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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005 to 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Tsukuba Breeding Center, Charles River Laboratories Japan, Inc. (Yokohama, Japan).

- Age at study initiation: P x ca. 5 weeks; (F1) x 3-4 weeks

- Weight at study initiation: not indicated

- Housing: animals were housed individually in suspended aluminum/stainless steel cages, except during the acclimation, mating and nursing periods. From day 17 of pregnancy to the day of weaning, individual dams and litters were reared using wood chips as bedding (White Flake, Charles River Laboratories Japan, Inc.).

- Diet (e.g. ad libitum): powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo,Japan),

- Water (e.g. ad libitum): Filtered tap-water, ad libitum.

- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC

- Humidity (%): 50 ± 20%

- Air changes (per hr): 10-15 per hour

- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: Day of selection To: Males - After the partuition of paired females; Females - After the weaning of pups.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan)
Details on exposure:
DIET PREPARATION
Dosed diet preparations were formulated by mixing HBCD into an appropriate amount of a powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) for each dietary concentration. The control rats were fed a basal diet only.

- Rate of preparation of diet (frequency): likely every 3 weeks, because analysis showed that the test substance was homogeneous in the diet and stable for at least 21 days at room temperature

- Mixing appropriate amounts with (Type of food): powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan).

- Storage temperature of food: likely room temperature
Details on mating procedure:
- M/F ratio per cage: 1:1

- Length of cohabitation: 3 weeks

- Proof of pregnancy: The presence of sperm in the vaginal smear and/or a vaginal plug was considered as evidence of successful mating.

- After 3 weeks of unsuccessful pairing replacement of first male by another male with proven fertility (F0 females).

- Further matings after two unsuccessful attempts: not reported.

- After successful mating each pregnant female was caged (how): individually. From day 17 of pregnancy to the day of weaning, individual dams and litters were reared using wood chips as bedding (White Flake, Charles River Laboratories Japan, Inc.).

- Any other deviations from standard protocol:
- Haematology, clinical chemistry and hormone levels: on the day of scheduled sacrifice, blood samples were collected from the abdominal aorta of adult rats under ether anesthesia. Hematological examinations (white blood cell (WBC) count and differential leukocyte count) were performed for 10 males and 10 females of F0 and F1 rats randomly selected from each group. Blood biochemical evaluations (total protein, albumin and globulin) were performed in 10 males and 10 females of F0 and F1 rats randomly selected from each group.
On the day of the scheduled sacrifice, blood samples were collected from the abdominal aorta of adult rats. Eight males and eight proestrous females
of F0 and F1 generations from each group were selected randomly for blood collection. Hormone levels were determined by Panapharm Laboratories Co., Ltd. (Uto, Japan). Serum levels of testosterone, 5α-dihydrotestosterone (DHT), luteinizing hormone (LH) and follicle stimulating hormone (FSH), thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) in males, and estradiol, progesterone, LH, FSH, T3, T4 andTSHin females were measured with a radioimmunoassay kit. Double antibody kits were used for measurement of testosterone, estradiol, progesterone, T3 and T4 concentration (Diagnostic Products Corp., Los Angeles, CA) and DHT concentration (Diagnostic Systems Laboratories Inc., Webster TX). Serum concentrations of LH, FSH and TSH were measured using (rat LH)[125I], (rat FSH)[125I] and (rat TSH)[125I]assay systems (Amersham Biosciences Ltd., Little Chalfont, Buckinghamshire, UK), respectively.
- Behavioural tests: spontaneous locomotor activity was measured with a multi-channel activity monitoring system (Supermex; Muromachi Kikai Co., Ltd., Tokyo, Japan) in 10 male and 10 female F1 rats selected from each group at 4 weeks of age. Rats were placed individually in transparent polycarbonate cages (27.6W×44.5D×20.4Hcm, CL-0108-1, CLEA Japan Inc., Tokyo, Japan), which were placed under an infrared sensor that detects thermal radiation from animals. Spontaneous motor activity was determined for 10 min intervals and for a total of 60 min.
A test in a water-filled multiple T-maze was conducted in 10 male and 10 female F1 rats selected from each group at 6 weeks of age. The apparatus was similar to that described by Biel. The water temperature of the maze was kept 21–22 ◦C. As a preliminary swimming ability test, each rat was allowed to swim three times in a straight channel on the day before the maze trial, and then tested in the maze with three trials per day for the next three consecutive days. The elapsed time between entry into the water at the starting point and touching the goal ramp and number of errors were recorded. To prevent the exhaustion of the rats, no animal was allowed to remain in the water for more than 3 min in any trial.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity and stability of the test substance were verified by analysis using liquid chromatography before and after the study. Analysis showed that the HBCD was homogeneous in the diet and stable for at least 21 days at room temperature, and was administered at the desired feed
concentrations throughout the study.
Duration of treatment / exposure:
F0 test animals were fed diet containing HBCD for 10 weeks prior to mating period, and then throughout the mating, gestation and lactation periods.
F1 selected animals were weaned onto diet containing HBCD until scheduled termination and necropsy.
Frequency of treatment:
Daily in diet.
Details on study schedule:
Twenty-four F0 rats (5-week-old males and females)/sex/group were fed a diet containing HBCD at 0, 150, 1500 or 15,000 ppm for 10 weeks prior to the mating period. Administration of HBCD was continued throughout the mating, gestation and lactation periods. Twenty-four male and 24 female F1 weanlings (1 male and 1 female in each litter) in each group were selected as F1 parents on PNDs 21–25 to equalize the body weights among groups. The day on which F1 parental animals were selected was designated as 0 week of dosing for the F1 generation. The administration of HBCD in the diet was not suspended during PNDs 21–25. F1 selected rats were administered HBCD in the diet of their respective formulations in the same manner as described for F0 rats. Administration of HBCD in the diet was continued throughout the mating, gestation and lactation periods. On PND 26, unselected F1 weanlings and all F2 weanlings were necropsied.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
15,000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
48 (24 male/24 female)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dosage levels were determined based on the results of a previous 90-day oral repeated dose toxicity study in male and female Crl:CD(SD)IGS BR rats given HBCD at 0, 100, 300 or 1000 mg/kg bw/day for 90 days. The author concluded that all test article-related changes, even at 1000 mg/kg bw/day were reversible, not associated with specific target organ damage or diminished function.

- Rationale for animal assignment: random.

Positive control:
Not applicable.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. For females exhibiting evidence of successful mating, body weight of dams were recorded on days 0, 7, 14 and 20 of pregnancy and days 0, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes. Same schedule as body weight.
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):
Daily vaginal lavage samples of each F0 and F1 female were evaluated for estrous cyclicity throughout the 2-week pre-cohabitation period and during cohabitation until evidence of copulationwas detected. Females having repeated 4–6 day estrous cycles were judged to have normal estrous cycles. After weaning their pups, parental female rats were necropsied at the proestrous stage of the estrous cycle.
Sperm parameters (parental animals):
Sperm parameters were determined for all F0 and F1 male adults on the day of the scheduled sacrifice. The right testis was used to count testicular homogenization-resistant spermatid heads. The right cauda epididymis was weighed and used for sperm analysis. Sperm motility was analyzed using a computer-assisted cell motion analyzer (TOX IVOS, Hamilton Thorne Biosciences, Beverly, MA). The percentage of motile sperm and progressively motile sperm, and the swimming speed and pattern were determined. After recording sperm motion, the cauda epididymal fluid was diluted and the sperm were enumerated using a hemacytometer under a light microscope. Sperm count per gram of epididymal tissue was obtained by dividing the total count by the gram weight of the cauda epididymis. Sperm were stained with eosin and mounted on a slide glass. Two hundred sperm in each sample were examined under a light microscope, and the percentage of morphologically abnormal sperm was calculated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). No adjustment was made for litters of fewer than eight pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 & F2 offspring:
All F1 and F2 pups were observed for pinna unfolding on PND 3, incisor eruption on PND 11, and eye opening on PND 14. One male and one female F1 and F2 pup selected from each dam were evaluated for the surface righting reflex on PND 5, negative geotaxis reflex on PND 8, and mid-air righting reflex on PND 18. All F1 offspring selected as F1 parents were observed daily for male preputial separation beginning on PND 35 or female vaginal opening beginning on PND 25. Body weight of the respective F1 rats was recorded on the day of preputial separation or vaginal opening. The anogenital distance (AGD) was measured using calipers on PND 4 in all F1 and F2 pups, and the normalized value of AGD to body weight, AGD per cube root of body weight ratio, was calculated.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not reported for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After the parturition of paired females
- Maternal animals: After weaning their pups, parental female rats were necropsied at the proestrous stage of the estrous cycle.

GROSS NECROPSY
A complete necropsy was performed on all rats found dead and those killed at the scheduled sacrifice. Live rats were euthanized by exsanguination under ether anesthesia. The external surfaces of the rats were examined. The abdomen and thoracic cavities were opened, and a gross internal examination was performed. Weights of the brain, pituitary, thyroid, thymus, liver, kidney, spleen, adrenal, testis, epididymis, seminal vesicle (with coagulating glands and their fluids), ventral prostate, uterus and ovary were recorded. Weights of the thyroid and seminal vesicle were measured after fixation. Major organs were stored in 10% neutral-buffered formalin. The testis and epididymis were fixed with Bouin’s solution and preserved in 70% ethanol.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluation of F0 and F1 adults was performed on the tissues specified below after fixation, paraffin embedding, and sectioning and staining with hematoxylin and eosin: the pituitary, liver, thymus, kidney, spleen, adrenal, bone marrow, mesenteric lymph node, Peyer’s patches, testis, epididymis, seminal vesicle, coagulating gland, ventral prostate, ovary, uterus, vagina and mammary gland of all males and females in the control and highest dose (15,000 ppm) groups and of females with abnormal estrous cycles, males and females without evidence of copulation or insemination and females with abnormal delivery or totally dead pups in all groups. Any organs or tissues of F0 and F1 adults showing gross alterations were evaluated histopathologically. The thyroid in all rats in all groups was examined histopathologically. In ten F1 females of each group, the number of primordial follicles was counted. The right ovary was fixed in 10% neutral-buffered formalin and then dehydrated and embedded in paraffin in a longitudinal orientation by routine procedures. Sections were cut serially at 5 µm and every 20th section was serially mounted on a slide and stained with hematoxylin and eosin. About 40 sections per ovary were used to determine the primordial follicles.
Postmortem examinations (offspring):
SACRIFICE
The weanlings not selected to become parents were euthanized and necropsied as described for the adults. Organ weights of one male and one female F1 and F2 weanling selected from each dam were measured as described above for adults. The weights of the pituitary, thyroid and seminal vesicle were not determined. All pups found dead before weaning were also necropsied. In all male and female F1 and F2 weanlings whose organs were collected, histopathological evaluations of the liver, in the control and 15,000 ppm groups, and thyroid, in all groups, were performed after fixation, paraffin embedding, and sectioning and staining with hematoxylin and eosin. On PND 26, unselected F1 weanlings and all F2 weanlings were necropsied.
Statistics:
Statistical analysis was performed according to the methods of Gad. Data on offspring before weaning were statistically analyzed using the litter as the experimental unit. Body weight, body weight gain, food consumption, length of estrous cycle, pre-coital interval, gestation length, numbers of implantations and pups delivered, delivery index, sperm parameters, hematological and blood biochemical parameters, hormone levels, organ weight, organ/body weight ratio (relative organ weight), number of primordial follicles, reflex response time, age and body weight at sexual maturation, parameters of behavioral tests, AGD, AGD/cube root of body weight ratio, and viability of pups were analyzed for statistical significance using the following method. Bartlett’s test of homogeneity of variance was used to determine if the groups had equivalent variances. If the variances were equivalent, the groups were compared by one-way analysis of variance (ANOVA). If significant differences were found, Dunnett’s multiple comparison test was performed. If the groups did not have equivalent variances, the Kruskal–Wallis test was used to assess the overall effects. Whenever significant differences were noted, pairwise comparisons were made by the Mann–Whitney U test.
The incidence of pups with changes in clinical and gross internal observations, and completion rate of developmental landmarks and reflexes were analyzed by the Wilcoxon rank sum test.
Reproductive indices:
The incidence of parent animals with changes in clinical, gross internal and histopathological findings, the incidence of females with normal estrous cycles, the copulation index, fertility index, gestation index, and completion rate of the reflex response test were analyzed by Fisher’s exact test. The 0.05 level of probability was significant. The probability was designated as the cut-off for statistical significance.
Offspring viability indices:
The incidence of weanlings with changes in histopathological findings, neonatal sex ratio were analyzed by Fisher’s exact test. The 0.05 level of probability was significant. The probability was designated as the cut-off for statistical significance.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant difference was seen between control and HBCD-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats during the pre-mating, mating, gestation, or lactation period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Fig. 1 shows the body weights of F0 males and females during dosing. In F0 males, the mean body weight and/or body weight gain were significantly higher than those of controls almost throughout the dosing period at 1500 ppm and in the first 5 weeks of dosing at 15,000 ppm. In F0 females, the mean body weight gain was significantly increased on days 0–4 of lactation at 150 ppm and during weeks 0–3 of dosing at 15,000 ppm compared to controls, and the mean body weight was significantly increased on week 2 of dosing at 15,000 ppm. The body weight gain was significantly decreased on days 0–14 of pregnancy at 15,000 ppm compared to controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean daily intakes of HBCD were 12.5, 125 and 1238 mg/kg bw during the pre-mating period, 9.6, 96 and 941 mg/kg bw during the gestation period, and 23.4, 240 and 2200 mg/kg bw during the lactation period in F0 females for 150, 1500 and 15,000 ppm, respectively. The mean daily
intakes of HBCD were 14.0, 138 and 1365 mg/kg bw during the pre-mating period, 9.7, 100 and 995 mg/kg bw during the gestation period, and 19.6, 179 and 1724 mg/kg bw during the lactation period in F1 females for 150, 1500 and 15,000 ppm, respectively. The mean daily intakes of HBCD
during the whole period were 10.2, 101 and 1008 mg/kg bw in F0 males, 14.0, 141 and 1363 mg/kg bw in F0 females, 11.4, 115 and 1142 mg/kg bw in F1 males, and 14.3, 138 and 1363 mg/kg bw in F1 females for 150, 1500 and 15,000 ppm, respectively.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
HBCD produced no significant deviations in estrous cycles, although a few control and HBCD-treated rats had extended estrus or diestrus. In F1 females, there were extended diestrus vaginal smears in a few control and HBCD treated rats, but no significant effect of HBCD was found on the
incidence of females with normal estrous cycles.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
A significantly lower number of epididymal sperm at 150 ppm and higher mean amplitude of lateral head displacement at 15,000 ppm was found in F0 males compared to controls. There were no significant changes in the sperm counts, the percentage of motile sperm and progressively motile sperm, swimming speed and pattern, and the percentage of morphologically abnormal sperm in F1 adults between control and HBCD-treated groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Results are summarised in Table 1. Copulation was not observed in two males and two females at 1500 ppmand two males and one female at 15,000 ppm. Two females each at 150 and 1500 ppm did not become pregnant and three females at 15,000 ppm neither. One pregnant female each at 150 and 15,000 ppm did not deliver live pups. There were significantly longer gestation length and lower sex ratio of live pups at 1500 ppm compared to controls. One dam experienced total litter loss by day 5 of lactation at 15,000 ppm; however, there were no significant differences in the copulation index, fertility index, gestation index, pre-coital interval, number of implantations, delivery index, number of F1 pups delivered, or viability of F1 pups during lactation between the control and HBCD-treated groups. Mean body weight of female F1 pups on PND 0 was significantly higher at 1500 ppm, and that of male F1 pups on PND 21 was significantly lowered at 15,000 ppm, compared to controls. In F1 generation, all pairs in all groups copulated. One female each in the control and 150 ppm groups, and three females each at 1500 and 15,000 ppm were not impregnated. One pregnant female did not deliver live pups at 1500 ppm. One dam experienced total litter loss by day 4 of lactation in the control group and by day 2 of lactation at 150 ppm. At 15,000 ppm, eight dams experienced total litter loss by days 4, 5, 7, 9, 11, 13 or 18 of lactation, and a significantly increased incidence of dams with total litter loss was noted. No clear clinical signs of toxicity were noted in these dams with total litter loss. No significant changes were observed in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of F2 pups delivered or the sex ratio of F2 pups. A significantly decreased viability index was noted in F2 pups on PNDs 4 and 21 at 15,000 ppm. Mean body weights were significantly lowered compared to controls in male F2 pups on PNDs 7, 14 and 21 and in female F2 pups on PNDs 4, 7, 14 and 21 at 15,000 ppm.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 males, there were a significantly decreased relative weight of the brain at 1500 ppm and decreased relative weight of the seminal vesicle at 1500 ppm and higher. On the other hand, there were significantly increased absolute and relative weights of the liver at 1500 ppm and higher and of the thyroid at 15,000 ppm. In F0 females, significant increases were found in the absolute weight of the thyroid, liver and adrenal, and relative weight of the liver at 15,000 ppm when compared with controls. In F1 parent animals, the relative weights of the brain and pituitary were significantly higher at 150 ppm compared to controls. At 15,000 ppm, absolute weight of the brain was significantly decreased, and absolute and relative weights of
the thyroid and liver were significantly increased compared to control. In F1 females, at 15,000 ppm there were a significant decrease in the absolute weight of the brain and a significant increase in absolute and relative weights of the thyroid and liver.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No compound-related gross lesions or microscopic alterations were observed in reproductive organs in male and female F0 and F1 adults showing reproductive difficulties, in male and female F0 and F1 adults of the highest dose group and in dead animals before scheduled sacrifice. There were no compound related gross lesions or remarkable microscopic alterations in other tissues and organs, except for the thyroid, in male and female F0 and F1 adults.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Decreased size of follicles in the thyroid was found in F0 and F1 adults at 1500 ppm and higher, and in F1 females at 150 ppm as well. A significant increased incidence of rats with decreased follicle size was noted in F0 males (25%) and females (21%) and F1 females (21%) at 1500 ppm and F0 males (87%) and females (48%) and F1 males (46%) and females (54%) at 15,000 ppm, compared to controls (0%). Background incidence of decreased follicle size in the laboratory performed current study was 0%in a total of 56 males and 56 females in 6 studies (5–12/sex/study) form 1998 to 2004. Hypertrophy of the follicular cells in the thyroid was also observed in F0 males at 1500 ppm and higher, and in F0 females at 1500 ppm.

OTHER FINDINGS (PARENTAL ANIMALS)
In male F0 and F1 and female F1 adults, no significant difference was noted in the total WBC or differential leukocyte count between control and HBCD-treated groups. In female F0 adults, there was a significantly lower percent of stabform and segmented neutrophils, and a higher percent of lymphocytes at 150 ppm compared to controls. Total protein and globulin were significantly higher in F0 males at 1500 and 15,000 ppm, in F0 females at 150 and 15,000 ppm and in F1 males at 15,000 ppm than those in controls.
There were no significant changes in T3 levels in F0 and F1 rats of both sexes. Lower levels of T4 compared to controls were observed at 15,000 ppm in F0 males and females. Significantly increased levels of TSH were found in F0 females at 150 ppm and higher, and F1 females at 1500 ppm and higher. In F0 adults, serum FSH levels were significantly decreased in males at 1500 ppm and increased in females at 15,000 ppm compared to controls. In F1 adults, significantly higher levels of DHT were observed in males at 1500 ppm. No significant differences in serum testosterone, estradiol, progesterone and LH levels were noted in F0 and F1 adults of both sexes between control and HBCD-treated groups

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
(parental toxicity)
Effect level:
150 ppm
Based on:
other: 10.2 mg/kg bw/day males; 14 mg/kg bw/day females
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
1 500 ppm
Based on:
other: 101 mg/kg/day males; 114 mg/kg/day females
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
(fertility)
Effect level:
15 000 ppm
Based on:
other: 1008 mg/kg bw/day males; 1363 mg/kg bw/day females)
Sex:
male/female
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
NOAEL
Remarks:
(development toxicity - postnatal development)
Effect level:
179 mg/kg bw/day
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
A significantly decreased viability index was noted in F2 pups on PNDs 4 and 21 at 15,000 ppm.

BODY WEIGHT (OFFSPRING)
Fig. 2 presents the body weights of F1 males and females during dosing. Significant decreases compared to controls were observed in the body weight during weeks 3–6 of dosing and body weight gain during the first 6 weeks of dosing in F1 males at 15,000 ppm. Compared with control group, a significantly lowered mean body weight was observed during weeks 3 and 6–10 of dosing, the whole period of gestation and days 0–14 of lactation, and a significantly reduced mean body weight gain was observed during weeks 0–10 of dosing at 15,000 ppm in F1 females. Food consumption was generally paralleled to the body weights/body weight gains during most of the study.

SEXUAL MATURATION (OFFSPRING)
Table 4 presents data on sexual development in F1 rats. No significant differences between control and HBCD-treated groups were noted in the age at preputial separation in males or vaginal opening in females, or body weight at the age of preputial separation or vaginal opening.

ORGAN WEIGHTS (OFFSPRING)
- F1 weanlings and adults:
Table 6 presents the organ weights of male and female F1 weanlings. The mean body weight at scheduled sacrifice was significantly lowered in males at 15,000 ppm compared to controls. In males, there were significant increases in the absolute and relative weights of the testis at 150 ppm, and relative weights of the testis and absolute and relative weight of the liver at 1500 ppm and higher. The absolute weights of the brain and kidney were significantly decreased at 15,000 ppm. In F1 females, significantly increased absolute and relative weights of the liver at 1500 ppm and higher, and decreased absolute weights of the brain and kidney at 15,000 ppm were observed.

- F2 weanlings:
Table 9 presents the organ weights of male F2 weanlings. The body weight at sacrificewas significantly reduced at 15,000 ppm compared to controls. A significant decrease was observed in the relative weight of the kidney at 150 ppm, and a significant increase was observed in the relative weight of the liver at 1500 and 15,000 ppm. There were significantly decreased absolute weight of the brain, kidney, spleen, adrenal, epididymis and ventral prostate and increased relative weight of the brain at 15,000 ppm. Table 10 also presents the organ weights of female F2 weanlings. At 15,000 ppm, a significant decrease compared to controls was found in the body weight at sacrifice. The absolute and relative weights of the ovary were significantly higher at 150 ppm. At 15,000 ppm, there were significantly reduced absolute weight of the brain, thymus, kidney, spleen, adrenal and uterus and increased relative weight of the brain, liver and ovary.

GROSS PATHOLOGY and HISTOPATHOLOGY (OFFSPRING)
There were no compound-related gross lesions and histopathological changes in male and female F1 and F2 pups and weanlings including dead pups

OTHER FINDINGS (OFFSPRING):
DEVELOPMENTAL LANDMARKS
Table 2 presents physical development of F1 and F2 pups. There was no significant difference in the incidence of male and female F1 and F2 pups that displayed pinna unfolding, or incisor eruption between the control and HBCD-treated groups. The incidence of male and female F1 pups showing completion of eye openingwas increased compared to controls at 1500 ppm. In F2 pups, the incidence of pups showing eye openingwas lowered compared to controls in males at 15,000 ppm and in females at 1500 and 15,000 ppm. The AGD and AGD per cube root of body weight ratio were not significantly different between control and HBCD-treated groups in male and female F1 and F2 pups. Table 3 shows reflex ontogeny in F1 and F2 pups. All male and female F1 pups in all groups completed the surface righting reflex, negative geotaxis reflex and mid-air righting reflex. No significant changes were observed in reflex response time, except for faster response in the surface righting in males at 15,000 ppm, in F1 pups of both sexes in HBCD-treated groups. In F2 pups, a few pups failed to complete the reflex response in HBCD-treated groups, and a significantly low incidence of females completed mid-air righting was noted at 15,000 ppm; however, there was no significant difference in the incidence of male and female pups with completed response in other reflexes and in the reflex response time between control and HBCD-treated groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
(other direct toxicity)
Generation:
F1
Effect level:
14.3 mg/kg bw/day
Based on:
other: maternal dose levels during lactation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The NOAEL of HBCD in this two-generation reproductive toxicity study was considered to be 150 ppm (10.2 mg/kg bw/day) in rats.

Applicant's summary and conclusion

Conclusions:
In conclusion, the NOAEL of HBCD in this two-generation reproductive toxicity study was considered to be 150 ppm (10.2 mg/kg bw/day) in rats. NCR estimated that the average oral dose rate in humans was 0.026 mg/kg bw/day. The estimated human intake of HBCD is well below the NOAEL in the present study.
Executive summary:

Male and female rats were fed a diet containing flame retardant hexabromocyclododecane (HBCD) at 0, 150, 1500 or 15,000 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through the mating, gestation and lactation periods for two generations. The mean daily intakes of HBCD during the whole period of administration were 10.2, 101 and 1008 mg/kg bw in F0 males, 14.0, 141 and 1363 mg/kg bw in F0 females, 11.4, 115 and 1142 mg/kg bw in F1 males, and 14.3, 138 and 1363 mg/kg bw in F1 females for 150, 1500 and 15,000 ppm, respectively. The incidence of rats with decreased thyroid follicles size was increased in F0 and F1 males and females at 1500 ppm and higher. Serum TSH levels were increased in F0 and F1 females at 1500 ppm and higher, and serum T4 levels were decreased in F0 males and females at 15,000 ppm. The number of the primordial follicles in the ovary of F1 females was reduced at 1500 ppm and higher. There were increases in the absolute and relative weights of the liver in male adults and male and female weanlings at 1500 ppm and higher, and in female adults at 15,000 ppm, and of the thyroid in male and female adults at 15,000 ppm. Decreased body weight and body weight gain associated with reduced food consumption were found in F1 males and females at 15,000 ppm. Decreases were found in the viability index of F2 pups and the body weight of male F1 and F2 pups and female F2 pups at 15,000 ppm. In F2 pups, there were low incidences of the completion of eye opening in males at 15,000 ppm and in females at 1500 ppm and higher, and of completed mid-air righting in females at 15,000 ppm. The data indicate that the NOAEL of HBCD in this study was 150 ppm (10.2 mg/kg bw/day). The estimated human intake of HBCD is well below the NOAEL in the present study.