Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-Generation Study:

rat, water: NOAEL systemic toxicity = 10 mg/kg bw/d (LOAEL = 50 mg/kg bw/d); NOAEL reproductive toxicity = 50 mg/kg bw/d (LOAEL = 150 mg/kg bw/d), (Dow, 2008)

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
other: JMAFF 12-8147, Guideline 2-1-17
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity: 99.85%
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Sex:
male/female
Details on test animals and environmental conditions:
Supplier and Location: Charles River Laboratories Inc. (Portage, Michigan)
Age at Study Start: Approximately six weeks at initiation of treatment.
Physical and Acclimation: Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed two-three per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately two weeks prior to the start of the study. During the second week of acclimation, all animals were acclimated to a water bottle containing acidified municipal water (see dose preparation).
Housing: After assignment, animals were housed one per cage in stainless steel cages, except during breeding (one male and one female) and during the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until completion of lactation. Cages had wire mesh floors and were suspended above absorbent paper. Cages contained a feed crock and a water bottle. During the study the room conditions were maintained as follows. Room humidity was measured outside of bounds at 70.6% on August 1, 2006. This transient increase in humidity was extremely minor, transient, had no noticeable impact on the animals, and did not affect the integrity of this study.
Temperature: 22 ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air changes: 12-15 times/hour
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Randomization and Identification: Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.
Feed and Water: Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. The results of the feed and water analyses indicated no contaminants that would interfere with the conduct of the study or interpretation of the results.
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
acidified pH 4
Details on exposure:
The drinking water solutions were prepared as a fixed percent and mixed periodically throughout the study based on stability data. The pH of each dose level was adjusted to pH 4 (+/- 0.5) with hydrochloric acid to ensure stability of bronopol. The control group was given municipal water adjusted to pH 4 (+/- 0.5). The pH was within limits throughout the study except for the water mixed on July 6, 2006 that was slightly elevated (pH 4.46-4.71) in all dose groups. This deviation was minor, transient, had no noticeable impact on the animals, and did not affect the integrity of this study.
To avoid potential overdosing during breeding, co-housed animals were given whichever dose (i.e., male or female) was of lower concentration. During gestation, females were given the same concentrations as provided during breeding. During lactation, dose concentrations were adjusted using historical control water consumption data for lactating females to account for the large and rapid increase in water consumption (2-3-fold) typical for rats in late lactation (Kirk et al., 1990). Dams awaiting necropsy received the same concentration of bronopol that was given during breeding. Weanlings received the same concentration of bronopol that was given to the P0 females during the third week of lactation until all litters had been weaned. Dose concentrations for the P1 generation were calculated as described for the P0 animals.
Details on mating procedure:
Breeding of the P0 adults commenced after approximately 10 weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were separated from the male and returned to their home cage. If a breeding male died or was removed from study, a substitute partner (from the same dose group) that already completed mating was provided. If mating did not occur after two weeks, the animals were separated without further opportunity for mating. Approximately 10 weeks after all F1 litters have been weaned, F1 offspring randomly selected to become P1 adults were bred as described above. Cohabitation of male and female littermates was avoided.
Duration of mating: 2 weeks
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of all dose solutions to determine concentration of the test material was conducted at least four times (at study start, P0 mating, P0 lactation and P1 pre-mating). The homogeneity of the low-dose and the high-dose test material solutions was determined concurrent with dose confirmation. The analysis was conducted using high performance liquid chromatography (HPLC) with ultraviolet detection and external standards.
Duration of treatment / exposure:
ten weeks prior to breeding, and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations
Frequency of treatment:
daily
Details on study schedule:
- Litters were standardized to 8/litter on PND4
- Litters were weaned on PND21
- One male and one female per litter were randomly selected as P1 animals to produce the second generation.
- F1 parental animals were mated 10 weeks after all F1 litters were weaned
Dose / conc.:
0 other: %
Dose / conc.:
0.01 other: %
Remarks:
10 mg/kg/day
Dose / conc.:
0.05 other: %
Remarks:
50 mg/kg/day
Dose / conc.:
0.15 other: %
Remarks:
150 mg/kg/day
No. of animals per sex per dose:
27
Control animals:
yes
Details on study design:
The high dose for bronopol in drinking water was chosen based upon a reproductive toxicity study (US EPA, 1995) showin NOELs for systemic and reproductive toxicity of 25 and 70 mg/kg/day, a palatability study (Dow, 2006), and a 90 day probe study (Dow, 2006) demonstrating a NOAEL of 0.15% for females and 0.075% for males. The high dose was expected to produce treatment related effects, while the intermediate and low dose were expected to produce dose response data.
Parental animals: Observations and examinations:
A cage-side examination was conducted at least twice daily. The examination was performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Clinical examinations were conducted on all males pre-exposure and weekly throughout the study. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 21. Females were observed for signs of parturition beginning on or about GD 20. Females that delivered litters were subsequently evaluated on LD 0, 1, 4, 7, 14, and 21. Clinical observations were not conducted on females that failed to mate or deliver a litter, unless deemed appropriate based on cage-side observations. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.
All rats were weighed during the pre-exposure period and weekly during the 10-week pre-breeding periods. Males continued to be weighed weekly after breeding until termination. Mated females were weighed on GD 0, 7, 14, and 21. Lactating females were weighed on LD 1, 4, 7, 14, and 21. Females that failed to mate and/or deliver a litter were weighed weekly during the subsequent gestation and/or lactation segments of the study. Body weight analyses during gestation and lactation were conducted for the following days: GD 0, 7, 14, 21 and LD 1, 4, 7, 14, and 21. Body weight gains of females were calculated for the following intervals in both generations: GD 0-7, 7-14, 14-21 and 0-21 and LD 1-4, 4-7, 7-14 and 14-21 and 1-21.
Feed and water consumption were determined weekly during the 10-week pre-breeding period for all animals by weighing feed containers or sipper bottles at the start and end of a measurement cycle. During breeding, feed and water consumption was not measured due to co-housing. Following breeding, feed or water consumption for males was measured weekly until termination. For mated females, feed and water consumption was measured on GD 0, 7, 14, and 21. For females delivering litters, feed and water consumption was measured on LD 1, 4, 7, 11, 14, 17, 19, and 21. Feed and water consumption was not measured for females that fail to mate or fail to deliver a litter.
Test material intake (TMI) was calculated for the P0 and P1 adults using test material concentrations in the drinking water, actual body weights, and measured water consumption.
Oestrous cyclicity (parental animals):
Vaginal lavage samples from all P0 and P1 females were collected daily for three weeks immediately prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two week mating period had elapsed. Lavage samples were collected by irrigating the vagina with water and transferring lavage fluid to a microscope slide to determine estrous cycle length and pattern. On the day of scheduled necropsy, vaginal lavage samples were collected from all P0 and P1 female rats for subsequent determination of the stage of the estrous cycle.
Sperm parameters (parental animals):
Weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters. Sperm parameters were evaluated in all P0 and P1 males at termination. Unless circumstances dictated otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis –counts; right testis – histopathology; left testis – counts.
Litter observations:
Females were observed periodically for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day that one or more delivered fetuses were noted, and was designated as LD 0. The following information was be recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21. Any pup found dead or sacrificed in moribund condition was sexed and examined grossly, to the extent possible, for external and visceral defects.
Postmortem examinations (parental animals):
The necropsy included an examination of the external tissues and all orifices. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The uteri of all females were stained and examined for the presence and number of implantation sites.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) were recorded, and the organ-to-body weight ratios calculated.
Histologic examination of the tissues was conducted on all control and high-dose adult rats. In addition, due to the apparent increase in the incidence of gross observations relating to stomach erosion-ulcer in bronopol treated P0 and P1 females, stomachs from all females of P0 and P1 generations were histologically examined. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose [kidneys (P0 and P1 males and females), liver, stomach (P1 and P2 females) and thyroid glands (P1 females)] and relevant gross lesions. Thyroid glands of all P0 females were histologically examined because mean relative thyroid weight of high-dose P0 females were increased and statistically identified compared to that of the controls. Adrenal glands from all P1 females were also examined due to the histologic finding of vacuolization of the zona glomerulosa in a few (3/27) P0 high-dose females. However, adrenal glands were not considered target organs because of the low isolated incidence and absence of this finding in the P1 high-dose females. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.
Histopathological examination of the right testis included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned, at 5 µm and stained with modified periodic acid Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Examination of the ovaries included enumeration of primordial follicles using a method similar to Bucci et al. (1997). From among the surviving post-lactational P1 females in the control and high-dose groups, 15 per group were randomly selected for this examination.
Postmortem examinations (offspring):
Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning were submitted on PND 22 for a complete necropsy. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative sample of grossly abnormal tissues and any known target organs were collected from all weanlings at the scheduled necropsy. In addition, one of the three pups/sex/litter was randomly selected from those examined grossly for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated.
Dead or moribund pups were examined in a similar manner for possible defects and/or cause of death.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) were recorded, and the organ-to-body weight ratios calculated.
Histopathology for F1 animals not selected for mating and F2 animals was the same as described for parental animals.
Statistics:
Parental body weights, gestation and lactation body weight gains, litter mean body weights, feed and water consumption, anogenital distance (absolute and relative to the cubed root of body weight), sperm count, follicle count, percent total and progressively motile sperm, mean estrous cycle length and organ weights (absolute and relative) were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA is significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) were performed.
Gestation length, age at vaginal opening (females), age at preputial separation (males), average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni’s correction was performed. Sperm morphology was arcsine transformed and analyzed using a parametric ANOVA. Slides containing less than 200 sperm were excluded from analysis. If the ANOVA was significant, the Dunnett’s test was performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and were excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction.
Reproductive indices:
Female and male mating indices, female and conception indices, female and male fertility index, gestation index, gestation survival index, post-implantation loss
Offspring viability indices:
day 1 or 4 pup survival index, and day 7, 14, or 21 pup survival index
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on behavior or demeanor were observed in P0 males at any dose level. Although one P0 male from the mid dose (0.05%) group was euthanized prior to scheduled termination, this death was not treatment related. This rat was observed with maloccluded incisors from test day 86 to 96. On test day (TD) 95, the rat sustained an apparent mechanical trauma to the left hindlimb, with associated swelling and a lame gait. By TD 96, the swelling and redness worsened with serous fluid noted on the paw; therefore, this rat was humanely euthanized. Overall, a number of additional incidental observations bearing no relation to treatment were observed.
Clinical observations of the P0 females were unremarkable during the pre-mating phase and most of gestation. However, one control dam delivered eight viable pups, but was observed on LD 1 with yellowish skin/mucous membranes, and subsequently lost all of her pups between LD 1 and 4. At the scheduled necropsy, the uterus was found dilated with bloody fluid. Other gross observations were pale liver and increased spleen size consistent with blood loss presumably from the uterus. Histopathologic examination of selected organs revealed extramedullary hematopoiesis in the adrenal gland, liver and spleen consistent with blood loss, which may have resulted in the loss of its litter.
Two dams from the 0.15% high-dose group experienced difficult birth (dystocia), manifested as protracted and/or incomplete delivery. One female from the 0.15% group was observed on GD 21 with red perinasal soiling. The following day (LD 0), this rat was found with one dead pup, and died spontaneously that day. At necropsy, eleven autolyzed fetuses and placental tissue were found in utero. Similarly, another high-dose female was found on LD 0 with red perinasal soiling, reddened eye membranes, pale mucous membranes, cold to the touch, slow and shallow breathing, apparently not eating or drinking, with decreased activity, and difficult birth. One male and one female pup were found dead, with the placenta still attached to the dead male. This rat also died spontaneously on LD 1. At necropsy, 14 autolyzed fetuses and placental tissue were found in utero. The deaths of these dams were attributed to secondary complications of dystocia. Although gross and histopathologic findings in both dams did not reveal a specific cause for dystocia, these two cases were considered to be treatment-related (see Reproductive Indices).
Mortality:
mortality observed, treatment-related
Description (incidence):
One P0 male from the mid dose (0.05%) group was euthanized prior to scheduled termination due to mechanical trauma of left hindlimb, this death was not treatment related. Two P0 females from the high dose (0.15%) group underwent spontaneous death on LD 0 or 1 due to complications from difficult birth (dystocia). This finding was deemed treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically identified differences in the body weights of any P0 male treated groups when compared to their respective controls or on body weights in P0 females during their pre-breeding, gestational, or lactation periods at any dose level. Despite the lack of statistically identified differences, there were sporadic slight decreases in body weight of animals administered 0.15% bronopol. These transient findings were likely due to decreased water consumption for this group.
Although body weight data were not statistically different for P0 females during gestation and lactation, body weight gain data during gestation and lactation were statistically different. The increased gestation body weight gains in the 0.01 and 0.05% groups during GD 0-7 were very likely spurious due to variability and outliers, and therefore judged not treatment related; however, the decreased gestation weight gains in the 0.15% group from GD 14-21 were consistent across the group. These data correlate with a significant decrease in food and water consumption for this group and timeframe. It is interesting to note that the gestation body weight effect occurred in the week just prior to parturition, temporally corresponding to the difficult deliveries seen in selected P0 females. This decrease in gestation body weight gain occurred in the absence of a pup body weight decrease (see Pup Body Weight section), suggesting that the decreased weight gain was a maternal effect, rather than a secondary consequence of fetotoxicity.
The increased lactation body weight gain in the 0.15% and, to a lesser extent, 0.05% groups, appeared to represent “catch up” to growth, as all groups had very similar weights by the end of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean feed consumption of P0 males was significantly decreased in the 0.15% group on TD 1-8, 36-43, 50-57, and 106-113. These differences were attributed to a corresponding decrease in water intake due to decreased palatability of the compound in drinking water.
Feed consumption of P0 females was significantly decreased in the 0.05% and 0.15% group on days 43-50 and 50-57. Feed consumption was also reduced in the P0 females 0.15% during days 1-8. In addition, feed consumption was significantly reduced in P0 females in the 0.15% group during days 14-21 of gestation and 4-7 of lactation. Overall, similar to the males, these slight reductions in feed consumption generally appeared to be secondary to decreased water intake, except for two females with more significant reductions in feed consumption that could have been related to maternal toxicity.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Male P0 rats given 0.05 and 0.15% bronopol in drinking water had statistically identified treatment-related decreases in water consumption from TD 1-57 or 1-120, respectively, attributed to decreased palatability. These animals consumed an average of 95.7 or 75.5% of control water intake, respectively, throughout the duration of the study.
Similar to males, female P0 rats given 0.05 or 0.15% bronopol in drinking water had statistically identified treatment-related decreases in water consumption from TD 1-50 or 1-71 of the premating phase, respectively, attributed to decreased palatability. These P0 females in the 0.05 and 0.15% groups consumed an average of 87.3 and 69.9% of control water intake before mating.
During gestation and lactation, P0 females in the 0.15% dose group had statistically identified treatment-related decreases in water consumption throughout gestation and lactation up to LD 14. In the 0.05% dose group, decreases in P0 female water consumption were not statistically identified during gestation and lactation, except for LD 4-7. Overall, these P0 females in the 0.05 and 0.15% dose groups had 94.9 and 71.9% of control intake during gestation, 91.6 and 78.5% of control intake during lactation days 1-14, and 96.2 and 94.2% of control intake during lactation days 14-21. The sharp increase in water consumption during the second half of lactation for the 0.15% P0 females corresponds with their increased weight gain during this period. Overall, although there was generally less water consumed in the 0.05 and 0.15% groups, the lack of body weight loss throughout the study strongly indicates that this reduced water intake was due to decreased palatability.
Test material intakes over the course of the study were 8.8, 41 and 104 mg/kg/day for P0 males. Time weighted averages of test material intakes for P0 females were 11.5, 53.2 and 129 mg/kg/day in the premating phase, 11.5, 56.5 and 129 mg/kg/day during gestation and 14.2, 67.8, and 183 mg/kg/day during lactation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The kidney was identified as a target organ in males and females of both P0 and P1 generations. In the kidney, P0 and P1 males and females given the high-dose of 0.15% bronopol had a higher, treatment-related incidence of very slight or slight, multifocal tubular degeneration with regeneration and very slight or slight, multifocal tubular dilatation. There was also a higher treatment-related incidence of very slight (males and females) or slight (males only) multifocal, subacute to chronic interstitial inflammation in the high-dose P0 rats.
These histologic changes in the kidney were consistent with very slight or slight nephropathy. P1 males given the mid-dose of 0.15% also had a higher, treatment-related incidence of very slight tubular degeneration with regeneration and very slight multifocal tubular dilatation consistent with a very slight nephropathy. Other associated treatment-related nephropathic changes variably present in the kidney were thickening of the tubular basement membranes, thickening of the Bowman’s capsule of the glomeruli, presence of eosinophilic proteinaceous material in isolated tubular lumens and interstitial fibrosis in the affected areas of the kidney.
The livers of P0 and P1 high-dose females had a higher incidence of very slight-slight multifocal hepatocyte vacuolization, consistent with fatty change, compared to the respective controls and was considered treatment-related. This minor change was characterized by the presence of intracytoplasmic lipid vacuoles in scattered individual hepatocytes, which may likely reflect lower feed consumption, particularly in the P0 generation.
There were isolated occurrences of very slight focal or multifocal erosions in the glandular stomach mucosa of females in all bronopol treated groups of P0 and P1 generations. However, they were not dose-related, and were within historical control incidence and hence, considered unrelated to treatment. A low incidence of stomach erosions of very slight severity is not uncommon in these rats as can be seen from historical control ranges derived from similar 2-generation studies conducted and reported in this laboratory in the past 5 years. However, multifocal erosions in the glandular mucosa of increased severity (graded as slight) and interpreted to be treatment-related were present in 3 P0 females and 2 P1 females given the high-dose of 0.15% bronopol. Focal ulcers (erosions extending deep into the muscularis mucosa) in the glandular stomach were also present in 2 P0 females given 0.15% bronopol, which was considered treatment-related. In addition, 7 P0 females and 5 P1 females given 0.15% bronopol had treatment-related slight acute multifocal inflammation glandular submucosa, characterized by the presence of slight edema and mixed inflammatory cell infiltrates composed of neutrophils, lymphocytes and lesser numbers of mast cells within the glandular submucosa. Focal or multifocal, acute inflammation of the submucosa of the glandular stomach of lesser severity (graded as very slight, characterized by very slight edema and presence of small numbers of mixed inflammatory cell infiltrates within the glandular submucosa) was present in P0 and P1 females across all dose groups including control, but the incidence was not dose-related and hence, considered unrelated to treatment.
Histologic examination of the thyroid glands of all P0 adult females were conducted because of the slightly elevated and statistically identified relative thyroid weight in the high-dose females (see Organ Weights section). Females given 0.05% and 0.15% bronopol had a very slight, treatment-related, overall increase in the numbers of dilated thyroid follicles and many of these dilated follicles were lined by flattened epithelial cells. However, there was no appreciable hyperplasia/hypertrophy or differences in the colloidal staining properties compared to that of the controls. In the absence of a thyroid weight effect in P1 adults, thyroid histopathology was not triggered.
In summary, treatment-related histologic changes were present in: P0 males (kidneys) and females (kidneys, liver, stomach and thyroid) given the high-dose of 0.15%; P0 females (thyroid) given 0.05%; P1 males (kidneys) and females (kidneys, liver, and stomach) given 0.15%; and P1 males (kidneys) given 0.05% bronopol. There were no treatment-related histologic changes in adult males and females given the low-dose of 0.01% in both P0 and P1 generations.
Two high-dose P0 females were found dead on LD 0 with one pup in the vaginal canal and multiple pups in the uterus (dystocia or difficult birth). Histopathologic examination did not reveal a specific cause for dystocia that was attributed as the cause of death in these two animals. Among other findings one of these females, there was acute neutrophilic inflammation in the adipose tissue surrounding the ovary on one side interpreted to be secondary to dystocia. The uterus contained necrotic placental material that was not considered abnormal given the presence of fetuses in the uterus observed at necropsy. Additionally this animal also had signs of non-specific stress such as slight hypertrophy of the zona fasciculata of adrenal glands and moderate atrophy of the thymus. The other female had very slight acute multifocal inflammation within the endometrium of the uterus interpreted to be secondary to dystocia. Among other findings, this animal also had signs of nonspecific stress such as slight hypertrophy of the zona fasciculata of adrenal glands and moderate atrophy of the thymus.
One mid-dose P0 male was euthanized for humane reasons due to severe trauma to its left hind limb. There was severe diffuse inflammation, edema and hemorrhage in the skin and subcutis at the site of trauma. Acute inflammation and edema extended to the muscles and connective tissues surrounding the bones of the left hind limb. However, no bone fracture was confirmed histologically. All these histologic changes were consistent with traumatic injury to the left hind limb and were not treatment-related.
One low-dose P1 male and one high-dose female were euthanized for humane reasons due to fracture of the maxilla on days 51 and 64 respectively. Associated with the fracture of the maxillary bone, there was marked multifocal acute inflammation with edema, hemorrhage and fibrin deposition in the soft tissues surrounding the maxilla and nasal cavity. In addition, the low-dose P1 male had fracture of the enamel and dentine of the incisor root with associated hemorrhage and inflammation and also multifocal subarachnoid hemorrhages in the ventral aspect of the thalamus and cerebellum. All these changes were consistent with traumatic injury in both animals and were not associated with bronopol treatment.
One low dose P1 male was found dead on day 129 with diffuse liver congestion at necropsy. Histopathologic evaluation, however, could not determine the cause of death in this animal. The cause of death for this animal was, however, considered unrelated to treatment due to the isolated occurrence.
Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects of treatment. The finding of low sperm count in one high-dose male which mated but failed to sire a litter correlated with the histologic finding of moderate multifocal degeneration of seminiferous tubules in the testis. The epididymides of this animal contained little or no mature spermatozoa, but had degenerated cells and debris. This was considered unrelated to treatment due to its isolated occurrence.
Tension lipidosis, a spontaneous change in the liver (observed at necropsy as a small, pale focus at the margin of the liver between the left and right medial lobes, at the site of attachment of the falciform ligament) was confirmed histologically. This was characterized by the presence of a discrete, subcapsular focus of vacuolated hepatocytes consistent with fatty change. Although there was an apparent increase in its incidence in the P0 and P1 high-dose males, this was not related to bronopol treatment. Additionally, there was no clear dose-response relationship in the incidence of tension lipidosis in the P0 and P1 males and there were no differences in its incidence in the females. Tension lipidosis is likely due to the ligament attachment impeding blood supply to the subjacent hepatic parenchyma by exerting tension on the capsule resulting in focal fatty change (Maclachlan and Cullen, 1995).
All other histologic observations not discussed herein were considered spontaneous or incidental alterations, unassociated with exposure to bronopol.
There were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females given 0.15% bronopol as compared to control females.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no evidence of an effect on estrous cyclicity at any dose level of bronopol.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no effects of bronopol on sperm morphology, sperm motility or progressive sperm motility at any dose level. There were no effects on P0 sticular sperm counts or epididymal sperm counts.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no effects of treatment at any dose level on conception, gestation index, time to mating, gestation length, pup survival indices, or pup sex ratio in either generation. Although there was some slight variability in the P0 mating, conception, or fertility indices, it was considered to be random and unrelated to treatment for the following reasons: 1) none of the values were statistically identified, 2) the values for these parameters were either equal to the control or within the historical control range, and 3) there was no dose response relationship for the changes in these parameters.
Additional discussion in the reproductive performance (P1) section.
There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size in the F1 litter in any dose group.
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
water consumption and compound intake
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects on behavior or demeanor were observed in P1 males at any dose level. Two males from the low dose group, underwent non-treatment related unscheduled death. One male was observed on test day 50 with blood in the cage, mechanical trauma to the nose and muzzle, and maloccluded incisors. Due to this accidental self-inflicted trauma, this animal was euthanized for humane reasons on the following day (TD 51). One male underwent spontaneous death on the day of scheduled necropsy (TD 129). This animal had no remarkable observations prior to its death and necropsy was unable to determine the cause of death. These deaths were considered spurious and unrelated to treatment.
No treatment-related effects on behavior or demeanor were observed in P1 females at any dose level, apart from two females in the high dose group with complete litter loss. One dam started labor on GD 21, was observed with red perinasal soiling, and gave birth to 15 live and three dead pups. The following day (LD 1), this dam was observed with red vulvar discharge, red perinasal soiling, perineal soiling, and twelve of the remaining pups died. By the following day (LD 2) the remaining pups died and the dam was observed with bilateral red periocular soiling, red perinasal soiling, bilateral partially closed eyelids, red vulvar discharge, perineal soiling, exhibiting aggressive behavior, and was attempting to bite. Another dam began labor on GD 21, and gave birth to one live and 13 dead pups. The live female pup died two days later on LD 2.
In addition, a female in the 0.15% dose group, was observed on TD 64 with blood in cage, mechanical trauma of the nose and muzzle, and maloccluded incisors. Based on this self-inflicted trauma, this animal was euthanized for humane reasons on TD 64. This death was considered spurious and unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two P1 males from the low dose (0.01%) group did not survive due to scheduled termination due to reasons unrelated to treatment. One P1 female in the high dose (0.15%) group was euthanized prior to scheduled termination due to mechanical trauma to the nose and muzzle, this death was not treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of P1 males in the high dose group were slightly decreased throughout the treatment period and statistically identified on TD 57. This decrease in body weight correlated with a statistically identified decrease in water consumption throughout the treatment period. Therefore, this decrease in body weight was considered secondary to the reduced water intake associated with the decreased palatability of 0.15% bronopol in acidified water. Similar to the P1 males, P1 female body weight was slightly decreased throughout the pre-breeding period and statistically identified on TD 8. This overall decrease in body weight correlated with a statistically significant decrease in water consumption throughout treatment. Again, this decrease in body weight was considered secondary to reduced water at the high dose level. There were no statistically significant changes in P1 female body weight during the gestation or lactation periods at any tested dose level, nor any statistically significant changes in body weight gain during lactation; however, there was a statistically significant decrease in body weight gain in the 0.15% dose group from GD 14-21, which resulted in a significant decrease in the amount of weight gained throughout gestation. This decrease in gestation body weight gain correlated with a decrease in water consumption during this same period. As in the P0 generation, the gestation body weight effect occurred in the week just prior to parturition, temporally corresponding to the total litter losses that occurred shortly after birth in selected P1 females. As in the P0 generation, this decrease in gestation body weight gain occurred in the absence of a pup body weight decrease (see Pup Body Weight section), suggesting that the decreased weight gain was a maternal effect, rather than a secondary consequence of fetotoxicity. Similar to the first generation, lactation body weight gain was increased in the 0.15% dose group, such that all groups had similar body weights by the end of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption of P1 males and females was slightly, but significantly, decreased in the 0.15% group on days 1-4 and 4-11. There were no statistically or treatment-related changes in P1 female feed consumption during gestation or lactation periods. Similar to the first generation, these early reductions in feed consumption are not attributed to treatment toxicity, but secondary effects of treatment from reduced water intake due to decreased palatability of the drinking water provided.
Description (incidence and severity):
Male P1 rats in the 0.15% dose group had statistically identified treatment-related decreases in water consumption throughout the dosing period; however, unlike the first generation, animals in the 0.05% dose group had marginal and only transient statistically identified decreases in water consumption that were deemed spurious and unrelated to treatment. Overall, male P1 rats given 0.05 or 0.15% of test material in drinking water consumed an average 97.6 and 80.5% of the water of controls, respectively, throughout the study. Similar to the male rats, female P1 rats had increased water consumption compared to the first generation with statistically identified treatment-related decreases in water consumption only in the 0.15% dose group from TD 1-74 and GD 14-21. There were no treatment-related decreases in water consumption for animals in the 0.05% dose group. Overall, female P1 rats in the 0.05 or 0.15% groups consumed an average of 92.2 and 78.3% of control water before mating, 100.6 and 88.2% of control water during gestation, 100.8 and 93.0% of controls during LD 1-14, and 100.5 and 85.3% of control water during LD 14-21. The higher water consumption of 0.05 and 0.15% bronopol by the second versus first generation animals is likely due to increased tolerance for the taste of the water, as these second generation animals were never given standard water. As before, although there was generally less water consumed in the 0.15% group, the lack of body weight decrements throughout the study strongly indicates that this reduced water intake was due to decreased palatability.
Test material intakes over the course of the study were 10.5, 52.6 and 141 mg/kg/day for P1 males. Time weighted averages of test material intakes for P1 females were 14.8, 67.1 and 181 mg/kg/day in the premating phase, 11.9, 58.3 and 156 mg/kg/day during gestation, and 13.9, 65.1 and 187 mg/kg/day during lactation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically identified differences in the final body weights of P1 males or females of any treated group compared to their respective controls.
The mean relative kidney weight of P1 adult males given 0.15% was marginally higher (5.7%) than that of the controls, statistically identified, and was outside of the historical control range of means. The mean absolute kidney weight of high-dose P1 adult females was also marginally increased by 6% and was outside of the historical control range of means, but was not statistically identified. However, the mean relative kidney weight, which was increased 6.5% over that of the control value, was statistically identified. These kidney weight changes were considered treatment-related and were associated with treatment-related histopathologic changes (see Histopathology section).
The mean absolute brain weight of females given 0.15% was statistically identified as decreased (4%) compared to the controls. However, the mean absolute and relative brain weights of the high-dose P1 females were within the historical control range, and therefore, these differences were considered spurious and unrelated to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The incidence of grossly observed focal/multifocal, erosion-ulcers of the glandular mucosa of the stomachs in P1 females given all doses of bronopol was slightly increased relative to respective controls. Analysis of historical data on the gross pathology incidence of focal or multifocal erosion-ulcers in control females from 2-generation dietary studies conducted and reported from this laboratory in the last five years showed that these observations were not uncommon. Therefore, the slightly higher incidence of erosions-ulcers in P1 bronopol treated females of all doses was considered unrelated to treatment. Isolated cases of focal erosion/ulcer were also noted in P1 control females, which were also considered unrelated to treatment due to the isolated occurrence. However, microscopic examination of these lesions was necessary for confirmation and also to assess if there were any treatment-related histopathologic differences in the stomach (see Histopathology section).
One P1 adult male given 0.01% bronopol was found dead on day 129 and presented with diffuse congestion of the liver at necropsy. Although the cause of death could not be determined, it was considered unrelated to exposure to bronopol due to its isolated occurrence. One P1 male given 0.01% and one P1 female given 0.15% bronopol were euthanized for humane reasons on days 51 and 64, respectively, due to traumatic fracture to the maxillary bone (upper jaw).
Livers of some males and females across all dose groups had small, pale, circumscribed focus along the margin of the right median lobe at or near the junction with the left median lobe. This is a spontaneous change, also known as ‘tension lipidosis’, due to the attachment of the falciform ligament at this site and characterized by the focal presence of lipid filled hepatocytes. Although the high-dose P1 males had a slightly higher incidence compared to respective controls (7 vs 3 respectively, in both generations), this spontaneous change (confirmed microscopically, see Histopathology section) was not a treatment-related change. Tension lipidosis is likely due to the ligament attachment exerting tension on the capsule, impeding blood supply to the subjacent hepatic parenchyma resulting in focal fatty change (Maclachlan and Cullen, 1995). All other gross pathologic observations not discussed herein were also considered to be spontaneous alterations, unassociated with exposure to bronopol.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The kidney was identified as a target organ in males and females of both P0 and P1 generations. In the kidney, P0 and P1 males and females given the high-dose of 0.15% bronopol had a higher, treatment-related incidence of very slight or slight, multifocal tubular degeneration with regeneration and very slight or slight, multifocal tubular dilatation. There was also a higher treatment-related incidence of very slight (males and females) or slight (males only) multifocal, subacute to chronic interstitial inflammation in the high-dose P0 rats.
These histologic changes in the kidney were consistent with very slight or slight nephropathy. P1 males given the mid-dose of 0.15% also had a higher, treatment-related incidence of very slight tubular degeneration with regeneration and very slight multifocal tubular dilatation consistent with a very slight nephropathy. Other associated treatment-related nephropathic changes variably present in the kidney were thickening of the tubular basement membranes, thickening of the Bowman’s capsule of the glomeruli, presence of eosinophilic proteinaceous material in isolated tubular lumens and interstitial fibrosis in the affected areas of the kidney.
The livers of P0 and P1 high-dose females had a higher incidence of very slight-slight multifocal hepatocyte vacuolization, consistent with fatty change, compared to the respective controls and was considered treatment-related. This minor change was characterized by the presence of intracytoplasmic lipid vacuoles in scattered individual hepatocytes, which may likely reflect lower feed consumption, particularly in the P0 generation.
There were isolated occurrences of very slight focal or multifocal erosions in the glandular stomach mucosa of females in all bronopol treated groups of P0 and P1 generations. However, they were not dose-related, and were within historical control incidence and hence, considered unrelated to treatment. A low incidence of stomach erosions of very slight severity is not uncommon in these rats as can be seen from historical control ranges derived from similar 2-generation studies conducted and reported in this laboratory in the past 5 years. However, multifocal erosions in the glandular mucosa of increased severity (graded as slight) and interpreted to be treatment-related were present in 3 P0 females and 2 P1 females given the high-dose of 0.15% bronopol. Focal ulcers (erosions extending deep into the muscularis mucosa) in the glandular stomach were also present in 2 P0 females given 0.15% bronopol, which was considered treatment-related. In addition, 7 P0 females and 5 P1 females given 0.15% bronopol had treatment-related slight acute multifocal inflammation glandular submucosa, characterized by the presence of slight edema and mixed inflammatory cell infiltrates composed of neutrophils, lymphocytes and lesser numbers of mast cells within the glandular submucosa. Focal or multifocal, acute inflammation of the submucosa of the glandular stomach of lesser severity (graded as very slight, characterized by very slight edema and presence of small numbers of mixed inflammatory cell infiltrates within the glandular submucosa) was present in P0 and P1 females across all dose groups including control, but the incidence was not dose-related and hence, considered unrelated to treatment.
Histologic examination of the thyroid glands of all P0 adult females were conducted because of the slightly elevated and statistically identified relative thyroid weight in the high-dose females (see Organ Weights section). Females given 0.05% and 0.15% bronopol had a very slight, treatment-related, overall increase in the numbers of dilated thyroid follicles and many of these dilated follicles were lined by flattened epithelial cells. However, there was no appreciable hyperplasia/hypertrophy or differences in the colloidal staining properties compared to that of the controls. In the absence of a thyroid weight effect in P1 adults, thyroid histopathology was not triggered.
In summary, treatment-related histologic changes were present in: P0 males (kidneys) and females (kidneys, liver, stomach and thyroid) given the high-dose of 0.15%; P0 females (thyroid) given 0.05%; P1 males (kidneys) and females (kidneys, liver, and stomach) given 0.15%; and P1 males (kidneys) given 0.05% bronopol. There were no treatment-related histologic changes in adult males and females given the low-dose of 0.01% in both P0 and P1 generations.
Two high-dose P0 females were found dead on LD 0 with one pup in the vaginal canal and multiple pups in the uterus (dystocia or difficult birth). Histopathologic examination did not reveal a specific cause for dystocia that was attributed as the cause of death in these two animals. Among other findings one of these females, there was acute neutrophilic inflammation in the adipose tissue surrounding the ovary on one side interpreted to be secondary to dystocia. The uterus contained necrotic placental material that was not considered abnormal given the presence of fetuses in the uterus observed at necropsy. Additionally this animal also had signs of non-specific stress such as slight hypertrophy of the zona fasciculata of adrenal glands and moderate atrophy of the thymus. The other female had very slight acute multifocal inflammation within the endometrium of the uterus interpreted to be secondary to dystocia. Among other findings, this animal also had signs of nonspecific stress such as slight hypertrophy of the zona fasciculata of adrenal glands and moderate atrophy of the thymus.
One mid-dose P0 male was euthanized for humane reasons due to severe trauma to its left hind limb. There was severe diffuse inflammation, edema and hemorrhage in the skin and subcutis at the site of trauma. Acute inflammation and edema extended to the muscles and connective tissues surrounding the bones of the left hind limb. However, no bone fracture was confirmed histologically. All these histologic changes were consistent with traumatic injury to the left hind limb and were not treatment-related.
One low-dose P1 male and one high-dose female were euthanized for humane reasons due to fracture of the maxilla on days 51 and 64 respectively. Associated with the fracture of the maxillary bone, there was marked multifocal acute inflammation with edema, hemorrhage and fibrin deposition in the soft tissues surrounding the maxilla and nasal cavity. In addition, the low-dose P1 male had fracture of the enamel and dentine of the incisor root with associated hemorrhage and inflammation and also multifocal subarachnoid hemorrhages in the ventral aspect of the thalamus and cerebellum. All these changes were consistent with traumatic injury in both animals and were not associated with bronopol treatment.
One low dose P1 male was found dead on day 129 with diffuse liver congestion at necropsy. Histopathologic evaluation, however, could not determine the cause of death in this animal. The cause of death for this animal was, however, considered unrelated to treatment due to the isolated occurrence.
Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects of treatment. The finding of low sperm count in one high-dose male which mated but failed to sire a litter correlated with the histologic finding of moderate multifocal degeneration of seminiferous tubules in the testis. The epididymides of this animal contained little or no mature spermatozoa, but had degenerated cells and debris. This was considered unrelated to treatment due to its isolated occurrence.
Tension lipidosis, a spontaneous change in the liver (observed at necropsy as a small, pale focus at the margin of the liver between the left and right medial lobes, at the site of attachment of the falciform ligament) was confirmed histologically. This was characterized by the presence of a discrete, subcapsular focus of vacuolated hepatocytes consistent with fatty change. Although there was an apparent increase in its incidence in the P0 and P1 high-dose males, this was not related to bronopol treatment. Additionally, there was no clear dose-response relationship in the incidence of tension lipidosis in the P0 and P1 males and there were no differences in its incidence in the females. Tension lipidosis is likely due to the ligament attachment impeding blood supply to the subjacent hepatic parenchyma by exerting tension on the capsule resulting in focal fatty change (Maclachlan and Cullen, 1995).
All other histologic observations not discussed herein were considered spontaneous or incidental alterations, unassociated with exposure to bronopol.
There were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females given 0.15% bronopol as compared to control females.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no evidence of an effect on estrous cyclicity at any dose level of bronopol.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects of bronopol on sperm morphology, sperm motility or progressive sperm motility at any dose level. There were no effects on P1 testicular sperm counts. Examination of epididymal sperm counts of the P1 control and high-dose males initially revealed a statistically significant decrease in the high-dose male total sperm count, but not sperm concentration per gram of epididymis, thus triggering an analysis of the 0.01 and 0.05% dose groups. Subsequent analysis of the combined data from all groups indicated that the decrease at the high-dose was no longer statistically significant. In addition, there were no effects on epididymal sperm concentration per gram tissue at any tested dose, nor were any effects observed in the first generation (P0) males. Also, there were no corroborating effects on testicular or epididymal weights or histopathology. Analysis of individual animal data in the high dose group revealed two males with low values of 32.9 and 81.6 (10E6) epididymal sperm that played a role in decreasing the mean values. Interestingly, when comparing these values to historical control, P1 epididymal total sperm count and sperm concentration values in the control group of this study were below the historical control range. Taken together, this weight of evidence indicated a lack of a treatment related effect on sperm counts.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
P1 mating and fertility indices, although not statistically identified, were slightly lower in the high dose group than in controls. The reduced fertility index was a cumulative effect of four females that didn’t mate (vs. three in controls), and three females that mated but did not litter (vs. two in controls). One of the latter cases was due to a male with a low sperm count associated with small testes, an isolated finding which was considered spurious. Viewing each component separately, one can see that the incidence relative to controls is not very different. In addition, there was no evidence of altered estrous cycles, decreased reproductive organ weights or histopathology, decreased sperm number or quality in animals from this group. Based on the overall weight of evidence, the slight differences in mating and fertility indices were considered spurious and unrelated to treatment.
There was a slight, but statistically identified increase in percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) in the high-dose F2, but not F1,litters. Most of this increase was attributable to three dams, one of which gave birth to 13 dead pups, and two others that had 8-9 implantation sites without corresponding pups or fetuses. This slight increase in postimplantation loss affected other dependent end points, namely a slight decrease in gestation survival index (total live born pups/total pups delivered) and a slight, non-statistically significant increase in number of pups born dead (1.1 vs. 0.1). The increase in postimplantation loss was considered treatment related for the following reasons. First, the values for postimplantation loss and gestation survival were outside their respective historical control ranges. Secondly, the increase in postimplantation loss was temporally correlated with other findings associated with the perinatal period, such as an additional total loss of a high-dose F2 litter shortly after birth.
It is important to note that the reproductive observations discussed thus far were essentially confined to two high-dose P0 dams with dystocia (3426 and 3437) and four high-dose P1 dams with increased postimplantation loss and/or total litter loss (5426, 5428, 5433 and 5441). These effects were interestingly not identified in a previous rat two-generation, two litters per generation, reproductive toxicity study that evaluated similar dose levels (US EPA, 1995). The details of these dams, discussed below, suggest a possible relationship to maternal toxicity in at least several instances.
P0 dams 3426 and 3437 had dystocia and underwent spontaneous death during parturition. Necropsy of these animals revealed that both had one pup in the vaginal canal and ten (#3426) or 13 (#3437) more autolyzed fetuses in the uterus. Interestingly, dam 3426 gained only 8.5 g, compared to a mean of 61.7 for the group, from GD 14-21. Given that the fetuses remaining in the uterus appeared to be of normal size, this would indicate maternal toxicity of this dam in the week prior to delivery. Unfortunately, dam 3437 showed no evidence of mating, so gestation body weight data were not available. These two cases of dystocia were considered treatment related based on the fact that dystocia is generally quite rare. In fact, there was only one case of dystocia among control dams in the laboratory’s ten most recent two-generation studies, although this case occurred in a study conducted within the same year as the present study.
Dams 5426, 5428, and 5433 from the P1 generation drove the increased postimplantation loss effect, while dams 5433 (again) and 5441 had total litter loss shortly after delivery. The individual postimplantation loss for dams 5426, 5428, 5433 and 5441 were: 9/18 (lost embryos or fetuses/implantation sites), 6/14, 15/16 and 4/19, respectively. Dam 5433 (15/16 postimplantation loss) delivered 1 live and 13 dead pups, and lost the remaining pup by LD 2. Dam 5441 (4/19 postimplantation loss) delivered 15 live and 3 dead pups, but lost all live pups by LD 2. In at least two of these dams (5433 and 5441) there were concomitant signs of maternal toxicity in the form of decreased body weight gain from GD 14-21 of 2.4 and 19.6 g, respectively, compared to a group mean of 60.4 g. Given that both of these dams gave birth to 14 and 18 full term fetuses, the decrease in gestation body weight gain was not a secondary consequence of fetotoxicity. However, gestation body weight gains were unaffected in the other two dams.
Consistent with the previously described effects on increased postimplantation loss and decreased gestation survival, the number of F2 pups born dead in the high-dose group (0.15%) was increased, concomitant with a decrease in live pups born. While not statistically significant, this effect was deemed treatment related because it was outside the historical control range and correlated with statistically significant decrease in gestation survival, increase in postimplantation loss, and two total litter losses in the 0.15% dose group.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Observations made on F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings, which included a small number of malformed pups in the control, low- and middle-dose groups but none in the high-dose groups, were seen with no evidence of a dose-response relationship.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Pup body weights at postnatal days 7 and 14 for the 0.15% group were significantly decreased, and below or at the cusp, respectively, of historical control data for these time-points. It is important to note that pup body weights for the 0.15% group were not significantly different at postnatal days 1, 4 or 21; however, the 0.15% group body weights as a percentage of control pup body weight is approximately 97% at postnatal day 1, but 92, 91, and 88% controls at postnatal days 4, 7, and 14, and back to 92% by postnatal day 21. In addition, the increase in body weight of the 0.15% pups by postnatal day 21 correlates with increased water consumption and body weight gain of lactating females from this same group. Therefore, these pup bodyweight differences are judged as secondary to treatment, likely due to reduced water consumption and not treatment related toxicity.
Sexual maturation:
no effects observed
Description (incidence and severity):
Age at vaginal opening and age at preputial separation were similar in all groups, indicating no influence of treatment.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically identified changes in the organ weights of F1 male weanlings. In the F1 female weanlings, body weights were decreased by 12%, resulting in a 4% decrease in absolute brain weight. Effects were secondary to decreased body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to bronopol.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Observations made on F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings, which included a small number of malformed pups in the control, low- and middle-dose groups but none in the high-dose groups, were seen with no evidence of a dose-response relationship.
Body weight and weight changes:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In F2 male weanlings, body weights were decreased by 9% relative to controls, with absolute brain weights decreased by 3%. These slight decreases in absolute brain weight were considered secondary to the decrease in body weight. The only other statistically identified change in F2 weanling male organ weights was a decrease in absolute thymus weight. Again this was attributed to decreased body weight. Based on the minor degree of change in these organ weights and their association with decreased body weight, they were not considered to be toxicologically significant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to bronopol.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Reproductive effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

Table 1. P0 Gestation/Lactation Body Weight Gain

 

Mean Body Weight Gain (G)

Dose Level (%):

0

0.01

0.05

0.15

GD 0-7

28.2

34.8*

35.9*

30.9

GD 7-14

32.2

33.2

30.5

32.6

GD 14-21

83.8

90.7

79.5

61.7$

GD 0-21

144.2

159.2$

146.0

125.2$

LD 1-4

11.4

15.5

19.5*

19.1*

LD 1-21

17.0

16.1

20.0

33.7*

* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

$Statistically different from control mean by Wilcoxon’s test, alpha = 0.05.

Bold typeindicates the effects judged to be treatment related.

Table 2. P1 Gestation/Lactation Body Weight Gain

 

Mean Body Weight Gain (G)

Dose Level (%):

0

0.01

0.05

0.15

GD 0-7

36.3

38.7

34.8

38.6

GD 7-14

27.8

31.3

30.2

32.0

GD 14-21

88.2

84.7

82.7

60.4*

GD 0-21

152.2

154.8

147.7

131.0*

LD 1-21

9.2

13.3

10.3

14.1

* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Bold typeindicates the effects judged to be treatment related.

Table 3. P0 Average Water Consumption Data

 

Mean g/Animal/Day relative to controls (%)

Dose Level (%):

0

0.01

0.05

0.15

Males days 1-120

100

99.9

95.7

75.5

Females days 1- 64

100

94.2

87.3

69.9

Females GD 0-21

100

96.4

94.9

71.9

Females LD 1-14

100

94.7

91.6

78.5

Females LD 14-21

100

101

96.2

94.2

Females LD 1-21

100

98.1

94.0

86.7

Bold type indicates the effects judged to be treatment related.

Table 4: P1 Average Water Consumption Data

 

Mean g/Animal/Day relative to controls (%)

Dose Level (%):

0

0.01

0.05

0.15

Males days 1-123

100

93.7

97.6

80.5

Females days 1- 74

100

97.7

92.2

78.3

Females GD 0-21

100

100.6

100.6

88.2

Females LD 1-14

100

107.6

100.8

93.0

Females LD 14-21

100

102.1

100.5

85.3

Females LD 1-21

100

104.6

100.7

88.8

Bold typeindicates the effects judged to be treatment related.

Table 5: P0 Adult Organ Weight Changes

MALES

Dose level (%)

0

0.01

0.05

0.15

 

Kidney – absolute (g)

3.931

3.967

3.922

3.983

 

Kidney – relative (g/100)

0.681

0.667

0.689

0.717

 

Adrenal – absolute (g)

0.064

0.062

0.057*

0.058

 

FEMALES

Kidneys – absolute (g)

2.195

2.213

2.249

2.356

 

Kidneys – relative (g/100)

0.730

0.742

0.756

0.789*

 

Thyroid – absolute (g)

0.0169

0.0186

0.0183

0.0191

 

Thyroid – relative (g/100)

0.0056

0.0062

0.0061

0.0064*

 

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

Bold typeindicates the effects judged to be treatment related.

Table 6: P1 Adult Organ Weight Changes

MALES

Dose level (%)

0

0.01

0.05

0.15

 

Kidneys – absolute (g)

4.029

3.952

4.049

4.118

 

Kidneys – relative (g/100)

0.685

0.682

0.679

0.724*

 

FEMALES

Kidney – absolute (g)

2.299

2.272

2.338

2.440

 

Kidney – relative (g/100)

0.734

0.740

0.743

0.782*

 

Brain – absolute (g)

2.007

1.993

1.994

1.925$

 

Brain – relative (g/100)

0.643

0.651

0.634

0.619

 

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

$Statistically Different from Control Mean by Wilcoxon’s Test, Alpha = 0.05

Bold type indicates the effects judged to be treatment related.

Table 7: P0 Histopath

Sex

Male

Female

Dose (%)

0

0.01

0.05

0.15

0

0.01

0.05

0.15

Kidney

Number Examined

 

Degeneration with regeneration, tubule unilateral/bilateral (combined), multifocal      

                                                                  Very slight

27

27

27

27

27

27

27

27

7

8

7

21

3

2

4

16

                                                                  Slight

0

0

0

3

0

0

0

4

Dilatation, tubule, unilateral/bilateral (combined), multifocal                                                 Very slight

2

2

4

8

0

2

1

5

                                                                 Slight

0

0

0

11

0

0

0

2

Inflammation, subacute to chronic, interstitium, unilateral/bilateral (combined), multifocal 

                                                                 Very slight

 

8

 

2

 

2

 

19

 

2

 

1

 

0

 

6

                                                                Slight

0

0

0

4

0

0

0

0

Liver

Number Examined

 

Vacuolization; consistent with fatty change; hepatocyte; individual cells; multifocal

                                                                 Very slight

 

27

 

7

 

7

 

27

 

27

 

27

 

27

 

27

 

 

5

 

 

0

 

 

0

 

 

4

 

 

7

 

 

2

 

 

3

 

 

16

Stomach

Number Examined

 

 

Erosion; glandular mucosa; focal   Very slight                                                 

 

 

 

0

 

1

 

1

 

2

 

27

 

27

 

27

 

27

 

0

 

0

 

0

 

0

 

1

 

0

 

1

 

2

 

Erosion; glandular mucosa; multifocal   Very slight                                                 

 

0

 

0

 

0

 

0

 

0

 

1

 

3

 

1

                                                                

                                                                Slight

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

3

Inflammation; acute; glandular submucosa; focal/multifocal (combined)                   Very slight                                                                                                                                                       

 

0

 

0

 

0

 

2

 

8

 

9

 

12

 

3

                                                              

                                                                Slight

 

0

 

0

 

0

 

0

 

3

 

1

 

3

 

7

 

Ulcer; glandular mucosa; focal              Slight

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

2

Thyroid Gland

Number Examined

 

Dilatation; follicle       

                                                                  Very slight

0

0

1

0

26

27

26

27

0

0

0

0

2

2

6

10

Bold typeindicates effects judged to be treatment related.

Table 8: P1 Histopath

Sex

Male

Female

Dose (%)

0

0.01

0.05

0.15

0

0.01

0.05

0.15

Kidney

Number Examined

 

Degeneration with regeneration, tubule unilateral/bilateral (combined), multifocal      

                                                                  Very slight

27

27

27

27

27

27

27

27

9

11

19

21

2

4

1

15

                                                                  Slight

0

0

1

5

0

0

0

3

Dilatation, tubule, unilateral/bilateral (combined), multifocal                                                 Very slight

3

4

8

9

1

1

0

4

                                                                 Slight

0

0

2

6

1

0

0

3

Inflammation, subacute to chronic, interstitium, unilateral/bilateral (combined), multifocal 

                                                                 Very slight

 

7

 

6

 

6

 

8

 

1

 

1

 

4

 

0

                                                                Slight

0

1

0

0

0

0

0

0

Liver

Number Examined

 

Vacuolization; consistent with fatty change; hepatocyte; individual cells; multifocal

                                                                 Very slight

 

27

 

5

 

3

 

27

 

27

 

27

 

27

 

27

 

 

9

 

 

0

 

 

0

 

 

6

 

 

3

 

 

4

 

 

4

 

 

7

 

                                                                 Slight

 

0

 

0

 

0

 

1

 

0

 

0

 

0

 

2

Stomach

Number Examined

 

Erosion; glandular mucosa; focal            

                                                                 Very slight                                                 

 

 

 

0

 

2

 

0

 

0

 

27

 

27

 

27

 

27

 

0

 

0

 

0

 

0

 

0

 

3

 

3

 

1

 

Erosion; glandular mucosa; multifocal   Very slight

 

0

 

0

 

0

 

0

 

0

 

1

 

0

 

0

                                                                

                                                                Slight

 

0

 

0

 

0

 

0

 

0

 

0

 

1

 

2

Inflammation; acute; glandular submucosa; focal/multifocal (combined)                    Very slight                                   

 

0

 

0

 

0

 

0

 

6

 

4

 

4

 

7

 

                                                                Slight

 

0

 

0

 

0

 

0

 

2

 

1

 

2

 

5

Bold type indicates effects judged to be treatment related.

Table 9: P0 Reproductive Indices

 

Dose Level (%)

Parameter (mean %)

0

0.01

0.05

0.15

Male Mating Index (%)

96.3 (26/27)

96.3 (26/27)

100.0 (27/27)

88.9 (24/27)

Female Mating Index (%)

96.3 (26/27)

96.3 (26/27)

100.0 (27/27)

88.9 (24/27)

Male Conception Index (%)

100.0 (26/26)

88.5 (23/26)

96.3 (26/27)

100.0 (24/24)

Female Conception Index (%)

100.0 (26/26)

88.5 (23/26)

96.3 (26/27)

100.0 (24/24)

Male Fertility Index (%)

96.3 (26/27)

85.2 (23/27)

96.3

(26/27)

88.9 (24/27)

Female Fertility Index (%)

96.3 (26/27)

85.2 (23/27)

96.3

(26/27)

88.9 (24/27)

Table 10: P1 Reproductive Indices

 

Dose Level (%)

Parameter (mean %)

0

0.01

0.05

0.15

Male Mating Index (%)

92.6 (25/27)

96.2 (25/26)

100.0 (27/27)

84.6 (22/26)

Female Mating Index (%)

92.6 (25/27)

96.2 (25/26)

100.0 (27/27)

84.6 (22/26)

Male Conception Index (%)

92.0 (23/25)

100.0 (25/25)

100.0 (27/27)

86.4 (19/22)

Female Conception Index (%)

92.0 (23/25)

100.0 (26/26)

100.0 (27/27)

86.4 (19/22)

Male Fertility Index (%)

85.2 (23/27)

96.2 (25/26)

100.0

(27/27)

73.1 (19/26)

Female Fertility Index (%)

85.2 (23/27)

96.2 (26/27)

100.0

(27/27)

73.1 (19/26)

Table 11: Gestation Survival and Postimplantation Loss

Dose Level (%):

0

0.01

0.05

0.15

F1 Gestation Survival (%)

99.2

99.7

100.0

98.4

F2 Gestation Survival (%)

99.4

98.9

99.4

91.9*

F1 Postimplantation Loss (%)

10.71

6.41

8.66

7.97

F2 Postimplantation Loss (%)

6.81

9.77

6.67

18.80*

Bold indicates effects considered to be treatment related

Table 12: F1 Litter Size

 

Mean litter size

Dose Level (%):

0

0.01

0.05

0.15

Born Live

13.5

14.5

14.3

13.0

Born Dead

0.1

0.0

0.0

0.2

Day 1

13.1

14.3

14.3

13.7

Day 4

13.0

14.3

14.2

13.3

Day 7

7.7

8.0

8.0

7.7

Day 14

7.7

8.0

8.0

7.7

Day 21

7.7

8.0

8.0

7.7

Table 13: F2 Litter Size

 

Mean litter size

Dose Level (%):

0

0.01

0.05

0.15

Born Live

13.9

13.5

12.9

11.9

Born Dead

0.1

0.2

0.1

1.1

Day 1

13.8

13.4

12.9

11.2

Day 4

13.3

13.3

12.8

10.8

Day 7

7.7

8.0

7.6

7.1

Day 14

7.7

8.0

7.6

7.1

Day 21

7.7

8.0

7.6

7.1

Bold type indicates the effects judged to be treatment related.

Table 14: F1 Pup Body Weights (g)

 

Dose Level (%)

Pup Age and Sex

0

0.01

0.05

0.15

PND 1 Female

PND 1 Male

6.6

6.9

6.7

7.0

6.6

7.0

6.4

6.6

PND 4 (BC) Female

PND 4 (BC) Male

9.6

9.9

9.4

9.9

9.3

9.8

8.8

9.1

PND 4 (AC) Female

PND 4 (AC) Male

9.6

9.9

9.4

9.8

9.3

9.8

8.8

9.1

PND 7 Female

PND 7 Male

15.8

16.4

15.5

16.1

15.6

16.2

14.0*

14.3*

PND 14 Female

PND 14 Male

33.6

34.5

32.8

34.0

33.3

34.3

30.6$

31.3*

PND 21 Female

PND 21 Male

53.1

54.4

51.6

53.1

52.7

54.6

48.8

49.7

* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

$Statistically different from control mean by Wilcoxon’s test, alpha = 0.05.

BC = Before culling; AC = After culling.

Table 15: F2 Pup Body Weights (g)

 

Dose Level (%)

Pup Age and Sex

0

0.01

0.05

0.15

PND 1 Female

PND 1 Male

6.6

7.1

6.8

7.2

7.1

7.4

6.5

7.1

PND 4 (BC) Female

PND 4 (BC) Male

9.6

10.1

9.9

10.4

10.2

10.6

9.9

10.6

PND 4 (AC) Female

PND 4 (AC) Male

9.6

10.1

10.0

10.5

10.2

10.6

9.9

10.5

PND 7 Female

PND 7 Male

15.9

17.0

16.3

17.1

16.5

17.3

15.7

16.6

PND 14 Female

PND 14 Male

33.7

35.4

34.0

35.1

33.8

34.9

32.7

34.2

PND 21 Female

PND 21 Male

52.8

56.3

53.5

55.5

53.2

55.7

51.0

53.2

BC = Before culling; AC = After culling.

Conclusions:
Administration of 0.15% bronopol resulted in decreased water consumption (attributed to decreased palatability) throughout most of the study, along with secondary, minor decreases in feed consumption. Decreases in body weights occurred sporadically in both sexes and generations, although there was a consistent decrease in body weight gain in P0 and P1 females in the week prior to delivery. Microscopic examination of tissues from adult animals revealed minor changes in the kidneys, thyroid, stomach, and liver. Kidney effects were found in males and females of both generations and consisted of very slight-slight nephropathy corresponding with increased absolute and relative kidney weights. There was an increased incidence of very slight follicle dilatation of the thyroid correlating with increased absolute and relative thyroid weight for P0 females. Other microscopic effects were of minor severity and restricted to P0 and P1 females. These changes included erosions, ulcer and/or inflammation of the glandular stomach, and vacuolization of scattered individual hepatocytes (fatty change) with no corresponding liver weight changes.
Similar to the high dose group, administration of 0.05% bronopol resulted in statistically identified decreased water consumption (attributed to decreased palatability) in P0 males and females during the first half of the study (days 1-57 and 1-50, respectively). Water consumption for P1 animals was not decreased in the 0.05% dose group. Histopathologic effects were limited to an increased incidence of very slight- slight nephropathy restricted to P1 males, but no change in organ weight. Also, histopathologic examination of the thyroid in P0 females revealed a marginal increase in the incidence of very slight follicular dilatation, without corresponding thyroid weight change. There were no other systemic or histopathologic treatment related effects in the mid-dose group. There were no treatment related systemic or histopathologic effects in the low-dose animals administered 0.01% bronopol.
Reproductive effects occurred only at the 0.15% dose level and were limited to two cases of difficult delivery (dystocia) in the P0 dams, and increased postimplantation loss (18.8 versus 6.8% in controls) and associated decreases in gestation survival and stillborn pups, along with two total litter losses in the P1 generation. These reproductive effects were attributable to six high dose dams, several of which exhibited clear signs of maternal toxicity in late gestation. There were no effects on any parameter of reproductive performance or offspring growth and survival at 0.01 or 0.05% bronopol.
The NOEL for systemic toxicity was 0.01% (10 mg/kg/day) bronopol in drinking water. The NOEL for reproductive toxicity was 0.05% (50 mg/kg/day) bronopol.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Several studies including one- and two-generation studies using bronopol as test material were available. The most recent study according to current guideline was used in order to get to a reliable conclusion regarding the effects on fertility of Bronopol. The results were supported in a weight of evidence approach by the other available studies. 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

A GLP conform 2-generation study was conducted according to OECD guideline 416 by Dow (2008). Bronopol (purity 99.85%) was administered to Crl:CD(SD) rats via drinking water at different target doses of 0.01%, 0.05% and 0.15% corresponding to concentrations of 0, 10,50 and 150 mg/kg bw/day.

At each dose level 27 animals per sex were treated for approximately 10 weeks prior to mating. Then animals were mated in both P0 and P1 generation. Mating duration was 2 weeks. Approximately 10 weeks after all F1 litters have been weaned, F1 offspring randomly selected to become P1 adults were bred as described above. The concentration of the test material in the dosing solutions was periodically analyzed by HPLC measurement.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. Oestrous cyclicity and sperm parameters were evaluated in P0 and P1. Vaginal opening and anogenital distance was recorded in pubs.

Administration of 0.15% bronopol resulted in decreased water consumption (attributed to decreased palatability) throughout most of the study, along with secondary, minor decreases in feed consumption. Decreases in body weights occurred sporadically in both sexes and generations, although there was a consistent decrease in body weight gain in P0 and P1 females in the week prior to delivery. Microscopic examination of tissues from adult animals revealed minor changes in the kidneys, thyroid, stomach, and liver. Kidney effects were found in males and females of both generations and consisted of very slight-slight nephropathy corresponding with increased absolute and relative kidney weights. There was an increased incidence of very slight follicle dilatation of the thyroid correlating with increased absolute and relative thyroid weight for P0 females. Other microscopic effects were of minor severity and restricted to P0 and P1 females. These changes included erosions, ulcer and/or inflammation of the glandular stomach, and vacuolization of scattered individual hepatocytes (fatty change) with no corresponding liver weight changes. Similar to the high dose group, administration of 0.05% bronopol resulted in statistically identified decreased water consumption (attributed to decreased palatability) in P0 males and females during the first half of the study (days 1-57 and 1-50, respectively). Water consumption for P1 animals was not decreased in the 0.05% dose group. Histopathologic effects were limited to an increased incidence of very slight- slight nephropathy restricted to P1 males, but no change in organ weight. Also, histopathologic examination of the thyroid in P0 females revealed a marginal increase in the incidence of very slight follicular dilatation, without corresponding thyroid weight change. There were no other systemic or histopathologic treatment related effects in the mid-dose group. There were no treatment related systemic or histopathologic effects in the low-dose animals administered 0.01% bronopol. Reproductive effects occurred only at the 0.15% dose level and were limited to two cases of difficult delivery (dystocia) in the P0 dams, and increased postimplantation loss (18.8 versus 6.8% in controls) and associated decreases in gestation survival and stillborn pups, along with two total litter losses in the P1 generation. These reproductive effects were attributable to six high dose dams, several of which exhibited clear signs of maternal toxicity in late gestation. There were no effects on any parameter of reproductive performance or offspring growth and survival at 0.01 or 0.05% bronopol. The NOEL for systemic toxicity was 0.01% (10 mg/kg/day) bronopol in drinking water. The NOEL for reproductive toxicity was 0.05% (50 mg/kg/day) bronopol.

This GLP-conform guideline study is classified as acceptable (key study)and was used for classification.

In another 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 (supporting 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. The study was not used for classification, since a GLP conform two-generation study according to OECD TG 416 was available.

 

 

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 (supporting 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, 10, 50 or 150 mg/kg/day. Treatment-related effects referring to systemic toxicity were seen at mid and high doses of Bronopol but were particularly pronounced at the highest dose tested. With regard to reproductive or litter parameters, significant treatment-related effects were reported for the high dose group; in fact the effects reported at the highest test dose of 150 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 that was conducted according to OECD TG 416.
Furthermore another two-generation toxicity study, the corresponding range-finding study and a previous one-generation study (supporting studies) are listed, which support the findings of the key study.

Effects on developmental toxicity

Description of key information

Developmental toxicity rat, p.o., gestation day 6-15: NOAEL maternal toxicity ≥ 80 mg/kg bw/day; NOAEL embryotoxic / teratogenic effects ≥ 80 mg/kg bw (GLP; EPA OPP 83 -3; Toxicology Laboratories Limited, 1995)

Developmental toxicity rabbit, p.o., gestation day 7 -27: NOAEL maternal toxicity = 10 mg/kg bw/day; NOAEL embryotoxic / teratogenic effects= 10 mg/kg bw (GLP; OECD 414; Dow, 2007)

Link to relevant study records

Referenceopen allclose all

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
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
28 mg/kg bw/day
Dose / conc.:
80 mg/kg bw/day
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.
Clinical signs:
no effects observed
Description (incidence and severity):
no treatment-related clinical symptoms were reported
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No mortality was observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
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 and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar in all groups and therefore inconspicuous
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy of the dams revealed no treatment-related abnormalities
Details on maternal toxic effects:
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:
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:
no effects observed
Developmental effects observed:
no
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2006 - 6 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: EEC, ISSN 0378-6978 and JMAFF Guideline 21-1-18, Teratogenicity Study (2000)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity: 99.9%
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Species and Sex: Rabbits, time-mated females
Strain and Justification: New Zealand White rabbits were selected because of their general acceptance and suitability for toxicity testing, the availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Covance Research Products, Inc. (Kalamazoo, Michigan)
Age and Weight at Study Start: Sexually mature adult, five to six months of age and weighing 2500-3500g
Physical Acclimation: Each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least six days prior to the start of the study.
Housing: Animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). The room relative humidity was maintained within a range of 38-60%. The average room temperature was maintained at 20 ± 1°C (with a maximum permissible excursion of ± 3°C). A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour. Cages had flattened tube grid floors and were suspended above catch pans with absorbent non-contact bedding. Cages contained a J-type feeder and a pressure activated lixit valve-type watering system.
Randomization and Identification: Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.
Feed and Water: Upon receipt, rabbits received approximately 2 oz. of LabDiet® Certified Rabbit Diet #5325 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. The amount of feed was increased in 2 oz. per day increments up to approximately 8 oz. to avoid gastrointestinal disturbances during the acclimation period. Municipal water was provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. The results of these analyses indicated no contaminants at levels that would interfere with the conduct of this study or interpretation of the results. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
pH 4
Details on exposure:
Bronopol solutions were prepared in an aqueous vehicle of acidified deionized water
(final pH~4) at concentrations of 0.75, 2.5, and 7.5 mg/ml and administered a dose volume of 4 ml/kg body weight in order to achieve the targeted dose levels. Dose volumes were adjusted daily based on individual body weights. Dose solutions were prepared every two weeks throughout the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The low- and high-dose solutions from the first mix were anlayzed prior to the start of dosing to verity homogeneous distribution of the test material in vehicle. Analysis of all dosing solutions from the first mix were initiated prior to the start of dosing using HPLC with ultraviolet detection and external standards to determine concentration. Stability in vehicle for at least 40 days was determined as part of a separate study.
Details on mating procedure:
Breeding Procedures: Sexually mature, adult virgin females, weighing approximately 2500-3500 grams were naturally mated with males of the same strain at CRP. The observed day of breeding was considered GD 0. GD 0 body weights and records of mating pairs were provided by CRP and maintained in the study record. Rabbits were shipped GD 0 or 1 and arrived in this laboratory on the same day.
Duration of treatment / exposure:
GD 7-27
Frequency of treatment:
daily
Duration of test:
until GD 28
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
26
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels for this study were selected on the basis of a developmental toxicity probe study (Dow, 2006). The high-dose of 30 mg/kg/day was expected to induce signs of maternal toxicity. The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose rabbits.
Maternal examinations:
Body weight: gestation day 0, 7-28 (daily)
Food consumption: gestation day 4-28
Clinical signs: at least once daily; mortality twice daily
Sacrifice on GD28 included necropsy of all external tissues and all orifices. The stomach, liver (with gallbladder), and kidneys were dissected out and incised. Gross pathologic alterations were recorded and the weight of the liver (with incised gallbladder), kidneys, and gravid uterus were recorded. Relative weight of kidneys and liver were calculated.
Ovaries and uterine content:
Gravid uterine weight, Number of corpora lutea (for females with >/=1 foetus), Number and position of implantations, Early and late resorptions, Litter size, Number of alive and dead fetuses, Fetal weight, Sex ratio
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, about half of the fetuses
Statistics:
Bartlett’s test for equality of variances followed by parametric or nonparametric analysis of variance (ANOVA); followed by Dunnett's test or Wilcoxon Rank-Sum test with Bonferroni's correction: maternal body weights, maternal body weight gains, organ weights (absolute and relative), fetal body weights and feed consumption
Censored Wilcoxon test with Bonferroni’s correction: Frequency of pre- and post-implantation loss, and fetal alterations
Nonparametric ANOVA followed by Wilcoxon Rank-Sum test with Bonferroni's correction: no. of corpora lutea, implantations, litter size
Fisher exact probability test with Bonferroni’s correction: pregnancy rates
Binomial distribution test: foetal sex ratios
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The animals that were euthanized had decreased or absent feces, and the high-dose animal was additionally cold to the touch.
In the 30 mg/kg/day dose group, sixteen of twenty-six animals had treatment-related decreased/absent fecal output. The decreases in fecal output were consistent with slight decreases in feed consumption (GD 7-11) and body weight gain (GD 7-10) in these animals. Other incidental observations of decreased, soft, and/or absent feces noted in does from the control, 3 or 10 mg/kg/day dose groups were not considered treatment related, as they did not correspond with feed consumption or body weight gain effects, and did not exhibit a dose-response relationship.
Five animals given 30 mg/kg/day had sporadic incidents of noisy respiration, and one of these five also had labored respiration that was interpreted to be related to treatment. The cause for these observations was not determined; however, they could possibly have been related to aspiration of small amounts of test material, which is known to have irritant properties. Other sporadic incidences of blood in cage (related to totally resorbed litter from doe #8022), head tilt, red vulvar discharge, slow respiration, ungroomed appearance, skin/mucous membranes pale, excessive hairloss, or perioral, perineal, or perinasal soiling were not considered to be related to treatment with bronopol.
Mortality:
mortality observed, treatment-related
Description (incidence):
One mortality was noted, and attributed to gavage error. One animal given 10 mg/kg and one given 30 mg/kg were euthanized on GD 22 due to severe inanition (GD 15-22). All three animals were noted with normal appearing fetuses
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically identified differences in body weights or body weight gains for any treated groups when compared to their respective controls. Although not statistically identified, 13/26 animals in the 30 mg/kg/day dose group had a mean body weight loss (with 6 animals losing >150 grams) at the start of treatment (GD 7-10), and slightly lower gains over the entire treatment period (GD 7-28), which correlated with decreased feces and feed consumption. The slight mean body weight loss and lower gain were considered related to the administration of bronopol.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no statistically identified differences in body weights or body weight gains for any treated groups when compared to their respective controls. Although not statistically identified, 13/26 animals in the 30 mg/kg/day dose group had a mean body weight loss (with 6 animals losing >150 grams) at the start of treatment (GD 7-10), and slightly lower gains over the entire treatment period (GD 7-28), which correlated with decreased feces and feed consumption. The slight mean body weight loss and lower gain were considered related to the administration of bronopol.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically identified differences in any of the measured parameters for any treated groups when compared to their respective controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to bronopol.
The animal that died due to gavage error had mottled and edematous lungs, congestion of the nasal turbinates, blood-tinged soiling on the external nares and lower jaw, congestion of the tracheal mucosa with froth in the trachea.
The animals that were euthanized had decreased ingesta, a hairball in the stomach (one animal) and accentuated lobular pattern in the liver.

Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
One animal given 3 mg/kg/day had a totally resorbed litter with late resorptions that was not considered treatment related because it was a single occurrence, and due to the lack of a dose-response. This doe had blood in its cage, red vulvar discharge, and appeared pale the day before being necropsied. At necropsy, it had decreased ingesta, hemoperitoneum, hydrothorax, dark lungs, and a dark vagina.
Early or late resorptions:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
There were no significant treatment related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent pre-implantation loss, percent post-implantation loss, fetal sex ratios, fetal body weights or gravid uterine weights in rabbits given 3, 10, or 30 mg/kg/day.
One animal given 3 mg/kg/day had a totally resorbed litter with late resorptions that was not considered treatment related because it was a single occurrence, and due to the lack of a dose-response. This doe had blood in its cage, red vulvar discharge, and appeared pale the day before being necropsied. At necropsy, it had decreased ingesta, hemoperitoneum, hydrothorax, dark lungs, and a dark vagina.
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
effects observed, treatment-related
Description (incidence and severity):
In the 10 mg/kg/day group, there was one fetus with a hypoplastic tail, which was not considered treatment related due to the lack of corroborating skeletal findings (i.e., normal caudal vertebrae and centra), as well as its low incidence.
There also was one fetus with a filamentous tail in the 30 mg/kg/day group that was confirmed by skeletal findings of missing caudal vertebrae and centra. Based on an increased incidence of other axial skeleton alterations in this dose group (see skeletal examination below), a relationship to treatment could not be ruled out.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
In the control group there were three malformed fetuses, including bilateral 12th thoracic hemivertebra, and two fetuses with extra lumbar ribs on the 4th, 5th, and/or 6th lumbar vertebrae.
There was one malformed fetus in the 3 mg/kg/day dose group that had fused thoracic ribs (8th and 9th). Based on the low incidence, which was similar to controls, this skeletal alteration was not considered treatment related.
In the 10 mg/kg/day dose group there were two malformed fetuses; one with a forked 8th thoracic rib, and another fetus with an extra lumbar rib on the 6th vertebra. Based on the low incidence, which was similar to controls, none of the skeletal alterations in the 10 mg/kg/day group were considered treatment related.
In the high dose-group (30 mg/kg/day), there were seven malformed fetuses from four different does. One fetus from doe #8063 had fused cervical centra. Doe #8064 had two malformed fetuses; one fetus had multiple skeletal malformations consisting of: a thoracic hemivertebra, a forked thoracic rib with fused centra, and missing caudal vertebrae and centra that were correlated to a filamentous tail observation in external examination. The other fetus from the same doe had two extra ribs with one of them fused, three fused thoracic centra, and two extra thoracic hemivertebrae. Both fetuses from doe #8065 and both from doe #8070 had extra lumbar ribs on the 5th and/or 6th vertebrae.
While the incidence of any one of these malformations was not statistically significant, the incidence of most of them was near or slightly above the upper boundary of the historical control range. A very slight increase in axial skeletal malformations also was noted in an earlier study at a slightly higher dose (80 mg/kg/day; USEPA, 1993). In addition, these skeletal malformations all involve patterning of the axial skeleton, suggesting a common etiology. Based on the weight of evidence, the axial skeletal effects at 30 mg/kg/day were considered to represent a marginal increase over the normally occurring background incidence of these skeletal findings. Maternal toxicity appeared to be a critical factor, based on the fact that six of the seven fetuses with axial skeleton malformations came from just three litters (#8064, #8065, #8070), all of which exhibited significant maternal toxicity, including body weight losses and marked reductions in feed consumption during the period of axial skeleton pattern formation (i.e., somitogenesis). Therefore, the marginal increase in axial skeletal malformations in the 30 mg/kg/day group was considered to be the result of maternal toxicity exacerbating the normal background incidence of these axial skeletal changes.
There was also an increased incidence of crooked hyoid (variation) in the 30 mg/kg/day dose group. Seven out of ninety-nine fetuses (7.1%) had crooked hyoids in this dose group, as compared to 2/107 fetuses (1.9%) in the control group. The incidence of crooked hyoid was within the range of historical control data (7.7% of fetuses, 26.9% of litters) and there was no increase in the incidence of crooked hyoid noted in an earlier study at a slightly higher dose (80 mg/kg/day; USEPA, 1993). Therefore, this was considered to be unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One control fetus had ectopic, fused kidneys and bilateral hydroureter.
In the 3 mg/kg/day dose group, one fetus was missing its gallbladder. This finding was not considered treatment related based on its low incidence, lack of a dose response, and because the incidence was within the historical control range.
One fetus in the 10 mg/kg/day dose group had a hypoplastic heart. This finding was not considered treatment related due to the low incidence, the lack of a dose response, and absence of corroborating cardiovascular findings.
In the 30 mg/kg/day group, there were three malformed fetuses, all from one doe. Two of the fetuses had right ectopic kidneys and ureters, and right unilateral hydroureter. The third fetus had ectopic, fused kidneys and bilateral hydroureter. The fetal incidence of kidney/ureter alterations in the 30 mg/kg/day group was outside the normal historical control range, whereas the litter incidence was essentially identical to both the historical and concurrent control incidence. There were also seven fetuses from three different litters in the 30 mg/kg/day dose group that had the variation, right-sided esophagus, a subtle variation whose significance is unclear. Prior data showing that bronopol at 80 mg/kg/day did not increase the incidence of any of these abnormalities (US EPA, 1993) indicating that the visceral alterations in the 30 mg/kg/day group of the present study were not treatment-related.
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Remarks on result:
other: at maternal toxic concentrations
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
marginal increase in axial skeletal malformations
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Table 1. Clinical Observations

 Dose Level (mg/kg/day)

0

3

10

30

Clinical Observations

No. of Animals Affected

Feces Abnormal Quantity – Decreased/Absent

7

5

7

16*

Feces Abnormal Consistency - Soft

0

1

0

0

*Considered treatment related

Table 2. Number of Fetuses/Litters with Visceral Variations/Malformations

Dose Level (mg/kg/day)

 

0

3

10

30

Ectopic/fused kidneys/ureters

F

L

1/224 (0.4)

1/25 (4.0)

0/200 (0.0)

0/24 (0.0)

0/209 (0.0)

0/24 (0.0)

3/215 (1.4)a,b,c

1/25 (4.0)

Hydroureter

F

L

1/224 (0.4)

1/25 (4.0)

0/200 (0.0)

0/24 (0.0)

0/209 (0.0)

0/24 (0.0)

3/215 (1.4)a,b,c

1/25 (4.0)

Missing gallbladder

F

L

0/224 (0.0)

0/25 (0.0)

1/200 (0.5)

1/24 (4.2)

0/209 (0.0)

0/24 (0.0)

0/215 (0.0)

0/25 (0.0)

Hypoplastic heart

F

L

0/224 (0.0)

0/25 (0.0)

0/200 (0.0)

0/24 (0.0)

1/209 (0.5)

1/24 (4.2)

0/215 (0.0)

0/25 (0.0)

Right-sided Esophagus

F

L

0/224 (0.0)

0/25 (0.0

2/200 (1.0)

2/24 (8.3)

0/209 (0.0)

0/24 (0.0)

7/215 (3.3)

3/25 (12.0)

a,b,c: same superscript indicates malformations common to a single fetus

Table 3. Five-year historical control for visceral variations/malformations

 

 

1

2001

2

2002

3

2002

4

2004

5

2005

6

2005

Ectopic kidney

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

1/179 (0.6)

1/24 (4.2)

0/206 (0.0)

0/25 (0.0)

0/223 (0.0)

0/25 (0.0)

Hydroureter

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/206 (0.0)

0/25 (0.0)

0/223 (0.0)

0/25 (0.0)

Missing gallbladder

F

L

0/202(0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

3/206 (1.5)

1/25 (4.0)

0/223 (0.0)

0/25 (0.0)

Hypoplastic heart

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/206 (0.0)

0/25 (0.0)

0/223 (0.0)

0/25 (0.0)

Right-sided esophagus

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

1/206 (0.5)

1/25 (4.0)

0/223 (0.0)

0/25 (0.0)

Bold indicates single study incidence

Table 4. Number of Fetuses/Litters with Skeletal Variations/Malformations

 

 

1

2001

2

2002

3

2002

4

2004

5

2005

6

2005

Ectopic kidney

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

1/179 (0.6)

1/24 (4.2)

0/206 (0.0)

0/25 (0.0)

0/223 (0.0)

0/25 (0.0)

Hydroureter

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/206 (0.0)

0/25 (0.0)

0/223 (0.0)

0/25 (0.0)

Missing gallbladder

F

L

0/202(0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

3/206 (1.5)

1/25 (4.0)

0/223 (0.0)

0/25 (0.0)

Hypoplastic heart

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/206 (0.0)

0/25 (0.0)

0/223 (0.0)

0/25 (0.0)

Right-sided esophagus

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

1/206 (0.5)

1/25 (4.0)

0/223 (0.0)

0/25 (0.0)

        a,b,c Same superscript indicates malformations common to a single fetus

Bold indicates effects considered to be treatment related

Table 5. Five-year historical control for skeletal variations/malformations

 

 

1

2001

2

2002

3

2002

4

2004

5

2005

6

2005

Hemivertebrae

F

L

1/202 (0.5)

1/24 (4.2)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

1/192 (0.5)

1/24 (4.2)

1/223 (0.4)

1/25 (4.0)

Fused

centra

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/192 (0.0)

0/24 (0.0)

1/223 (0.4)

1/25 (4.0)

Fused rib

F

L

2/202 (1.0)

2/24 (8.3)

0/221 (0.0)

0/26 (0.0)

2/192 (1.0)

2/25 (8.0)

0/179 (0.0)

0/24 (0.0)

1/192 (0.5)

1/24 (4.2)

0/223 (0.0)

0/25 (0.0)

Forked rib

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

1/192 (0.5)

1/24 (4.2)

1/223 (0.4)

1/25 (4.0)

Missing caudal vertebrae & centra

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/192 (0.0)

0/24 (0.0)

0/223 (0.0)

0/25 (0.0)

Extra lumbar rib

F

L

0/202 (0.0)

0/24 (0.0)

0/221 (0.0)

0/26 (0.0)

0/192 (0.0)

0/25 (0.0)

0/179 (0.0)

0/24 (0.0)

0/192 (0.0)

0/24 (0.0)

0/223 (0.0)

0/25 (0.0)

Crooked hyoid

F

L

4/95 (4.2)

4/24 (16.7)

8/104 (7.7)

7/26 (26.9)

2/88 (2.3)

2/24 (8.3)

1/82 (1.2)

1/23 (4.3)

2/97 (2.1)

2/25 (8.0)

3/106 (2.8)

2/25 (8.0)

Bold indicates maximum single study incidence

Conclusions:
Oral administration of bronopol to time-mated female rabbits resulted in maternal and developmental effects at 30 mg/kg/day. The maternal effects consisted of body weight loss at the start of treatment, sporadic occurrences of slightly reduced feed consumption, slightly decreased body weight gains over the entire treatment period (GD 7-28), decreased fecal output, and noisy respiration. There were no maternal effects at lower doses. Treatment-related developmental effects at the 30 mg/kg/day dose level consisted of a marginal increase in the overall incidence of axial skeletal malformations. However, the incidence of each individual axial skeleton malformation was near or slightly above the upper boundary of the historical control range. Furthermore, six of the seven fetuses with axial skeleton malformations came from just three litters, all of which exhibited significant maternal toxicity during the period of axial skeleton pattern formation (i.e., somitogenesis). Therefore, the marginal increase in axial skeletal malformations in the 30 mg/kg/day group was considered to be the result of maternal toxicity exacerbating the normal background incidence of these axial skeletal changes. The maternal and fetal NOAEL were 10 mg/kg/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
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 (supporting 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 (supporting 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 studies

In a GLP conform developmental toxicity study according to OECD 414, groups of pregnant female New Zealand White rabbits (26/group) received 3, 10 or 30 mg test substance /kg bw/ day per gavage during gestation (GD 7-27; Dow, 2007). A standard dose volume of 4 mL/kg body weight was used. The control group was dosed with the vehicle only (acidified deionized water, pH 4). Food consumptions and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On day 28 p.c., all females were sacrificed and assessed by gross pathology. For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external findings. Thereafter, fetuses of each litter were examined for soft tissue findings and skeletal findings.Oral administration of bronopol to time-mated female rabbits resulted in maternal and developmental effects at 30 mg/kg/day. The maternal effects consisted of body weight loss at the start of treatment, sporadic occurrences of slightly reduced feed consumption, slightly decreased body weight gains over the entire treatment period (GD 7-28), decreased fecal output, and noisy respiration. There were no maternal effects at lower doses. Treatment-related developmental effects at the 30 mg/kg/day dose level consisted of a marginal increase in the overall incidence of axial skeletal malformations. However, the incidence of each individual axial skeleton malformation was near or slightly above the upper boundary of the historical control range. Furthermore, six of the seven fetuses with axial skeleton malformations came from just three litters, all of which exhibited significant maternal toxicity during the period of axial skeleton pattern formation (i.e., somitogenesis). Therefore, the marginal increase in axial skeletal malformations in the 30 mg/kg/day group was considered to be the result of maternal toxicity exacerbating the normal background incidence of these axial skeletal changes.

Thus, the NOAEL for maternal toxicity was 10 mg/kg bw/day and the NOAEL for embryotoxic / teratogenic effects was 10 mg/kg bw/day, too.

Conclusively, developmental toxicity was observed at a test dose, which was shown to be toxic to the maternal animals

This study is classified as acceptable (key study). The test was conducted according to OECD TG 414 and followed GLP.

 

The effects of Bronopol on the embryonic and fetal development during organogenesis of rabbits following oral daily administration to pregnant females was investigated in another developmental toxicity study (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 (supporting 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 (supporting 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 studies represent guideline studies conducted in accordance with GLP (reliability: 1). Furthermore, the corresponding range-finding studies and previous studies about development of rat and rabbit are listed, which support the findings of the key studies.

Since the test substance 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 developmental toxicity in a developmental toxicity study in rabbits according to OECD 414 was only observed at a test dose, which was shown to be toxic to the maternal animals, Bronopol is not considered to be classified for reproductive toxicity.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.