kg bw group. For both, the F1 males and females, the decrease in water consumption seen in the treated groups was a dose-dependent, treatment-related effect.
F1 females over the gestation period, mating F2a and F2b:
The mean daily water consumption for the F1 control females during day 0 to day 20 of gestation was about 40 g/animal/day for mating F2a and F2b respectively. Considering the treated groups, at mating F2a, the mean water consumption was about 34, 30 and 22 g/animal/day respectively for the 25, the 70 and the 200 mg/kg bw group; at mating F2b, the water consumption respectively was about 41, 36 and 29 g/animal/day. These results indicate that during the F2a gestation period, the water consumption was decreased in comparison to control in all treated groups; during the F2b gestation period, only the females of the 70 and the 200 mg/kg bw group showed a decrease in water consumption when compared to control. The reduced water consumption during gestation was seen as a dose-dependent, treatment-related effect.
F1 females over the lactation period, mating F2a and F2b:
The mean daily water consumption for the F1 control females from day 0 to day 21 of lactation was about 69 and 78 g/animal/day for mating F2a and F2b respectively. Considering the treated groups, at mating F2a, the mean water consumption was about 68, 60 and 52 g/animal/day respectively for the 25, the 70 and the 200 mg/kg bw group; at mating F2b, the water consumption respectively was about 78, 74 and 69 g/animal/day. These results indicate that during the F2a lactation period, the water consumption was decreased in comparison to control in the 70 and the 200 mg/kg bw groups; in the 25 mg/kg bw group, water consumption was similar to control. This also was true for the F2b lactation period. The reduced water consumption during lactation was seen as a dose-dependent, treatment-related effect.

F1a mating:
Reproductive performance and gestation data:
The copulatory index was 92.3% for the 25 mg/kg bw group, 96% for control, and 100 % for each of the 70 and the 200 mg/kg bw groups. The mean copulatory interval for the control group was about 2.4; for the treated groups, intervals of 3.6, 3.0 and 3.1 days were respectively reported for the 25, the 70 and the 200 mg/kg bw group. The fertility indices for the treated males were > 90% and were within control range. The mean gestation length was similar for all groups and had a mean duration of 22.4 days. The fertility indices of the treated females were similar to control for the 25 and the 70 mg/kg bw groups (>90%); in contrast, the fertility index of the females treated with 200 mg/kg bw bronopol was slightly decreased when compared to control (75% versus 87%). Excepted for the fertility index of the 200 mg/kg bw females, all the parameters mentioned above were not affected by the treatment.
Necropsy of offspring:
23 pups were found dead between lactation day 0 and 7 (control: 5, low dose: 6, mid dose: 3, high dose: 9). 19 missing pups (day 0 to 19 of lactation; control: 2, low dose: 4, mid dose: 5, high dose 8) were presumed to having been cannibalized by the dams. Necropsy of dead pups and of pups sacrificed after the lactation period revealed no treatment-related abnormalities.

F1b mating:
The copulatory index was 96.2% for the 25 mg/kg bw group, 100% for the 70 mg/kg bw group and 95.7% for the 200 mg/kg bw group, versus 96% for control. The mean copulatory interval for the control group was about 3.2; for the treated groups, intervals of 2.8, 4.0 and 3.7 days were respectively reported for the 25, the 70 and the 200 mg/kg bw group. The fertility indices for the treated males were > 90% and were within control range. The mean gestation length was similar for all groups and had a mean duration of 22 days. The fertility indices of the treated females were similar for the control group and the 25 and the 70 mg/kg bw groups (respectively 87.5, 88 and 92.3%); for the 200 mg/kg bw group, the fertility index of the females was slightly decreased when compared to control (81.8% versus 87.5%). Excepted for the fertility index of the 200 mg/kg bw females, all the parameters mentioned above were not affected by the treatment.
79 pups were found dead between lactation day 0 and 21 (control: 6, low dose: 10, mid dose: 23, high dose: 40). 21 missing pups (day 0 to 14 of lactation; control: 3, low dose: 3, mid dose: 4, high dose: 11) were presumed to having been cannibalized by the dams. Necropsy of dead pups revealed pale hepatic lobes in two female pups of the same litter in the mid-dose group; one of these females further showed undeveloped renal papilla whereas the second one had distended ureters. In the high dose group, 3 dead pups were small in size.
Excepted for a decrease in size reported for 22 pups of the high dose group during lactation and which clearly was associated to the decreased body weights, necropsy of the pups that survived the lactation period and were sacrificed revealed no more conspicuous abnormalities.

F2a mating:
The copulatory index was > 90% in all groups. The mean copulatory interval for the control group was about 3.5; for the treated groups, intervals of 3.9, 4.3 and 5.7 days were respectively reported for the 25, the 70 and the 200 mg/kg bw group. The fertility indices for the treated males were 100% for each of the low, the mid and the high dose, versus 85% for control. The mean gestation length was similar for all groups and had a mean duration of 22.3days. Fertility indices of 88% for respectively the control and the mid dose group, 96% for the low dose group and 100% for the high dose group were reported. All the parameters mentioned above were not affected by the treatment.
Necropsy:
24 pups were found dead between lactation day 0 and 11 (control: 10, low dose: 7, mid dose: 4, high dose: 3). 13 missing pups (day 0 to 9 of lactation; control: 4, low dose: 2, mid dose: 4, high dose: 3) were presumed to having been cannibalized by the dams.
Necropsy of dead pups revealed no treatment-related abnormalities. Necropsy of the pups that had survived and were sacrificed at the end of the lactation period revealed one case of abnormal eye in the mid dose group, sparse haircoat in ten pups of the same litter in the 25 mg/kg bw group, and missing distal tail in one pup of respectively the mid and the high dose group.

F2b mating:
The copulatory index was about 92% for each of the control, the 70 mg/kg bw and the 200 mg/kg bw groups; for the 25 mg/kg bw group, a copulatory index of 84% was reported. The mean copulatory interval for the control group was about 3.7; for the treated groups, intervals of 3.3, 3.3 and 4.6 days were respectively reported for the 25, the 70 and the 200 mg/kg bw group. The fertility indices for the males were 92.3% for respectively the control, the mid and the high dose group, whereas a value of 84.6% was reported for the low dose group. The mean gestation length was within the same range for all groups and had a mean duration of 22.5 days. The fertility indices of the treated females ranged between 82.6 and 95.7%, whereas a value of 79.2% was reported for the control group. All the parameters mentioned above were not affected by the treatment.
Necropsy:
19 pups were found dead between lactation day 0 and 21 (control: 4, low dose: 7, mid dose: 4, high dose: 4). 9 missing pups (control: 0, low dose: 5, mid dose: 1, high dose: 3) were presumed to having been cannibalized by the dams.
Necropsy of dead pups revealed no treatment-related abnormalities. At necropsy of the pups that had survived and were sacrificed at the end of the lactation period, one control pup, two pups of the low dose group and 14 pups of the high dose groups were found to be small in size. Furthermore, 20 pups of the low dose group (two litters concerned, 10 pups per litter) showed sparse haircoat and one pup of the same group was discoloured purple. One high dose pup showed a mass with hair loss on the top of its head.

Pathology:
F0 generation:
Macroscopic findings at terminal sacrifice: Two males and four females of the 200 mg/kg bw group had kidneys with granular appearance; the finding was seen as treatment-related but was not a direct effect of the test substance as such. No further conspicuous findings were reported.
Organ weights: A dose-related increase in kidney weight was reported for the treated females of all groups; however, the difference between the low-dose group and the control was minimal.
Microscopic findings: A treatment-related increase in incidence of progressive nephropathy was seen in the mid-and high-dose groups, which further was dose-related and more prominent in the females. The finding was in accordance with the macroscopic findings described above as well as with the increase in kidney weight.
F1 parental generation:
Macroscopic findings at terminal sacrifice: One male and two females of the 200 mg/kg bw group had kidneys with granular appearance; the finding was seen as treatment-related but was not a direct effect of the test substance as such. No further conspicuous findings were reported.
Organ weights: The treated males of the high-dose group showed significant decreases in mean body weight, mean absolute heart weight and mean absolute liver. The changes in liver and body weight were seen as apparently treatment-related whereas the changes in heart weight were considered as possibly treatment-related.
Microscopic findings: Progressive nephropathy, which is a common spontaneous lesion seen in the rats of the strain used, was found in all groups including the control; however, in the high dose group, the incidence of this lesion was slightly more increased and was therefore considered to be treatment-related.

Effect levels (P0)

open allclose all
Dose descriptor:
other: LOAEL parental sytemic toxicity
Effect level:
25 mg/kg bw/day
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day

Results: F1 generation

Details on results (F1)

Pathology:
F1b pups:
Macroscopic findings at terminal sacrifice: No treatment-related abnormalities were seen.
Organ weights: No treatment-related changes in organ weights could be evidenced.
Microscopic findings: No treatment-related abnormalities were seen.
F2b generation:
Macroscopic findings at terminal sacrifice: No treatment-related abnormalities were seen.
Organ weights: A treatment-related decrease in mean absolute liver weight was reported for the males of the 200 mg/kg bw group; the females of the same group showed significant decreases in absolute kidney and liver weights.
Microscopic findings: No treatment-related changes were found.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Necropsy of the animals that died or were sacrificed in extremis (F0 parents).

Animal

Clinical signs

Necropsy findings

Control female (died)

Malaligned upper incisors, dry dark matter around eyes, nose and forelimbs

Fracture of a bone in the dorsal nasal area

25 mg/kg bw/day female (died)

Vagina surrounded by wet red matter, which probably was blood

Blood in the lumen of the uterus, four normally developing fetuses, five implantation scars

200 mg/kg bw/day male (died)

Dry red matter around the nose, thin appearance

No abnormalities

200 mg/kg bw/day female one (died)

Cool to touch

No abnormalities

200 mg/kg bw/day female two (died)

No visible symptoms

Diffuse, tan foci on the heart, moderate hepatic congestion, black foci on the stomach mucosa, red foci on the ileum mucosa, mild focal congestion of the caecum mucosa

200 mg/kg bw/day female three (sacrificed)

Dry red matter around the nose, thin appearance, labored breathing, emaciation, absence of righting reflex, cool touch, moribund appearance

Mild hydronephrosis of one kidney, clear fluid in both uterine horns.

200 mg/kg bw/day female four (died)

Dry red matter around the nose, thin appearance

Cloudy cornea in one eye

200 mg/kg bw/day female five (sacrificed)

Thin, dehydrated and moribund appearance, dry, black matter around nose and eyes, labored breathing, pale eyes.

Enlarged adrenals.

Table 2: Group mean body weights (F0 parents).

F0 male, group mean body weights (g)

Study Week

Bronopol (mg/kg bw/day)

0

25

70

200

0

178 +/- 8.7 (N=13)

179 +/- 7.0 (N=13)

181 +/- 8.8 (N=13)

179 +/- 8.2 (N=13)

1

222 +/- 19.4 (N=13)

227 +/- 8.3 (N=13)

223 +/- 14.2 (N=13)

213 +/- 15.8 (N=12)

20

528 +/- 40.2 (N=13)

563 +/- 48.1 (N=13)

563 +/- 33.3 (N=13)

503 +/- 46.3 (N=12)

21

532 +/- 41.1 (N=13)

563 +/- 48.1 (N=13)

566 +/- 31.2 (N=13)

508 +/- 40.2 (N=12)

30

567 +/- 44.3 (N=13)

608 +/- 51.5 (N=13)

610 +/- 40.8 (N=13)

554 +/- 38.3 (N=12)

31

573 +/- 45.5 (N=13)

613 +/- 51.4 (N=13)

616 +/- 46.4 (N=13)

559 +/- 38.1 (N=12)

F0 female, group mean body weights (g)

0

129 +/- 6.8 (N=26)

134 +/- 6.4 (N=26)

134 +/- 7.7 (N=26)

133 +/- 7.4 (N=26)

1

154 +/- 7.7 (N=26)

158 +/- 7.6 (N=26)

156 +/- 17.4 (N=26)

151 +/- 14.5 (N=25)

20

300 +/- 16.2 (N=25)

300 +/- 19.7 (N=26)

309 +/- 27.7 (N=26)

282** +/- 25.6 (N=23)

21

308 +/- 18.6 (N=25)

305 +/- 21.4 (N=26)

314 +/- 27.2 (N=26)

284** +/- 23.4 (N=23)

30

321 +/- 15.9 (N=21)

325 +/- 20.9 (N=22)

325 +/- 24.2 (N=24)

295** +/- 32.3 (N=17)

31

321 +/- 17.3 (N=21)

327 +/- 21.1 (N=22)

326 +/- 25.8 (N=24)

300* +/- 24.5 (N=17)

Table 3: On the basis of the water consumption, the mean intake of bronopol for the F0 animals that survived up to study termination was calculated to be as follows.

Calculated mean bronopol intake (mg/kg bw/day)

F0 animals

Nominal doses (mg/kg bw/day)

25

70

200

F0 males (week 1  - 31 excluding mating on week 13 & 23)

18.1

46.7

121

F0 females (week 1 – 12)

31.3

73.1

189

F0 females, F1a mating, gestation

26.0

62.4

160

F0 females, F1a mating, lactation*

71.1

177

455

F0 females, F1b mating, gestation

25.4

63.2

170

F0 females, F1b mating, lactation*

61.5

157

411

*, the values reported for the lactation period are very increased and can not be

seen as reliable as pups may have contributed to the consumption values

Table 4: Group mean body weights (F1 parents).

F1 male, group mean body weights (g)

Age (weeks)

Bronopol (mg/kg bw/day)

0

25

70

200

4

119 +/- 10.1 (N=13)

121 +/- 9.5 (N=13)

114 +/- 15.9 (N=13)

93** +/- 1912 (N=13)

10

423 +/- 29.8 (N=13)

414 +/- 35.0 (N=13)

407 +/- 43.3 (N=13)

354** +/- 45.7 (N=13)

15

520 +/- 53.0 (N=13)

521 +/- 43.9 (N=13)

517 +/- 52.2 (N=13)

456* +/- 60.3 (N=13)

20

561 +/- 62.6 (N=13)

560 +/- 47.5 (N=13)

570 +/- 61.3 (N=13)

493* +/- 56.3 (N=13)

21

563 +/- 63.5 (N=13)

571 +/- 50.4 (N=13)

582 +/- 60.0 (N=13)

501* +/- 60.6 (N=13)

22

577 +/- 63.7 (N=13)

583 +/- 50.9 (N=13)

595 +/- 60.0 (N=13)

515* +/- 60.0 (N=13)

25

597 +/- 77.1 (N=13)

610 +/- 58.7 (N=13)

619 +/- 69.2 (N=13)

536 +/- 68.2 (N=13)

30

605 +/- 78.3 (N=13)

615 +/- 61.7 (N=13)

621 +/- 70.9 (N=13)

542 +/- 63.3 (N=13)

41

680 +/- 96.2 (N=12)

688 +/- 90.2 (N=12)

707 +/- 90.7 (N=12)

608 +/- 76.9 (N=12)

F1 female, group mean body weights (g)

4

105 +/- 11.2 (N=26)

102 +/- 10.9 (N=26)

97 +/- 13.2 (N=26)

80** +/- 14.0 (N=26)

10

239 +/- 19.5 (N=26)

238 +/- 22.1 (N=26)

238 +/- 23.1 (N=26)

217** +/- 28.6 (N=26)

14

271 +/- 27.1 (N=26)

269 +/- 22.4 (N=26)

270 +/- 27.9 (N=26)

252* +/- 30.9 (N=26)

15

274 +/- 28.1 (N=26)

273 +/- 23.7 (N=26)

279 +/- 30.5 (N=26)

255 +/- 32.3 (N=26)

16

280 +/- 28.8 (N=25)

273 +/- 28.0 (N=26)

280 +/- 29.7 (N=23)

264 +/- 31.3 (N=24)

*, p<0.05; **, p<0.01, N = Number of animals used for mean calculation

Table 5: On the basis of the water consumption, the mean intake of bronopol for the F1 animals that survived up to study termination was calculated to be as follows.

Calculated mean bronopol intake (mg/kg bw/day)

F1 animals

Nominal doses (mg/kg bw/day)

25

70

200

F1 males (age: 4 to 41 weeks, excl. mating periods)

18.3

50.4

135.6

F1 females (age: 4 to 17 weeks)

30.5

78.3

202.4

F1 females, F2a mating, gestation

21.6

52.4

131.2

F1 females, F2a mating, lactation*

58.3

133.5

367.8

F1 females, F2b mating, gestation

24.4

60.3

154.0

F1 females, F2b mating, lactation*

61.7

162.1

466.0

*, the values reported for the lactation period are very high and can not be seen

as reliable as pups may have contributed to the consumption value

Table 6: Mean body weights of live pups during lactation (F1a mating).

Group mean body weight during lactation (g +/- SD)

Day

Bronopol test group (mg/kg bw/day)

0

25

70

200

0

6.3 +/- 0.54

6.1 +/- 0.60

6.2 +/- 0.54

5.9 +/- 0.87

4 (BR)

10.3 +/- 1.23

9.7 +/- 1.18

9.8 +/- 0.70

9.4 +/- 1.84

4 (AR)

10.3 +/- 1.23

9.7 +/- 1.14

9.8 +/- 0.71

9.5 +/- 1.82

7

15.5 +/- 1.67

15.2 +/- 1.41

15.1 +/- 1.40

14.0* +/- 2.30

14

29.8 +/- 2.16

29.5 +/- 1.81

29.7 +/- 2.82

25.9** +/- 3.39

21(males)

45.9 +/- 4.08

45.5 +/- 3.59

45.0 +/- 4.98

38.5** +/- 6.42

21(females)

44.6 +/- 3.61

44.4 +/- 3.51

44.5 +/- 5.39

36.0** +/- 5.40

BR, before reduction; AR, after reduction, SD, standard deviation; *, p<0.05; **, p<0.01

Table 7: Mean body weights of live pups during lactation (F1b mating).

Group mean body weight during lactation (g +/- SD)

Day

Bronopol test group (mg/kg bw/day)

0

25

70

200

0

6.2 +/- 0.78

6.0 +/- 0.69

6.1 +/- 0.82

5.3** +/- 0.93

4 (BR)

10.5 +/- 2.30

10.0 +/- 2.13

9.9 +/- 1.87

8.3** +/- 2.15

4 (AR)

10.5 +/- 2.31

10.0 +/- 2.10

9.9 +/- 1.87

8.3** +/- 2.14

7

16.1 +/- 3.19

15.8 +/- 2.62

15.1 +/- 2.32

12.7** +/- 3.03

14

31.4 +/- 5.34

31.5 +/- 3.41

30.1 +/- 3.76

26.1** +/- 4.26

21(males)

53.5 +/- 7.67

52.4 +/- 5.30

49.9 +/- 6.81

42.6** +/- 6.82

21(females)

50.5 +/- 8.17

50.4 +/- 6.26

47.2 +/- 5.84

39.1** +/- 6.14

BR, before reduction; AR, after reduction, SD, standard deviation; *, p<0.05; **, p<0.01

Table 8: Mean body weights of live pups during lactation (F2a mating).

Group mean body weight during lactation (g +/- SD)

Day

Bronopol test group (mg/kg bw/day)

0

25

70

200

0

6.0 +/- 0.40

6.1 +/- 0.60

6.6* +/- 0.94

5.8 +/- 0.73

4 (BR)

10.2 +/- 0.98

9.6 +/- 1.14

10.9 +/- 2.05

9.2* +/- 1.56

4 (AR)

10.2 +/- 0.90

9.7 +/- 1.11

10.9 +/- 2.04

9.2** +/- 1.56

7

14.8 +/- 1.24

14.2 +/- 1.74

16.1* +/- 2.39

13.4* +/- 2.46

14

27.8 +/- 2.26

26.4 +/- 3.82

28.8 +/- 3.36

24.1** +/- 4.41

21(males)

44.8 +/- 3.86

42.0 +/- 6.80

45.3 +/- 6.07

36.6** +/- 6.66

21(females)

43.2 +/- 3.76

40.9 +/- 6.26

43.0 +/- 5.68

34.7** +/- 5.83

BR, before reduction; AR, after reduction, SD, standard deviation; *, p<0.05; **, p<0.01

Table 9: Mean body weights of live pups during lactation (F2b mating).

Group mean body weight during lactation (g +/- SD)

Day

Bronopol test group (mg/kg bw/day)

0

25

70

200

0

6.7 +/- 0.76

6.3 +/- 0.66

6.4 +/- 0.72

5.8** +/- 0.72

4 (BR)

11.5 +/- 2.54

10.4 +/- 1.34

11.0 +/- 2.01

9.1** +/- 1.53

4 (AR)

11.6 +/- 2.51

10.7 +/- 1.26

11.1 +/- 1.98

9.2** +/- 1.49

7

17.7 +/- 3.34

16.3 +/- 1.96

16.8 +/- 2.54

13.8** +/- 2.33

14

32.5 +/- 4.35

31.4 +/- 2.71

31.6 +/- 4.14

26.3** +/- 3.63

21(males)

53.1 +/- 5.69

51.5 +/- 5.49

51.3 +/- 6.80

42.5** +/- 5.55

21(females)

51.8 +/- 6.91

48.5 +/- 4.18

49.9 +/- 6.39

41.0** +/- 5.65

BR, before reduction; AR, after reduction, SD, standard deviation; *, p<0.05; **, p<0.01

Applicant's summary and conclusion