Registration Dossier

Administrative data

Description of key information

The following information is presented for this endpoint:
Chengelis C. P. (1996). A 28 Day Repeated-Dose Oral Toxicity Study of HBCD in Rats. Report no.: WIL 186004. Report date: 1997-02-13. as amended by,
Chengelis C. P. (1998). Addendum to the final report A 28-day repeated dose oral toxicity study of HBCD in rats. Report date: 1998-05-28.
Chengelis C.P. (2001). An Oral(Gavage) 90 Day Toxicity Study of HBCD in Rats. Report no.: WIL-186012. Report date: 2001-12-14.
van der Ven, L. T. M., Verhoef, A., van de Kuil, T., Slob, W., Leonards, P. E. G., Visser, T. J., Hamers, T., Herlin, M., Håkansson, H., Olausson, H., Piersma, A. H. and Vos, J. G. (2006). A 28-day oral dose toxicity study enhanced to detect endocrine effects of Hexabromocyclododecane in Wistar rats. Toxicological Sciences (2006) Vol. 94(2), pp. 281-292.
Chengelis (2001) and Chengelis (1996) as amended by Chengelis (1998) were both awarded reliability scores of 1 according to the criteria of Klimisch et al. (1997) based upon the recognised study guidelines used within the methodologies, the GLP conditions which were in effect throughout the study period and depth of reporting of methodologies, results and substance identification information. Chengelis (2001) was selected as the key study for this endpoint as it is the only sub-chronic study available for the registered subtance, it is also the most recent and reliable study available.
Van der Ven et al. (2006) was also conducted according to recognised testing guidelines however information regarding GLP conditions was not reported and so reliability was reduced to a score of 2 in line with criteria set out by Klimisch et al. (1997).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
1 000 mg/kg bw/day
Study duration:

Additional information

Chengelis (2001) was selected as the key study for this endpoint. The study was conducted according to OECD Guideline 408 and EPA OPPTS 870.3100; no deviations were reported from these guidelines and GLP conditions were reported throughout the study period. Groups of 7 week old Crl: CD(SD) IGS BR rats were dosed by gavage for 90 days (daily dosing). The test material was administered at concentrations of 0, 100, 300 and 1000 mg/kg/day in corn oil. 30 animals (15 male/15 female) were used for each dose group with an additional 40 animals (20 male/20 female) used for each satellite group. Satellite groups were dosed at a concentration of 0 and 1000 mg/kg/day in corn oil in an identical manner to the main test groups. Animals in the main toxicology groups were observed twice daily throughout the study for mortality and morbidity. Body weights and food consumption were measured weekly. Blood was collected at study weeks 3 (n=5/sex/group), 13 (n=10/sex/group) and 17 (n=5/sex/group) for haematology, serum chemistry and hormone (T3, T4 and TSH) measurements. Urine was collected prior to each necropsy, at study weeks 13 and 17, for urinalysis. Ocular examinations were performed prior to initiation of dosing and during study weeks 12 and 15. Functional Observational Battery and Locomotor Activity evaluations were performed on 5 animals/sex/group prior to initiation of dosing, during the last week of test article administration (study week 13), and during the recovery period. An examination of vaginal cytology (for estrus cycle determinations) was performed on study days 69-90. At each necropsy, sperm motility/viability, morphology, and number were assessed. Complete necropsies were performed on all animals. Approximately 40 organs and/or tissues/animal were collected and preserved. The adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus, thyroids with parathyroids, and uterus with cervix were weighed. Paraffin sections of tissues stained with hematoxylin and eosin from the control and 1000 mg/kg/day dose groups and the liver, lungs, prostate glands and thyroid glands in the 100 and 300 mg/kg/day doses, and gross lesions from all animals were examined under the light microscope. The livers of five randomly chosen animals/sex from the control and 1000 mg/kg/day dose groups was examined microscopically using Oil Red O or periodic acid Schiff s (PAS) reagent for evidence of lipid accumulation or glycogen accumulation/depletion, respectively. Statistical comparisons by sex and treatment day were made between the control and treated animals where indicated (p <0.05).

No test article-related effect on mortality occurred. Clinical signs were non-specific, low in incidence, non-dose-related and not related to test article administration. No test article-related changes occurred in body weight, food consumption, Functional Observational Battery or Locomotor Activity. No test article-related effects on hematologic parameters were noted. No test article-related ocular lesions were detected at the ophthalmic exams. No test article-related changes were noted on the estrus cycle as determined by vaginal cytology, or on sperm motility/viability, morphology, and number. Instances of statistically significant differences between control and some treatment groups were detected at study week 13 in the clinical chemistry data, hormone data, organ weight data and histology findings. They were generally secondary to the inducing effects on the liver or were otherwise not considered adverse effects of treatment as discussed further below.

Statistically significant (p <0.05 or p <0.01) test article-related clinical chemistry changes at week 13 include an increase in albumin (all dose levels for males), total protein (all dose levels for females and 1000 mg/kg/day for males), globulin (300 and 1000 mg/kg/day for females), and chloride (all doses for both sexes). In addition, increased gamma glutamyltransferase levels were noted in the 1000 mg/kg/day group (p <0.01). Serum thyroxin (T4) levels were decreased at study week 13 compared to the control mean in all male dose groups and the 300 and 1000 mg/kg/day dose females (p <0.05 or p <0.01). There were no corresponding statistical effects on T3 and TSH. While potentially test article-related, the changes in serum chemistry parameters were not of sufficient magnitude to be adverse, occurred in otherwise clinically normal animals, tended to be within or close to historical control values, and were not present at the end of the recovery period. The increases in serum chloride and CGT levels were likely secondary to hepatic enzyme induction. The changes in serum chloride were considered to be due to interference with serum chloride measurement secondary to free bromide in the test article. The decrease in T4, which was also reversible, was considered secondary to hepatic enzyme induction, which is known to cause increased metabolism and clearance of T4 in the rat. The induction of T4 glucoronidation by HBCDD and cytochrome P450 levels, wa later demonstrated in livers obtained from rats treated for 28 days with HBCDD.

The incidence of observations noted at gross necropsy was low and there was no evidence of frank organ damage. Liver weights were increased. At week 13, mean liver weights in all dose levels of both sexes (absolute, relative to bodyweight and relative to brain weight) were increased compared to the male and female control means (p <0.05 or p <0.01). The increases in liver weight were considered a result of induction of hepatic enzymes and as such is considered indicative of toxicity in the absence of frank organ damage. Increases in mean prostate weight were noted in the 1000 mg/kg/day group males at the primary necropsy. However, the increases in prostate weight was not considered toxicologically meaningful since the increases did not persist to the recovery period, there were no correlating histological findings and no change in sperm production. No other changes in organ weights, including the thyroid, were observed.

On histopathologic examination of tissues, relatively mild findings occurred in both the control and treated groups. Potential test article-related histological changes were identified in the liver and thyroid glands but these would not be considered indicative of frank toxicity. These organs were examined microscopically in all groups at both necropsies. The liver changes in male rats at the 90-day necropsy (Study Week 13) were characterized as minimal hepatocellular vacuolation and occurred in 10% of control males and -50% of the males at 100, 300 and 1000 mg/kg/day. Minimal hepatocellular vacuolation was also detected in females in the control and test article treated groups without a clear dose response (3 to 6/10 animals per group) but, mild and moderate vacuolation was detected in females only in the 300 (1/10) and 1000 mg/kg/day (2/10) dose groups. Minimal to mild hepatocellular hypertrophy was also detected only in the 1000 mg/kg/day group (5/10) females. Minimal thyroid follicular cell hypertrophy was detected 1/10, 1/10, 5/10 and 7/10 males in the control, 100, 300 and 1000 mg/kg/day groups, respectively and in 4/10 and 3/10 females in the 300 and 1000 mg/kg/day groups, respectively. In addition, mild thyroid follicular hypertrophy was detected in 4/10 females and 1/10 males in the 1000 mg/kg/day group. The reversible histologic changes (vacuolation and hypertrophy) are often found to accompany increased liver weight caused by liver enzyme induction. At week 17, the liver changes (weight and histology) had at least partially, if not fully, resolved in all treated groups without delayed or long-term toxic effects. The histologic changes in the thyroid had also nearly completely resolved except in the 1000 mg/kg/day group females, where partial recovery occurred.

HBCD was detected in the adipose tissue of male and female rats treated with 1000 mg/kg/day for up to 90 days. Isomer-specific analysis showed that the relative isomer concentrations in adipose tissue at all time points were alpha>>gamma>beta which is in contrast to the test article composition (gamma>>alpha>beta). Steady state levels were achieved by study day 27. Levels in male and female rats were similar at all time points and declined during the recovery period.

All the test article-related changes at 100 and 300 mg/kg/day were mild, reversible and generally secondary to hepatic enzyme induction (which is an adaptive not a toxic change) and without effect on the clinical condition of the animals. The additional findings observed at 1000 mg/kg/day (increased gamma glutamyltransferase and additional increases in the size of the liver and prostate), were also reversible, not associated with specific target organ damage or diminished function and were, therefore, probably of limited, if any, toxicological significance.

On this basis the no-observed-adverse-effect level (NOAEL) of HBCD administered to Crl: CD®(SD) IGS BR rats by gavage in corn oil for 90 days is 1000 mg/kg/day.

The EU Risk Assessment Report (2008) took a more precautionary approach and derived a LOAEL from this study of 100 mg/kg bw/day based on increased relative liver weights (18% in males and 24% in females respectively) in this dose level and changes in thyroid hormone levels (T4 reduction, TSH increase).

The EU Risk Assessment Report used van der Ven et al. (2006) with a BMDL of 22.9 mg/kg bw/day as the reference dose for liver weight increase. However, the changes at mid-dose level in this study can also be considered as adaptive and a NOAEL of 200 mg/kg bw could also be derived.

For the van der Ven et al. study, it needs to be noted that the number of animals was low and for some parameters only very few animals were used with causes some doubts regarding the results. The choice of the CES (critical effect size) levels in this study is not transparent. It is still to be established which effects are to be judged as adverse. Given the low amount of animals used per dose group, the deaths that occurred by misdosing can significantly affect the statistical value of the study, in particular given the nature of the effects. It is therefore difficult to address the borderline between physiological and adverse reactions. In fact the changes in absolute liver weights in both sexes are rather small at the dose levels investigated (up to 200 mg/kg bw) and support the enzyme inducing character of the substance. There was no increase in ALAT in the treated animals indicating no pathological liver damage. All changes in clinical chemistry and histopathological parameters observed in this study seem to to be within the physiological range (corresponding historical data of the laboratory were not provided) and do not indicate adverse effects up to the highest dose tested (200 mg/kg bw). A NOAEL of 200 mg/kg bw could be derived from this study. In our opinion the thyroid effects cannot be considered as adverse. It should be noted that the levels of T3, the active hormone were not significantly altered in this study. Furthermore, rats are more sensitive than man with regard to changes in thyroid hormones. Both rats and humans have a non-specific low affinity carrier of thyroid hormone but humans and other primates, as well as dogs, also have a high affinity binding protein, thyroxin binding globulin (TBG) that predominantly binds T4. Rats lack TBG and as a consequence the biological half-life of thyroid hormones in rats is about 10 fold shorter than humans. Consequently, any interference with thyroxin metabolism, even a transient one, leads to more rapid depletion of the hormone in the rat followed by TSH secretion by the pituitary gland to stimulate the thyroid to synthesise more T3 and T4 (Zbinden (1987) TIPS 8: 511-514; Hard (1998) Environ Health Perspect 106: 427-433; Hill et al. (1998) Environ Health Perspect 106: 447-457). This would suggest that the rat is more sensitive than humans to changes in thyroid hormone levels. Indeed, rodents have been reported to be particularly sensitive to thyroid enlargement (Zbinden, 1987; Hard, 1998; Hill et al., 1998).

For the overall assessment of the risk to human health, the result of the two generation study (Ema et al., 2008) will also be considered.

Chengelis (1996) as amended by Chengelis (1998) was conducted according to OECD Guideline 407 (28 day exposure period) and testing was conducted utilising test concentrations of 125, 350, 1000 mg/kg/day in corn oil. The study reported a NOAEL value of 1000 mg/kg bw/day which supported the result of the sub-chronic key study.

Justification for classification or non-classification

Based upon the results of the key study (Chengelis, 2001), the registered substance does not meet the criteria for classification according to 67/548/EEC and EC Regulation 1272/2008.