Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
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
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.
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
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
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.
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.
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
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.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day
Reproductive effects observed:
not specified

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
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Three key studies were used in order to get to a reliable conclusion regarding the effects on fertility of Bronopol. The database for toxicity to reproduction (effects on fertility) is complete and is considered to meet data requirements of REACH.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction: Two-Generation study in rat

In a two-generation study, male and female Charles River COBS CD rats were applied drinking water containing 0, 25, 70 or 200 mg/kg/day of Bronopol ( International Research and Development Corporation, 1987). At each dose level 13 males and 26 females were treated for a minimum of 80 days prior to mating. Then animals were mated in both F0 and F1 generation (the F0 females were mated twice to produce a F1a and F1b generation, F1b parental animals were used to produce a F2a generation, and finally F1b females were subjected to a further mating to produce a F2b generation).

All parental animals were checked for mortality, clinical symptoms of toxicity, body weight, food consumption, water consumption and test substance intake; necropsy included gross pathology and histopathology. Reproduction, delivery and litter parameters included mating performance, fertility indices, gestation length, litter size, number of stillbirths, number of live births and gross abnormalities.

No treatment-related mortalities and symptoms were reported. Treatment-related reduced body weight and body weigh gain were reported for high dose animals.

An increased incidence of progressive nephropathy was reported for some high dose parental animals of both sexes (F0 and F1); the finding was seen as treatment-related but was not a direct effect of the test substance as such. In high dose F1 parents, changes in liver and body weight were reported as apparently treatment-related effects whereas changes in heart weight were reported as possibly treatment-related. A treatment-related decrease in mean absolute liver weight was reported for the F2b males of the high dose group; the F2b females of the same group showed significant decreases in absolute kidney and liver weights.

For the F0 generation, the fertility index of the high dose females was slightly reduced compared to control, especially for F1a mating; all other reproduction and delivery parameters were inconspicuous. For the F1 generation, all reproduction and delivery parameters were inconspicuous. Survival indices of all F1a pups, of the mid and high dose F1b pups, and of all F2a and F2b pups were inconspicuous; survival index of the high dose F1b pups was slightly decreased. The high dose F1a, F1b, F2a and F2b pups showed a treatment-related significant decrease in mean body weights during lactation; the mean body weight of the mid dose F1b pups was slightly decreased towards the end of the lactation period. Mean body weights of the remaining pups were inconspicuous. Necropsy of dead F1a, F2a and F2b pups revealed no treatment-related abnormalities.

Treatment-related effects referring to systemic toxicity were seen at all tested doses of Bronopol but were particularly pronounced at the highest dose tested. With regard to reproductive or litter parameters, no significant treatment-related effects were reported; in fact the effects reported at the highest test dose of 200 mg/kg bw/day rather resulted from the high systemic toxicity observed at this dose level than indicating reproductive toxicity.

The achieved mean doses of Bronopol for the F0 and the F1 males and females were 22.5, 55.2 and 147 mg/kg bw/day, respectively. The lower achieved dosages of Bronopol were due to the reduced water consumption, which was observed in all treated groups.

Overall, ingestion of Bronopol elicited signs of toxicity at all dosages, though the only reproductive or litter parameter affected at the 25 and 70 mg/kg bw/day dosages was a minimal (not significantly) decrease in the bodyweight of F1b pubs at weaning. Conclusively, these findings were regarded as non-relevant with respect to reproduction and offspring.

For reproduction (effects on fertility) NOAEL parental was 200 mg/kg bw/day and NOAEL offspring was 200 mg/kg bw/day. Furthermore, LOAEL parental sytemic toxicity was defined as 25 mg/kg bw/day.

This study is classified as acceptable (key study).The study was conducted according to the SOP of the International Research and Development Corporation (IRDC). The stability of the test substance over the experimental period was assured.

 

 

Toxicity to reproduction: Two-Generation study in rat (range-finder)

In a range-finding study (International Research and Development Corporation, 1986) rats of the Charles River COBS CD strain were randomly assigned to one control and four treatment groups of five males and five females each and used to determine dosage levels of Bronopol for a two-generation reproduction study. Concentrations of 0.025, 0.050, 0.1 and 0.2% to produce anticipated dose levels of 25, 50, 100 and 200 mg/kg bw/day were administered orally in the drinking water to males and females throughout the study.

Treatment was conducted from beginning of the study until sacrifice (on day 3 of lactation), except for the 10 day mating period (which corresponds to about 5-6 weeks of treatment (14 days pre-mating, ca. 21 days of gestation, 3 days post parturition). Control animals received drinking water alone. Mating was conducted after 14 days of treatment.

The parent animals were checked for mortality and clinical symptoms of toxicity, body weight, food consumption, water consumption and intake of test substance. Reproduction and delivery data included mating performance, fertility indices (males and females), gestation length, implantation sites, implantation losses, number of pups delivered. The litters were examined for litter size, pup survival indices, and gross abnormalities. Dead pups were recorded and subjected to necropsy. Surviving pups were checked for mortality, abnormalities and body weight. On day 3 of lactation, all parental animals and pups were sacrificed for the purpose of necropsy.

Water consumption was reduced during the first week for all treated groups of females, and throughout the study for all treated groups of males. Consequently, the dosages of Bronopol that the animals received were less than intended. Mean achieved dosages for the males were 17.2, 31.8, 64.3 and 113.3 mg/kg bw/day and, for the females, were 30.9, 52.7, 95.7 and 193.7 mg/kg bw/day.

One 200 mg/kg bw/day male was sacrificed in extremis during week 2; there were no treatment-related antemortem or necropsy findings. There were no deaths in any other group.

For the males, treated animals in all groups gained less weight than controls and the magnitude of the effect was generally dose-dependent. Some females from all groups, including controls, gained less weight than expected during the first two weeks of treatment; body weight data could not be assessed thereafter.

During the first week of the study, mean food intake for males and females given 200 mg/kg bw/day was reduced. Necropsy of the sacrificed animals was inconspicuous except for one female of the 100 mg/kg bw/day group that displayed a cavity in the liver. There was no indication of any treatment-related effects on reproductive performance or on the progeny. None of the considered litter parameters showed treatment-related effects and the pups showed no treatment-related abnormalities.

Conclusively, within the present range-finding study, treatment-related effects referring to systemic toxicity were seen at all tested doses of Bronopol. However, the tested doses affected neither the reproductive performance of the rats nor did they affect the offspring.

On the basis of these results, for the main two-generation reproduction study with rats, the nominal doses selected were 25, 70 and 200 mg/kg bw/day.

 

 

Toxicity to reproduction: One-Generation study in rat

The effects of Bronopol on the fertility and the reproduction of rat were investigated (Huntingdon Research Centre, 1973). Male and female rats of the CD strain were used; each of three test groups (control, 20 and 40 mg/kg bw Bronopol) consisted of 11 males and 22 females. The test animals received daily oral application of the test substance throughout an overall period of 19 weeks; treatment of the males was started 63 days prior mating whereas treatment of the females was started 14 days prior mating. For mating, the animals were caged 1 male to 2 females; during mating daily vaginal smears were taken for 20 days, and the day when sperm or a vaginal plug was found was considered day 0 of pregnancy. On day 13 of pregnancy 10 females per group were sacrificed for examination of the reproductive tract. Parent animals and litters were observed until 21 days after parturition. The parental animals were observed for mortality, clinical signs of toxicity and body weight. Considering the fertility and reproduction, following parameters were assessed: mating performance, pregnancy rate, duration of gestation and parturition, number of corpora lutea, pre- and post-implantation losses, resorptions, viability of the fetuses, litter loss, size and weight, pup survival, and weight, and abnormalities, which were subjected to histopathological examination.

Group mean values were calculated in 2 ways using the litter as the basic sample unit. Non-parametric methods, usually Wilcoxon test, were used for statistical assessment where necessary.

Parental animals: Five cases of death were reported (one male and one female in the 20 mg/kg bw/day group; one male and two females in the 40 mg/kg bw/day group), which however could not be attributed to the treatment. No treatment-related symptoms of toxicity were seen. Body weight changes of all treated females (pre-mating, gestation, lactation and post-lactation period) and of the males treated with 20 mg/kg bw/day Bronopol, were within control range and therefore inconspicuous. For the males of the 40 mg/kg bw group and starting from week 2 of treatment, weight gain was found to be slightly but clearly below control values.

Reproduction parameters: All considered parameters were within control range and showed no treatment-related effects; no treatment-related abnormalities were seen.

Conclusively, the fertility and reproductive performance of the rats as well as the pre-and post-natal development of the young were not adversely affected by the repeated oral treatment with Bronopol at doses up to and including 40 mg/kg bw/day.

This study is classified as acceptable (key study).The test was conducted according to FDA Guidelines; GLP was not compulsory at the time the study was conducted.


Short description of key information:
In a two-generation study the test substance was applied via dring water in doses of 0, 25, 70 or 200 mg/kg/day. Treatment-related effects referring to systemic toxicity were seen at all tested doses of Bronopol but were particularly pronounced at the highest dose tested. With regard to reproductive or litter parameters, no significant treatment-related effects were reported; in fact the effects reported at the highest test dose of 200 mg/kg bw/day rather resulted from the high systemic toxicity observed at this dose level than indicating reproductive toxicity.

Justification for selection of Effect on fertility via oral route:
The key study was selected which represents a GLP-conform 2-Generation-study in rat.
Furthermore the corresponding range-finding study and a previous one-generation study (both key studies) are listed, which supports the findings of the key study (International Research and Development Corporation, 1987)

Effects on developmental toxicity

Description of key information
In a rat developmental toxicity study, no evidence of developmental toxicity was obseerved at any of the dose levels tested (0, 10, 28 and 80 mg/kg bw/d Bronopol). Thus, the NOAEL for maternal toxicity was 80 mg/kg bw/day and the NOAEL for embryotoxic / teratogenic effects was 80 mg/kg bw/day, too.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The author had applied the criteria of 40 CFR 158.34 for flagging studies for potential adverse effects, to the results of the present study.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Age: 8 to 10 weeks
Mean weight: 201 to 225 g
Route of administration:
oral: gavage
Details on exposure:
The treatment period was followed by a post-exposure period of 5 days
Details on mating procedure:
Follow an acclimatisation period of 10 days, the females were mated within 3 consecutive days. The females were paired with the sexually mature males at a ratio of 2 females:1male. The presence of sperm in the vaginal smear indicated day 0 of pregnancy.
Duration of treatment / exposure:
Day 6 to 15 of pregnancy
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
10, 28, 80 mg/kg bw/day
Basis:

No. of animals per sex per dose:
24 females per test group
Control animals:
yes, concurrent no treatment
Details on study design:
Sex: female
Duration of test: 15 days (day 6 to day 20 of pregnancy)
Maternal examinations:
Body weight: Body weights were recorded on day 0, daily from day 6 to day 15 of pregnancy, and finally on day 20.
Food consumption: Food consumption was measured over following intervals: day 0 to day 6, day 6 to day 9, day 9 to day 12, day 12 to day 15 and day 15 to day 20 of pregnancy.
Clinical signs: The animals were checked daily from day 0 to day 20 of pregnancy for clinical symptoms and mortality.
Necropsy: On day 20 of pregnancy, the females were sacrificed for the purpose of necropsy; they were examined for gross abnormalities. Females, which were sacrificed prematurely in extremis, also were subjected to necropsy. Organs or tissues showing abnormalities were fixed in neutral buffered formaldehyde for further examination.
Ovaries and uterine content:
The dams were examined for pregnancy status, gravid uterus weight, number of corpora lutea, and number and distribution of implantation sites. The implantations were classified in early resorptions, late resorptions, dead fetuses and live fetuses; they were further separated in numbers for each horn.
Following indices were calculated:
Pre-implantation loss (%):
(Number of corpora lutea - Total number of implantation sites) x 100/Number of corpora lutea

Post-implantation loss (%):
(Total number of implantation sites - Number of live fetuses) x 100/Total number of implantation sites

Fetal examinations:
General: The live fetuses were weighed and were examined for sex. All fetuses were examined for external abnormalities. The mean fetal body weights were calculated for each litter and sex; group mean body weights were calculated from the litter means.
Soft tissue: About 2/3 of the live fetuses from each litter were placed in 70% alcohol for the purpose of subsequent dissection and examination for visceral abnormalities. The remaining fetuses from each litter were fixed in Bouin´s fluid and were then transferred in 70% alcohol for slight fixation for subsequent sectioning and/or dissection.
Skelet: The carcasses of the 2/3 fetuses from each litter, which were fixed in 70% alcohol and were then dissected and examined for visceral abnormalities, were further processed for skelet examination. For this purpose, the carcasses were cleared in a potassium hydroxide solution, stained with Alizarin red S and preserved in aqueous glycerol with thymol crystals. The bones were identified and examined for shape, size and extent of ossification.
Statistics:
Group means and standard deviations (SD) were calculated where appropriate, and the data were subjected to analysis of variance or to the Kruskal-Wallis test. When significance was achieved (p>95%) and depending on the test method (analysis of variance or Krustal-Wallis test), each treated group was then compared to control using either Dunnett´s test or Dunn´s multiple comparison test.
Maternal body weights, gravid uterus weights and food consumption: These data were subjected to analysis of variance.
Number of corpora lutea, live fetuses and implantation sites: These data were subjected to analysis of variance; the results for each group were compared by means of the Kruskal-Wallis test.
Fetal body weights: Group mean fetal body weights were calculated from the litter means and were compared by analysis of variance.
Sex ratio: The sex ratio was calculated for each litter and the results for each group were compared by means of the Kruskal-Wallis test.
Fetuses with abnormalities: The percentages of fetuses with abnormalities in each litter were calculated, and the group mean percentages, which were calculated from the litter percentages, were compared by means of the Kruskal-Wallis test.
Details on maternal toxic effects:
Details on maternal toxic effects:
Mortality and clinical symptoms of toxicity: No mortality was observed and no treatment-related clinical symptoms were reported.
Body weight: At the highest tested dose of 80 mg/kg bw/day, the maternal body weight gain was significantly below that of control animals over days 6 to 7 of pregnancy (1+/- 5 g versus 5 +/- 3 g for control). Thereafter, body weight gain in this group turned back to control level.
Food consumption: Food consumption was similar in all groups and therefore inconspicuous.
Necropsy: Necropsy of the dams revealed no treatment-related abnormalities.
Pregnancy and implantation data:
The number of pregnant females per test group was 24, 22, 24 and 24 for the control, the 10 mg/kg bw, the 28 mg/kg bw and the 80 mg/kg bw group respectively. None of the considered parameters was affected by the treatment.
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Fetal parameters: None of the considered fetal parameters (sex ratio, fetal weight, gravid uterus weight) was affected by the treatment.
Fetal major abnormalities: A total of 1, 0, 1 and 3 fetuses showing major abnormalities (skeletal and external/visceral combined) was reported for the control, the 10 mg/kg bw, the 28 mg/kg bw and the 80 mg/kg bw group respectively In fact, one case of pulmonary valvular atresia was reported for the control group and one case of microphthalmia was reported for the 28 mg/kg bw group; in the 80 mg/kg bw, two cases of exencephaly (with associated skull abnormalities, open eye, protruding tongue and partial cleft palate) and one case of microphthalmia were reported. No abnormalities were found in the 10 mg/kg bw group. The reported abnormalities were of the type occurring spontaneously in the used rat strain, and the incidences were within background range. The increased incidence seen in the 80 mg/kg bw group was mainly due to one litter containing two fetuses with similar abnormalities (exencephaly with associated skull abnormalities, open eye, protruding tongue and partial cleft palate),and therefore, the finding was considered to rather be genetic than due to treatment.
Fetal minor abnormalities: When compared to control, the incidence of fetuses with incomplete ossification of one or more sacral neural arches was found to be significantly lowed in the 80 mg/kg bw group than in control, indicating a slightly advanced ossification when compared to control. Comparison to background mean revealed that the advance was not unusual. Furthermore, in both the 28 and the 80 mg/kg bw groups, advanced ossification of the forelimb phalanges was seen when compared to control; however, when compared to background mean, the advance was not unusual. The authors concluded that there might have been an association between treatment and advanced ossification of the sacral neural arches and forelimb phalanges; however, the findings still were within background mean range and therefore not unusal.
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Five key studies (rat/rabbit) were used in order to get to a reliable conclusion regarding the developmental toxicity potential of Bronopol. This database on developmental toxicity is complete and is considered to meet the relevant data requirements of REACH.
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

Rat developmental toxicity study

The effects of Bronopol on the embryonic and fetal development of rat following oral daily administration to pregnant females by gavage during organogenesis were investigated (Toxicology Laboratories Limited, 1995). Pregnant Sprague-Dawley rats (24 rats/test group) were treated from day 6 to day 15 of pregnancy with 0, 10, 28 and 80 mg/kg bw/day of Bronopol (purity >= 99.5 %) by gavage. The treatment period was followed by a post-exposure period of 5 days, and on day 20 of pregnancy, the dams were sacrificed for the purpose of necropsy. The animals were observed for clinical symptoms of toxicity, mortality, body weight and for food consumption. At necropsy, the dams were examined for gross abnormalities and furthermore, they were examined for pregnancy status, gravid uterus weight, number of corpora lutea, and number and distribution of implantation sites. The implantations were classified in early resorptions, late resorptions, dead fetuses and live fetuses; they were further separated in numbers for each horn. The live fetuses were weighed and were examined for sex. All fetuses were examined for external abnormalities. The mean fetal body weights were calculated for each litter and sex; group mean body weights were calculated from the litter means. About 2/3 of the live fetuses from each litter were placed in 70% alcohol for the purpose of subsequent dissection and examination for visceral abnormalities. The remaining fetuses from each litter were fixed in Bouin´s fluid for further examinations.

The statistical assessment of the data was mainly based on the analysis of variance and the Kruskal-Wallis test. When a significance was achieved (p>95 %) and depending on the test method (analysis of variance or Krustal-Wallis test), each treated group was then compared to control using either Dunnett´s test or Dunn´s multiple comparison test.

Test substance formulation, analysis of Bronopol content: The weighed amount of test substance was added to the appropriate volume of purified water and was mixed. The pH of the test solution was adjusted to 4 by means of HCl. Fresh formulations were prepared daily and separate formulations were prepared for each dose level. Two samples were taken from each formulation prepared for the first and the last days of dosing; the samples were analysed for Bronopol content. The analysis of the samples taken on first day of dosing revealed a Bronopol recovery of 100 to 102% of nominal, confirming the accuracy of the preparation. The analysis of the samples taken on the last day of dosing revealed a Bronopol recovery of about 91 to 98 % of nominal concentration of the solutions used for the mid and high dose group respectively, and about 83 to 88 % for the solution used for the low dose group. As chromatography of the samples taken on the last day of dosing revealed secondary peaks, which were considered to be due to elevated pH, the accuracy of the preparation could not be confirmed for the formulations prepared for the last day of dosing. However, as a standard procedure was used and as the pH value of the solutions was confirmed, the formulations were considered as acceptable for use.

Maternal toxicity: At the highest tested dose of 80 mg/kg bw/day a significant but transient decrease in body weight gain was reported for days 6 to 7 of pregnancy (1+/- 5 g versus 5 +/- 3 g for control). Thereafter, body weight gain in this group turned back to control level. No further treatment-related effects were seen.

Pregnancy and implantation data: None of the considered pregnancy data was affected by the treatment.

Embryonic/Fetal development: No adverse effect of Bronopol treatment on embryonic and fetal development could be evidenced. In fact, advanced ossification of the sacral neural arches in the 80 mg/kg bw/day group, and advanced ossification of the forelimb phalanges in both the 28 and the 80 mg/kg bw/day groups were reported; these findings might have been related to the treatment, but as they still were within background mean range, they were not seen as conspicuous.

There was no evidence of developmental toxicity at any of the dose levels tested.

Thus, the NOAEL for maternal toxicity was 80 mg/kg bw/day and the NOAEL for embryotoxic / teratogenic effects was 80 mg/kg bw/day, too.

This study is classified as acceptable (key study). The test was conducted according to EPA OPP 83-3 and followed GLP.

 

 

Rat developmental toxicity, range-finding study

In a range-finding study the suitable dose levels to be used in a further main developmental study with rat were defined (Toxicology Laboratories Limited, 1993). Within an initial test defined as phase I, pregnant Sprague-Dawley rats (5/test group) were treated from day 6 to day 15 of pregnancy with 0, 3, 10, 30 and 100 mg/kg bw/day of Bronopol (purity >= 99.5%) by gavage. The initial test was complemented with a further test, defined as phase II, where additional groups of pregnant females were treated with 60, 80 and 100 mg/kg bw/day Bronopol. The treatment period was followed by an exposure period of 5 days, and on day 20 of pregnancy, the dams were sacrificed for the purpose of necropsy. Moribund animals were sacrificed in extremis prior test termination. The animals were observed for clinical symptoms of toxicity, mortality, body weight and for food consumption. At necropsy, the dams were examined for gross abnormalities and furthermore, they were examined for pregnancy status, gravid uterus weight, number of corpora lutea, and number and distribution of implantation sites. The implantations were classified in early resorptions, late resorptions, dead fetuses and live fetuses; they were further separated in numbers for each horn. The fetuses were taken and were examined for external abnormalities; live fetuses were weighed and were examined for sex. The statistical assessment of the data.was mainly based on the analysis of variance and the Kruskal-Wallis test. When a significance was achieved (p > 95 %) and depending on the test method (analysis of variance or Krustal-Wallis test), each treated group was then compared to control using either Dunnett´s test or Dunn´s multiple comparison test.

Test substance formulation: The weighed amount of test substance was added to the appropriate volume of purified water and was mixed. The pH of the test solution was adjusted to 4 by means of HCl. Samples were taken for analytical monitoring of the test substance contents. The results of the analysis (samples of phase II) revealed, that the formulation of the samples was correct, and that there were nor gross deviations from the nominal concentrations.

Maternal toxicity: Signs indicative of maternal toxicity were observed from 30 mg/kg bw/day, up to the highest test dose of 100 mg/kg bw/d of Bronopol. In fact, these signs mainly consisted of a reduction in body weight gain, a reduction in food consumption, a poor state of health (100 mg/kg bw/day) and impaired respiration (100 mg/kg bw/day). No signs of toxicity were seen at the lowest tested doses of 3 and 10 mg/kg bw/day respectively.

Three females of the 100 mg/kg bw/day group were sacrificed in extremis. Two of these females were sacrificed during phase I of the study, necropsy revealed red lung lobes in both cases and in one female haemorrhaging of the stomach glandular mucosa and gas in the caecum also were reported. The third 100 mg/kg bw female which was sacrificed in extremis during phase II of the study had an extensive ulceration within the glandular stomach mucosa and colon contents were dehydrated; this female was not gravid.

Developmental toxicity: No signs of developmental toxicity could be evidenced; in fact, all considered parameters were inconspicuous.

Conclusively, on the basis of the results of the present range-finding study, a dose between 80 and 100 mg/kg bw/ day is considered suitable as high dose level for a main developmental study with rats; as the low dose level should be a “no effect”- level, a dose between 3 and 10 mg/kg bw/day was taken; a mid dose level would be chosen between 20 and 30 mg/kg bw/day.

This study is classified as acceptable (key study). The test followed GLP; no guideline was mentioned, however the conduct of the study was very acceptable.

 

 

Effects of Bronopol on peri- and post-natal development of rat

The effects of Bronopol on the peri- and post-natal development of rat pups following daily oral administration of the test substance in pregnant females (CD rats) was investigated from day 15 of gestation to day 21 post-partum (Huntingdon Research Centre, 1973).

Preliminary test: A preliminary test was conducted for determination of the dose level to be used for the main test. Dosing was started on day 15 of pregnancy and was continued until day 12 post parturition. Four test groups were used, with 5 animals/group; following dose levels were tested: 0, 10, 20 and 40 mg/kg bw/day. The test solutions were prepared in distilled water. The control group received the vehicle alone. The animals were checked daily for clinical symptoms and mortality, Body weights were recorded on days 1, 7, 14 and 20 of gestation, and on day 0, 7and 12 post partum. The dams were examined for pregnancy rate, pregnancy duration and dystocia during parturition. The newborns were counted, weighed and examined for abnormalities. All litters were inspected daily for dead and/or abnormal pups. The pups were weighed again on day 4 and 12 post-partum; on day 12 post-partum, they were sacrificed and were examined for external and internal abnormalities as well as for the sex (gonadal inspection). Pups that died during the study period also were subjected to necropsy.

Main test: Dosing was started on day 15 of pregnancy and was continued until day 21 post parturition. Three groups were used, with 20 animals/group; following dose levels were tested: 0, 20 and 40 mg/kg bw/day. The animals were checked daily for clinical symptoms and mortality. Body weights were recorded on days 1, 7, 14 and 20 of gestation, and on day 0, 7, 14 and 21 post partum. The dams were examined for pregnancy rate, pregnancy duration and dystocia during parturition. The newborns were counted, weighed and examined for abnormalities. All litters were inspected daily for dead and/or abnormal pups. Cumulative group mean pup losses were calculated from individual litter percentages. The pups were weighed again on day 4, 12 and 21 post-partum; on day 21 post-partum, they were sacrificed and were examined for external and internal abnormalities as well as for the sex (gonadal inspection). Pups that died during the study period also were subjected to necropsy. Statistical assessment of intergroup differences mainly was based on Wilcoxon test.

Preliminary test: No treatment-related mortality was observed and no treatment-related clinical symptoms were reported. In fact, one dam of the 40 mg/kg bw group was sacrificed in extremis on day 24 because of dystocia; necropsy revealed one dead pup in the vagina whereas further 15 dead pups were found in the uterus which was filled with blood. No treatment-related effects were reported for body weight, pregnancy rate and pregnancy duration. Except for the dam of the 40 mg/kg bw/day group which was sacrificed in extremis, no litter losses were reported. Litter size, pup mortality, litter weights and mean pup weights showed no treatment related effects; pups were free from abnormalities.

Main test: Two cases of mortality were reported, which occurred in the 20 and the 40 mg/kg bw/day groups. In both cases, the dams died during the post-partum period; both dams had low initial body weights when compared to the remaining animals. The mortalities were not considered to be treatment-related. No treatment-related clinical symptoms were reported. Body weight, pregnancy rate and pregnancy duration were unaffected by the treatment. With regard to litter and pup data, one dam of the control group, two dams of the 20 mg/kg bw/day group and one dam of the 40 mg/kg bw/day group showed total litter loss. The litter losses were not treatment-related. Compared to control, pup mortality was slightly increased in the treated groups from day 4; in fact, the differences were statistically significant on day 12 and 21 for the 20 mg/kg bw group, and on day 21 for the 40 mg/kg bw group. These findings however were not considered to be of toxicological relevance as the pup mortality in the control group was unusually low when compared to laboratory standard range; furthermore, total litter losses were similar in the control and the high dose group. Litter and mean pup weights in the treated groups were slightly below control values from day 12 post-partum and were below the laboratory standard range on day 21; however, the differences were of no statistical significance. The pups showed no treatment-related abnormalities.

Conclusively, neither the dams nor the pups showed treatment-related effects at the dose levels tested in the present study.

Thus, the NOAEL for maternal toxicity was 40 mg/kg bw/day, the NOAEL for embryotoxic / teratogenic effects was 40 mg/kg bw/d and the NOAEL for post natal development was 40 mg/kg bw/day also.

This study is classified as acceptable (key study). Investigations were conducted prior to implementation of guidelines, and GLP was not compulsory at the time the study was done. However, the data reported within the present study are scientifically acceptable and are seen as complementary to the data obtained from the other available studies on developmental toxicity.

 

 

Rabbit developmental toxicity (teratogenicity) study

The effects of Bronopol on the embryonic and fetal development of rabbit following oral daily administration to pregnant females was investigated by gavage during organogenesis (Toxicology Laboratories Limited, 1991/TX92042). Each female was mated with a buck and was then given an intravenous injection of 25 IU chorionic gonadotropin to stimulates ovulation. Mating was conducted at the supplier´s premises, and at their arrival in the testing laboratories, the females were on day 2 of pregnancy. The test substance (purity 99.8%) was administered once daily by gavage from day 7 to 19, inclusive, of pregnancy. Each test group comprised 18 to 20 animals, and the test doses were as follows: 5, 20, 40 and 80 mg/kg bw/day. Control animals received vehicle only. During the treatment period and thereafter until day 28 of pregnancy, the dams were checked daily for clinical symptoms and mortality. Body weights were recorded on day 0 (by the supplier) and thereafter on day 3, 7 to 19 inclusive, 22, 25 and 28. Food consumption was measured every 2 days from day 3 to 27, and over one day from day 27 to 28. Except for two females, all dams were sacrificed on day 28 of pregnancy. In fact, one female of the 5 mg/kg bw/day group and one of the 80 mg/kg bw/day group were sacrificed in extremis because of bad state of health. At necropsy, the animals were examined for gross abnormalities, and organs/tissues with abnormalities were fixed for further examination. The dams sacrificed on day 28 of pregnancy were examined for gravid uterus weight, number of corpora lutea, and number/distribution of implantation sites. The implantations were classified in early and late resorptions, and in dead and live fetuses, and were separated in number per horn. Pre- and post-implantation losses were calculated.

The live fetuses were examined for external abnormalities before being sacrificed. They were weighed and were then slight fixed in alcohol. The fixed fetuses were prepared for and subjected to visceral examination and sex determination. Following evisceration, the fetuses were placed again in alcohol and after at least 12 hours, the brain was removed and examined. After brain removal, the carcasses were cleared in a potassium hydroxide solution, stained with Alizarin red S, and were then examined for skeletal variants and abnormalities. The skeletal specimens were stored in aqueous glycerol containing thymol crystals. The statistical assessment of the findings was based on group means and standard deviations calculation where appropriate; the data were subjected to analysis of variance or to the Kruskal-Wallis test. Where necessary, Dunnett´s test or Dunn´s multiple comparison test also were considered. Samples of the test solutions were analysed for verification of Bronopol content. Samples taken on day 1 of dosing revealed a good Bronopol recovery and confirmed the accuracy of the preparation. The analysis of the samples taken towards test ending revealed a Bronopol recovery of about 94.6% of nominal concentration for the test solution used for test group 3; for the remaining groups recovery was about 95% of nominal and was therefore within acceptable limits. The formulations were considered as acceptable for use.

Maternal toxicity: No treatment-related mortalities were reported. In fact, two moribund females (one of the 5 mg/kg bw group and one of the 80 mg/kg bw/day group) were sacrificed in extremis, but Bronopol treatment was not considered to be the direct cause of morbidity. With regard to the animals sacrificed at scheduled time (day 28 of pregnancy), the dams of the 80 mg/kg bw/day group showed a reduction in size and quantity of fecal pellets throughout most of the treatment period; this effect was related to the decreased food consumption of these animals. No further treatment-related symptoms were seen. Maternal body weight gain in the 80 mg/kg bw group was decreased compared to control (day 7 to 9:-0.06 +/- 0.11 kg versus 0.05 +/- 0.06 kg for control), mean food consumption was significantly lower than for control (day 7 to 11: 113 +/- 78 g/rabbit/day versus 181 +/- 31 g/rabbit/day for control). Necropsy of the dams that were sacrificed at scheduled time revealed no treatment-related abnormalities; necropsy of the 80 mg/kg bw female sacrificed in extremis revealed extensive ulceration of the gastric mucosa. As the deterioration of the health state of this female had commenced prior to starting Bronopol treatment; Bronopol was not considered to be the direct or primary cause for this state, but it can not be ruled out that the treatment might have exacerbed the bad state of health of the animal and might have contributed to the development of the gastric ulceration.

No signs of maternal toxicity were seen in the remaining test groups (5, 20 and 40 mg/kg bw/day).

Pregnancy and implantation data: All females including those, which were sacrificed in extremis were pregnant; each group comprised between 17 and 19 litters. No adverse treatment-related effects on corpora lutea, implantations, number of live fetuses, and sex ratio were reported.

Teratogenic/embryotoxic effects: Embryotoxicity was evidenced in the 80 mg/kg bw/day group. In this group, the mean fetal weight (both sexes) was significantly decreased compared to control. The decrease in fetal weight was indicative of embryonic growth retardation, which again probably related to the decreased food comsumption and body weight gain reported for the dams of this group. Gravid uterine weights showed no treatment-related effects. A mean incidence of 6.9% of fetuses showing major abnormalities was reported for the 80 mg/kg bw group (control: 0%; this conspicuous low incidence in control group was unusual and incidental); the difference however was not statistically significant. The major abnormalities were of great variability and belong to the most common fetal abnormalities occurring spontaneously in the rabbit strain used; however, the conspicuously elevated incidence reported for the 80 mg/kg bw/day group probably was due to treatment. Further treatment-related effects seen in the 80 mg/kg bw/day group included increased incidences of runted fetuses (related to the growth retardation observed in this group), fetuses with minor skeletal abnormalities, and of fetuses with non-ossification of the fore- and hind limb epiphyses; these abnormalities were indicative of a general retardation of the fetal skeletal ossification and growth.

Embryotoxicity could not be evidenced in the 5, 20 and 40 mg/kg bw/day groups.

Conclusively, developmental toxicity was observed at a test dose, which was shown to be toxic to the maternal animals (i.e. 80 mg/kg bw/day).

Thus, the NOAEL for maternal toxicity and the NOAEL for embryotoxic / teratogenic effects was 40 mg/kg bw/day, respectively.

This study is classified as acceptable (key study). The study was conducted according to the EPA Data Requirement EPA OPP 83-3 and followed GLP.

 

 

Rabbit teratology dose ranging study

A range-finding study was conducted in order to define the suitable dose levels of Bronopol to be used in a further main developmental study with rabbit (Toxicology Laboratories Limited, 1991/TX91104).

Each female was mated with a buck and was then given an intravenous injection of 25 IU chorionic gonadotropin to stimulate ovulation. Mating was conducted at the supplier´s premises, and at their arrival in the testing laboratories, the females were on day 2 of pregnancy. The test substance (purity 99.8 %) was administered once daily by gavage and following dosages were used: 1, 3, 10, 20, 40 and 80 mg/kg bw/day; control animals received vehicle only. The control group was defined as groups 1; the remaining groups were defined as group 2, 3, 4, 5,6 and 7. Each test group consisted of 5 mated females. Groups 1 to 6 were dosed from day 7 to day 19 (inclusive) of pregnancy. The females of group 7 were treated with 80 mg/kg bw/day of Bronopol from day 7 to 13 of pregnancy. On day 14, one moribund female was sacrificed in extremis. As the remaining 4 females showed no symptoms, it was decided to elevate the test dose to 160 mg/kg bw/d starting from day 14 of pregnancy. On day 17, one of these females was found dead; despite of the fact that the remaining 3 females were inconspicuous; dosing in this group was stopped on this day.

The dams were checked daily for clinical symptoms and mortality until day 28 of pregnancy. Body weights were recorded as well as food consumption.

One female of the control group was sacrificed in extremis on day 20 of pregnancy because of abortion. In group 7, one female was sacrificed in extremis on day 14 of pregnancy because of bad state of health. A further female was found dead on day 17 of pregnancy. The remaining 3 females which were inconspicuous and free of symptoms, also were sacrificed on day 17 of pregnancy. All remaining females survived the experiment until the scheduled time point of day 28 of pregnancy and were then sacrificed for the purpose of necropsy. At necropsy, the animals were examined for gross abnormalities, and organs/tissues with abnormalities were fixed for further examination. The dams sacrificed on day 28 of pregnancy were examined for gravid uterus weight, number of corpora lutea, and number/distribution of implantation sites. The implantations were classified in early and late resorptions, and in dead and live fetuses, and were separated in number per horn. Pre- and post-implantation losses were calculated. The live fetuses were examined for external abnormalities before being sacrificed. They were weighed and were then slight fixed in alcohol. The fixed fetuses were prepared for and subjected to visceral examination and sex determination. The statistical assessment of the findings was based on group means and standard deviations calculation. Samples of the test solutions were analysed for Bronopol content. The analysis revealed a good Bronopol recovery for most samples (within 5 % of theoretical value) and confirmed the accuracy of the preparations.

Maternal toxicity: The highest test dose of 160 mg/kg bw/day which was given to the females of group 7 starting from day of pregnancy, was found to be too high as this dosage resulted in mortality and body weight loss. No treatment-mortalities were reported for the remaining dosages including 80 mg/kg bw/d (group 7, day 7 to 13 of pregnancy). One moribund female treated with 80 mg/kg bw was sacrificed in extremis on day 14 of pregnancy; the bad state of health of this animal already was seen prior dosing and therefore was not treatment-related.

Signs indicative of maternal toxicity were seen in females treated with 80 mg/kg bw/day of Bronopol, and mainly consisted of loss in body weight gain, decrease in food consumption, and development of haemorrhages and ulceration in the gastric mucosa, as revealed by necropsy. The females of the groups treated with up to 40 mg/kg bw/day of Bronopol showed no treatment-related adverse effects.

Pregnancy and implantation data: No adverse treatment-related effects on pregnancy, corpora lutea, implantations, number of live fetuses, and sex ratio were reported.

Fetal data: No fetal data were reported for group 7 as the dams died or were sacrificed prior to day 28 of pregnancy. In the groups treated with up to 40 mg/kg bw/day of Bronopol, all considered data (embryonic growth, mean fetal weight, sex ratio, major and minor abnormalities) were not affected by the treatment.

On the basis of these findings, 80 mg/kg bw/day was retained as the dose that induces maternal toxicity and was therefore selected as highest dose to be tested within the main developmental toxicity study with rabbit. 5, 20 and 40 mg/kg bw/day were selected as low and mid dose levels for the main study.

This study is classified as acceptable (key study). The test followed GLP; no guideline was mentioned, however the conduct of the study was acceptable.


Justification for selection of Effect on developmental toxicity: via oral route:
The key study represents a guideline study conducted in accordance with GLP (reliability: 1). Furthermore the corresponding range-finding study and a previous study about development of rat are listed, which supports the findings of the key study (Toxicology Laboratories Limited, 1995). Moreover, a key study on rabbit developmental toxicity (teratogenicity) study and the corresponding range-finding study are presented.

Justification for classification or non-classification

Since Bronopol caused no developmental toxicity and teratogenicity in the rat in a developmental toxicity study according to EPA OPP 83-3 (Prenatal Developmental Toxicity Study) and due to the fact that no apparent adverse effects on reproductive organs or fertility were observed in rats in a subchronic study according to according to the SOP of the International Research and Development Corporation (IRDC), Bronopol is considered not to cause damage to the unborn child and is not subject to classification according to Directive 67/548/EEC and Regulation 1272/2008/EC.