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Key value for chemical safety assessment

Effects on fertility

Description of key information
Additional human exposure information relevant to the toxicity to reproduction endpoint is available in the exposure related observation section.
This additional information was gathered from literature sources and is presented as a weight of evidence approach. Studies conducted as World Health Organisation surveys were rated as reliability 2 according to the scale of Klimisch et al. (1997) as the studies were overseen by a reputable and internationally recognised organisation with defined quality standards. Other studies were available which were based either on WHO data or were not conducted according to WHO standards these studies were rated as reliability 4 as insufficient data was presented to properly assess the methodologies and results.
Effect on fertility: via oral route
Dose descriptor:
NOAEL
10.2 mg/kg bw/day
Additional information

Two Generation Reproduction, Rat. A GLP compliant two-generation reproduction study in rats according to OECD Guideline 416 (Ema et al., 2008). Male and female rats were fed a diet containing the test material at 0, 150, 1500 and 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 the test material during the study were 10.2, 10.1 and 1008 mg/kg bw in F0 males, 14.0, 115 and 1142 mg/kg bw in F1 makes and 14.3, 138 and 1363 mg/kg bw in F1 females for 150, 1500 and 15,000 ppm respectively. Due to the increase in daily food intake during lactation, the average daily dose levels during lactation were 23, 240 and 2200 mg/kg bw in the dams of the low, mid and high dose groups respectively. In the F1 generation the respective doses during lactation were 20, 179 and 1724 mg/kg bw.

Parental toxicity

F0 generation

Mortality

One F0 male and two F0 females in the high dose group died or were euthanised during the administration period. One F0 female of the high dose group died during parturition. The deaths were not treatment related.

Clinical observations

Clinical signs were comparable between controls and treated animals.

Bodyweight and bodyweight gain

No treatment related changes in body weights were observed in the F0 males and females, except for higher values in the mid dose males from week 2 of administration and males of the high dose group in weeks 2, 3 and 5 and females of the high dose group in week 2. Body weight gains were or tended to be higher in mid dose males from week one and high dose males between week 1 and 4 of administration. F0 females had significantly higher body weight gains in the low dose group on lactation days 1 to 4 and in the high dose group in weeks 1 to 3 and significantly lower body weight gains in the high dose group on lactation days 0 to 14.

Food consumption

Increased food consumption was observed in males of the low and high dose group in weeks 2 to 3, males of the mid dose group in weeks 2 to 4 and 6 to 8 of administration. No changes in food consumption were observed in F0 females.

Hematology and clinical chemistry

No clearly test substance related hematological or clinical changes were reported.

Blood hormone levels.

In the mid dose group males only significantly reduced FSH concentrations were reported, but as no dose response was observed and a similar finding was not reported in the F1 males, this finding was not considered treatment related. In the high dose males and females significantly lower T4 serum levels compared to controls were observed. In females of all dose groups TSH levels were significantly higher than control levels. In high dose females FSH levels were significantly higher than in controls. This finding was not reproduced in the F1 generation. T3 levels were comparable to those of controls in all dose groups in both sexes.

Gross pathological observations

No treatment related macroscopic findings were reported in the F0 animals of all dose groups.

Organ weights

In the mid dose group males absolute and relative liver weights were significantly increased compared to controls (11 and 5.5% respectively). Significantly lower values of relative brain and epididymis weights in this dose group were attributed to the increased body weights compared to controls. In the high dose group males significantly higher absolute and relative liver (27 and 25.6% respectively) and thyroid weights (22.3 and 20.7% respectively) were reported compared to controls. In females of the high dose group absolute and relative liver weights were significantly increased compared to controls (30.9% and 29.4% respectively). Absolute thyroid (14.2%) and adrenal weights (10.3%) were also increased in this dose group, but the relative weights were not significantly different from controls.

Histopathological findings

The incidence of rats with decreased thyroid follices size was increased in F0 males and females at 1500 ppm and 15,000 ppm. No other treatment related histopathological findings were reported in the F0 generation.

Observations in adult animals of the F1 generation

Mortality

One F1 male of the mid dose group died or was euthanised during the adiministration period. The death was not treatment related.

Clinical observations

Clinical signs were comparable between controls and treated animals.

Bodyweight and bodyweight gain

No treatment related changes in bodyweights were observed in the F1 males and females of the low and mid dose group. In the high dose group bodyweights of males were significantly lower than controls between week 3 to 6 of administration, bodyweight gains were reduced between week on and 4 of administration. In females of the high dose group bodyweights and bodyweight gains were significantly lower than controls from week 3 to 10 and during gestation and lactation.

Food consumption

Decreased food consumption was observed in males of the low dose group in weeks 7 and of high dose group in weeks 3 and 4 of administration. F1 females of the high dose group showed significantly lower food consumption compared to controls during weeks 1 to 5 of treatment and on lactation days 7 to 14.

Hematology and clinical chemistry

No clearly test substance relayed hemtaological or clinical chemical changes were reported.

Blood hormone levels

In the mid dose group males only significantly elevated DHT concentrations were reported, but as no dose response was observed and a similar finding was not reported in the F0 males, this finding was not considered treatment related. No changes in thyroid hormone levels and TSH compared to controls were observed in males of any dose group. In females of the mid and high dose groups TSH levels were significantly higher than control levels, but no clear dose response was observed. T4 levels in females were not significantly different from controls and. T3 levels were comparable to those of controls in all dose groups in both sexes.

Gross pathological observations

F1 males of the high dose group showed a higher incidence of renal pelvis enlargement compared to controls. No other findings were reported that were significantly different from controls.

Organ weights

F1 males of the low dose group were reported to have significantly higher relative brain and pituitary weights than control animals. However, as no organ weight changes compared to controls were observed in the mid dose males, this finding is not regarded to be treatment related. In the high dose group males significantly higher absolute and relative liver (14 and 18% respectively) and thyroid weights (19 and 23% respectively) and lower absolute, but not relative brain weights were reported compared to controls. In females of the high dose group absolute and relative liver (15.4% and 20.8% respectively) and thyroid weights (23.8% and 29.1%) were significantly increased compared to controls. Absolute brain weights were also increased in this dose group, but the relative weights were not significantly different from controls.

Histopathological findings

The incidence of rats with decreased thyroid follicles size was increased in F1 males of the high dose group and females at 1500 ppm and 15000 ppm. No other treatment related histopathological findings were reported in the F0 generation. Based on the findings of increased liver and thyroid weights a conservative NOEL for parental toxicity of 10 mg/kg bw/day for males and 14 mg/kg bw per day for female rats could be derived. However as the effects were mild in the mid dose group and within the range of physiological adaptation an NOAEL of 101 and 114 mg/kg bw/day can also be justified. For direct toxicity to F2 pups a NOAEL of 14.3 mg/kg bw per day based on the maternal dose during lactation can be derived based on the increased mortality in males of the F2 generation (PND 5 to 21).

Effects on fertility

In both generations no difference in estrous cycle were observed in treated groups compared to controls. No differences between treated and control groups were reported in both sexes and generations with regard to copulation ratio and fertility ratio, days required until copulation. The delivery ratio, number of implantations and parturition ratio in females of both generations was also comparable to controls. A statistically significantly longer pregnancy period in females of the mid dose F0 group only was not regarded treatment related as no such effect was observed in the high dose group and the F1 generation.

The number of sperm heads in the testis, sperm motility, favourable sperm ratio, swimming speed and the ratio of sperm with abnormal morphology were comparable between treated and control groups. The number of epididymal sperm was significantly lower than control values in the low dose F0 generation only. As similar effects were not reported in the mid and high dose F0 animals and the F1 generation, this observation was not considered treatment related. Similarly a significantly elevated number of sperm heads with higher lateral amplitude was observed in the high dose F0 generation only, but not in the F1 generation and therefore not considered a treatment related finding. In ovaries of F1 adult females of the mid and high dose group the number of primary follicles was reported to be significantly decreased compared to controls. However there was no dose response and the findings were in the range of the historical control data of the institute. In the absence of any fertility impairment and additional histopathological changes these findings are not considered of toxicological significance. The fact that the determination of primordial follicles was made in adult rats that had already weaned one litter and the ovaries contained therefore also primary, secondary and other developing follicles as well as deteriorating corpora lutea, the determination of primordial follicles is difficult and leads to large variation which is also reflected in the high variation of the findings in the individual animals of this study.

Conclusion on fertility

No indications of test substance related fertility impairment were observed in this study up to the highest dose tested. The NOAEL for male and female fertility is therefore 15000 ppm, the highest dose tested in this study. (1008-1042 mg/kg bw /day for males and 1363 mg/kg bw/day for females). The EU Risk Assessment Report (EC, 2008) by applying another method of statistical analysis claimed that statistical significant effects on fertility could be derived from the study. However the EU Risk Assessment Committee (RAC) in their evaluation of the study (ECHA, 2010) did not confirm this assessment. An independent analysis of suitable statistical methods to be applied to the data (unpublished report, Simon, 2010) revealed that suitable statistical analysis did not indicate a significant effect on fertility in this study. Based on this study no classification for fertility of HBCD is warranted. This is in principle confirmed by RAC (2010) in the background document.

Developmental endpoints

The number of implantation losses as well as the number of life pups and the number of pups with abnormal findings on PND 0 and between PND 1 to 4 was comparable between test and controls in the F1 and F2 generation. The sex ratio (total number of male pups / total number of pups) was comparable between treated and control groups, except for the F1 animals of the mid dose group, in which it was significantly lower. As this finding was observed only in one generation and not dose related, it is considered incidental rather than test substance related.

Pup mortality was significantly increased compared to controls in mid and high dose males and high dose females of the F2 generation between PND 5 and 21. A decrease in viability index compared to controls was reported in high dose animals of the F2 generation on PND 4 and 21.

Body weights were significantly lower compared to controls in F1 males of the high dose group on PND 21 and in the high dose animals of the F2 generation from PND 7 to PND 21 for males and from PND 4 to PND 21 for females.

There was no significant difference between exposed and control groups in the time to vaginal opening and preputial separation in the F1 generation. The percentage of animals with completed pinna detachment and incisor eruption (observations on PND 3, 11 and 14) was comparable between controls and treated groups in both generations. The percentage of pups with completed eye opening (observations PND 3, 11, 14) was reported to be significantly higher than controls in F1 pups (both sexes) in the F1 generation. This finding was regarded incidental as there was no dose response and the observation cannot be regarded as an adverse effect. Eye opening completion was lower than controls in F2 mid and high dose females and F2 high dose males. This finding was regarded secondary to the growth retardation in the high dose group animals by the authors.

Locomotor activity was comparable between treated and control group F1 animals. In the test on T-maze learning no significant difference was observed between females of the treated and control groups in the F1 generation. F1 males of the mid and high dose group were reported to have needed shorter times than control animals for completing the maze on day 3 only (comparable to controls on days 1,2, and 4) and high dose animals were reported to have significantly less errors on day 3 only. As the significant differences only occurred at a single day in one sex only and furthermore indicated a better performance of treated than control males, these changes were not attributed to treatment.

No significant differences between treated and control groups were observed with regard to surface righting reflex, negative geotaxis reflex in both males and females of the F1 and F2 generations, with the exception of a shorter response time for surface righting reported in F1 males of the high dose group. As this would rather indicate a better performance of the high dose F1 males in this test and the finding was only reported for one sex and one generation, it is not regarded treatment related. The mid air righting reflex success rate was significantly reduced in high dose females of the F2 generation only. This finding was related to the lower body weight of those animals compared to the controls.

Autopsy findings in F1 and F2 pups sacrificed or found dead on postnatal days 0 to 4 did not show any treatment related changes. Similarly in animals of the F1 and F2 generation sacrificed at PND 26 or found dead between PND 5 and 26, no differences were found between treated and control groups with the exception of renal pelvis dilatation in male rats of the high dose group in the F1 generation only. As this effect was not observed in the F2 generation it was considered of no toxicological significance.

Conclusions on developmental toxicity and/or lactational effects

The main treatment related effects in the offspring were increased mortality in the F2 generation in high dose males and females and mid dose males between PND 5 and 21. Lower body weights compared to controls were observed in the high dose F1 males on PND 21, in the high dose F2 males from PND 7 to 21 and in high dose F2 females from PND 4 to 21. All other effects reported were secondary to the growth retardation or not treatment related. No increases in pup mortality was observed at the day of birth and in the first 4 days of lactation, indicating that the effect was likely not a developmental effect, but probably related direct intake of the test substance by the pups in addition to lactational exposure, as rats start feeding the test substance containing diet during the interval in which mortality was recorded (PND 5 to 21; normally feeding starts between day 7 and 14). The lower body weight compared to controls in F1 male pups at PND 21 only can probably be attributed to direct intake of the test substance in late lactation, rather than a developmental or lactational effect. The lower body weights in F2 males and females compared to controls on PND 7 and 21 or 4, 7, and 21 respectively also have to be viewed in the context of the lower body weight and body weight gain compared to controls of the respective dams in the F1 generation during lactation concurrent with a reduced food consumption in those animals. This indicates that the effect on body weights in the F2 generation was probably secondary to maternal effects. Furthermore it needs to be taken into consideration that the dose levels during the lactation period considerably exceeded the accepted limit dose of 1000 mg/kg bw. Consequently a NOAEL for postnatal development of 179 mg/kg bw/day can be derived from this study based on the maternal mid-dose level of the F1generation.

The EU RAC committee in the evaluation of this study concluded that in the absence of a cross fostering study it could not unequivocally differentiate if the observed effects in particular on pup mortality in this study were due to exposure during the lactation period or secondary to effects initiated during gestation. However, considering the weight of evidence from the Ema et al. (2008) study and two developmental toxicity studies mentioned above showing no effects after gestational exposure, it can be concluded that it is unlikely that the effects observed in the offspring in the 2 generation study were due to gestational exposure.

van der Ven et al. (2009) and Lilienthal et al. (2008) are publications of the same study that was conducted , in a similar way to OECD Guideline 415 However the study design was modified to follow a bench mark dose approach using fewer animals per group (10 per sex per dose level) and a larger number of dose groups. This can however weaken the statistical power of the analysis. As the test substance was added to the diet in a corn oil vehicle, an additional vehicle control group was added to account for the effect of a lipid enhanced diet. The litter size was not standardized in the F1 generation which can again jeopardize the statistical analysis. For the immunological and neurological investigations the groups for neurobehavioral testing and immunization assays it is stated that the groups were “preferably composed of animals from different litters, but depending on the availability, occasionally supplemented with replicates from the same litter”. As individual data are not provided in the publication it can not be excluded that those data include a litter bias and are to be treated with caution. The study included additional parameters that are not normally included in the OECD protocol (immunological parameters, steroid analysis, retinoid analysis and bone analysis, as well as the auditory startle experiments of Lilienthal et al., 2008). For these additional investigations neither historical data nor the normal variations in this strain of rats were provided. The choice of the critical effect size is not transparently described as well which makes the results difficult to evaluate.

Because these investigations were conducted according to GLP or to ISO 9001 - 2000 standards, they resulted in reliability scores of 2 for all studies according to the criteria of Klimisch et al. (1997).

Results

Parental effects

The authors report a reduction in food intake in females during the third week of gestation. From the group data in the supplementary data it can be deduced that it was only observed at the highest dose level. Food and therefore also test substance intake increased considerably during lactation. The authors did not determine the test substance intake during lactation separately. High dose group males were reported to have lower body weights than controls only during the first 3 weeks of dosing. The authors derived a BMDL of 88 and 95.4 mg/kg bw/ day for this effect. 

Fertility

No effects were observed on fertility endpoints, mating success, time to gestation, gestation duration, number of implantation sites, litter size up to the highest dose tested. The NOAEL for fertility was therefore > 100 mg/kg bw/day.

Developmental endpoints

No change in sex ratios of F1 litters was observed. Pup mortality during lactation was comparable to controls in all dose groups. The authors reported a significant decrease in body weights during lactation for females on PND 4, 7, and 21 and males on PND 4, 14 and 21, with no significant dose relationship for the entire period of lactation (days 4 to 21), the lowest BMDL reported was 11.6 mg/kg bw/d in males on PND21. When looking at the group data provided in the supplementary information, a clear dose response cannot be distinguished and the claimed effect rather seems to be an artifact of the BMDL method used. Similarly the authors claim significant dose related reduction of body weights in weeks 4 to 11 after weaning in both sexes with the lowest BMDL being 25.9 mg/kg bw/day in week 4 females. Again no dose related differences were reported for the whole period (weeks 4 to 11). The group data in the supplementary information again show considerable non-dose related variation. Only in the highest dose group there seems to be an indication of lower body weights than controls indicating a tentative NOAEL of 30 mg/kg bw. The body weights seem to go very much in parallel with the food consumption, although for food consumption the authors only report significant reductions in females of week 9 and males in weeks 5 and 6.

The authors report an increase in anogenital distance (AGD) in male pups of the highest dose group on PND 4, but not on PND 7 and 21. This is not regarded of toxicological relevance as the effect disappeared with age. No changes in AGD were reported in female rats. The time to vaginal opening was reported to be delayed in the highest dose group which was concomitant with a decreased body weight and is thought to be secondary to the lower food intake and body weight observed in these animals.Odum et al., 2004 reported that age at vaginal opening in female rats is related to cumulative energy intake prior to puberty onset. In the high-dose group, female pups consumed 24% less diet at 5 weeks of age (earliest time point reported) and weighed 14% less than vehicle controls. In the intervals prior to puberty onset, high-dose female pups weight markedly less than vehicle controls: 15% less on day 14, 20% less on day 21, and 16% less on week 4.It can therefore be concluded that the delay in vaginal opening was not test substance related. No effects on preputial separation were observed in male F1 pups. Reproductive organ weights were not changed compared to controls at the time of weaning.

At the end of the exposure period no test substance related changes in cauda epididymis sperm counts and sperm morphology except for a reduction of the ratio of separate sperm heads for which the authors identified a significant dose response, but could not derive a BMDL due to the high variation in the data. Therefore this cannot be regarded as a test substance related finding. The authors report a decrease in absolute testes weights with a BMDL of 11.5 mg/kg bw /day. However, no relative organ weights were determined and analysis of the data given in the supplementary information shows that the testis weights were affected to a lesser or similar degree as the body weights: e.g. in the highest dose group, body weight decreased by 21% compared to the standard diet and by 12% compared to the high fat diet. F1 testis weights were only decreased by 13% and 14%. This also becomes obvious when evaluating the relative testis weights, as given in the following table, which are relatively constant. In conclusion, the absolute testis weight changes as detected in the van der Ven 2009 study are probably unspecific, body weight related effects.

 

Table 1: Body weight versus TESTIS weight in F1 males of the van der Ven et al. (2009) study

 

male F1

HBCDD

body weight

body weight change vs standard diet

body weight change vs fat diet

testis weight

testis weight change vs standard diet

testis weight change vs fat diet

testis weight rel to body weight

mg/kg bw

g

% change

% change

g

% change

% change

 

0 standard control

368

 

 

2.98

 

 

0.810

0 high fat control

329

-11 %

 

3.01

1 %

 

0.915

0.1

333

-10 %

1 %

2.91

-2 %

-3 %

0.874

0.3

340

-8 %

3 %

3.07

3 %

2 %

0.903

1

390

6 %

19 %

3.18

7 %

6 %

0.815

3

323

-12 %

-2 %

2.88

-3 %

-4 %

0.892

10

335

-9 %

2 %

2.82

-5 %

-6 %

0.842

30

380

3 %

16 %

2.97

0 %

-1 %

0.782

100

291

-21 %

-12 %

2.6

-13 %

-14 %

0.893

Other absolute organ weight decreases were reported by the authors for kidney and thymus in both sexes and adrenals, prostate, heart and brain in F1 males all with BMDLs of above 30 mg/kg bw/day indicating that they were confined to the high dose group. The changes were in the order of the body weight changes and thus not indicative of a specific effect. No remarkable pathological changes were reported in either of these organs.

Clinical chemistry and hormone levels

No test substance related clinical chemistry findings were reported. T3 and T4 hormone levels were unchanged in treated animals of either sex in both generations. No histopathological changes in the thyroid were observed.

Other parameters

The authors report a decrease in apolar retinoids in liver of in male and female F1 animals with BMDLs of 29.2 and 1.3 mg/kg bw/ day for the sum of apolar retinoids or 24.3 and 21.7 mg/kg day for the sum of apolar retinoids/g liver. However when regarding the supplementary group data there seems to be no clear dose response and the derivation of the BMDL dose seems rather arbitrary. Furthermore in the absence of any historical control data no conclusion can be drawn on the toxicological relevance of this finding. Similarly a reported decrease in trabecular bone mineral density in females with a BMDL of 0.056 mg/kg bw/d as well as decreased bone length in males and females and decreased total mineral content, total area, cortical area and cortical thickness in males with BMDLs above 50 mg/kg bw/day remain spurious given the high standard variations in those parameters, the fact that most values where within the standard controls (without corn oil vehicle). In the absence of any historical control data the toxicological relevance of these findings remains questionable. Similarly the authors report several findings with regard to immunological parameters, but the high variation of the data did not provide a sufficient basis to calculate a BMDL and no conclusions can be drawn on the toxicological relevance of these findings.

Murai et al. (1985) performed a developmental toxicity study in which 20 rats per group received 0, 0.01 %, 0.1 % or 1 % HBCDD in the diet during day 0-20 of gestation. Thus, this study also covered potential effects during the preimplantation stage. The doses are approximately equivalent to 0, 7.5, 75, and 750 mg/kg/day, respectively, based on a mean weight of 200g and a food consumption of 15 g/day. On day 20 of gestation 14 rats per group were killed by cervical dislocation. Major organs were examined macroscopically and organ weights were determined.Numbers of corpora lutea, implants, resorptions, and live fetuses were determined. The incidence of abnormalities on external examination was examined, sex was identified and both body weight and placental weight were measured for each live foetus. One third of the fetuses were examined for visceral anomalies and 2/3 for skeletal abnormalities. The remaining 6 dams per group were allowed to deliver naturally, and the pups were monitored through weaning. The number and body weight of delivered fetuses, number of live fetuses, and abnormalities resulting from external examination were recorded. From the third week, males and females were separated, and the new-borns growth and survival was observed until the 7th week. In the highest dose group the maternal food intake was slightly decreased, and both the absolute and relative maternal liver weights were significantly increased by 13 %. The mothers showed no signs of toxicity and were body-weight gain was not affected. No significant changes in number of implants, number of resorbed, dead or live fetuses were reported, nor external, visceral or skeletal anomalies of fetuses that could be attributed to exposure to HBCDD. No difference was seen in the number of live new-borns, or in the number of dead new-borns, and no abnormalities were observed based on external examination. No abnormalities were observed during parturition, during the weaning period, and after the weaning period. Normal body weight changes of both male and females of each administration group were observed. No significant difference in either the weaning index or the survival index, obtained when the experiment was over, was observed between administration groups and the control group. Although a somewhat lower number of animals per dose level was used (14) in comparison with the 20 individuals called for in OECD Guideline 414, about 150 fetuses per dose level were examined without any adverse effects being detected, and it is highly unlikely that the inclusion of an additional 6 dams per dose group would have given a significantly different result. This study gives a fetal NOAEL of 750 mg/kg/day, the highest dose tested, and a maternal NO(A)EL of 75 mg/kg/day, with a liver weight increase by 13 % and the next higher dose.


Short description of key information:
The following information was selected as the key study for toxicity to reproduction endpoint:

Ema, M. Fujii, S., Hirata-Koizumi, M. and Matsumoto, M. (2008). Two-generation reproductive toxicity study of the flame retardant hexabromocyclododecane in rats. Reproductive Toxicology (2008), Vol. 25, pp. 335-351.

Ema et al. (2008) was conducted in accordance with OECD Guideline 416 and under GLP conditions and was awarded a reliability score of 1 according to the criteria of Klimisch et al. (1997).

This same study was also chosen as a key study for the developmental endpoint, however the following study was also chosen as a key study due to the different method of investigation and testing guideline utilised:

Stump, D.G. (1999). A Prenatal Developmental Toxicity Study of hexabromocyclododecane (HBCD) in Rats. Report no.: WIL-186009. Report date: 1999-08-04.

Stump (1999) was conducted according to OECD Guideline 414 and EPA OPPTS 870.3700and was awarded a reliability score of 1 according to the Klimisch et al. (1997) criteria based upon the test methodology, GLP conditions and depth of reporting provided in the report.

Murai et al. (1985) was performed equivalent to a guidelines study and contained a subgroup of offspring that was observed until weaning. The study is therefore awarded a reliability score of 2 according to the Klimisch et al. (1997) criteria.

Effects on developmental toxicity

Description of key information
The following information was selected as the key study for toxicity to reproduction endpoint:
Ema, M. Fujii, S., Hirata-Koizumi, M. and Matsumoto, M. (2008). Two-generation reproductive toxicity study of the flame retardant hexabromocyclododecane in rats. Repruductive Toxicology (2008), Vol. 25, pp. 335-351.
Ema et al. (2008) was conducted in accordance with OECD Guideline 416 and under GLP conditions and was awarded a reliability score of 1 according to the criteria of Klimisch et al. (1997).
This same study was also chosen as a key study for the developmental endpoint, however the following study was also chosen as a key study due to the different method of investigation and testing guideline utilised:
Stump, D.G. (1999). A Prenatal Developmental Toxicity Study of hexabromocyclododecane (HBCD) in Rats. Report no.: WIL-186009. Report date: 1999-08-04.
Stump (1999) was conducted according to OECD Guideline 414 and EPA OPPTS 870.3700and was awarded a reliability score of 1 according to the Klimisch et al. (1997) criteria based upon the test methodology, GLP conditions and depth of reporting provided in the report.
Murai et al. (1985) was performed equivalent to a guidelines study and contained a subgroup of offspring that was observed until weaning. The study is therefore awarded a reliability score of 2 according to the Klimisch et al. (1997) criteria.
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
10.2 mg/kg bw/day
Additional information

Stump (1999) was conducted according to EPA OPPTS 870.3700 and OECD Guideline 414 under conditions of GLP. Testing was conducted on Sprague-Dawley rats at test concentrations of 10, 20 and 40 mg/ml in corn oil administered by gavage (oral route). The test material was administered to pregnant female rats on days 6 -19 of gestation at doses of 250, 500 or 1000 mg/kg bw daily. All maternal animals were sacrificed on day 20 of gestation and uterine contents were examined as well as gross necropsy. No developmental effects were noted at any of the test doses and so a NOAEL value of 1000 mg/kg bw/day (actual dose). In addition no maternal toxicity was noted at any test dose and so a NOAEL value of 1000 mg/kg bw/day (actual dose). Test doses were identified based upon a preliminary study (Stump, 1999), this study was conducted to recognised testing guidelines and under GLP conditions. It was considered that the reliability of this initial study further supports the reliability and relevance of the main study.

Ema et al. (2008) was conducted according to OECD Guideline 416 under conditions of GLP. The test substance was administered at doses of 150, 1500, 15,000 ppm in feed to Sprague-Dawley rats 10 weeks prior to mating, throughout the mating, gestation and lactation periods for F0 animals and from weaning until scheduled death for F1 animals. For details of the study see section on fertility. The main treatment related effects in the offspring were increased mortality in the F2 generation in high dose males and females and mid dose males between PND 5 and 21. Lower body weights compared to controls were observed in the high dose F1 males on PND 21, in the high dose F2 males from PND 7 to 21 and in high dose F2 females from PND 4 to 21. All other effects reported were secondary to the growth retardation or not treatment related. No increases in pup mortality was observed at the day of birth and in the first 4 days of lactation, indicating that the effect was likely not a developmental effect, but probably related direct intake of the test substance by the pups in addition to lactational exposure, as rats start feeding the test substance containing diet during the interval in which mortality was recorded (PND 5 to 21; normally feeding starts between day 7 and 14). The lower body weight compared to controls in F1 male pups at PND 21 only can probably be attributed to direct intake of the test substance in late lactation, rather than a developmental or lactational effect. The lower body weights in F2 males and females compared to controls on PND 7 and 21 or 4, 7, and 21 respectively also have to be viewed in the context of the lower body weight and body weight gain compared to controls of the respective dams in the F1 generation during lactation concurrent with a reduced food consumption in those animals. This indicates that the effect on body weights in the F2 generation was probably secondary to maternal effects. Furthermore it needs to be taken into consideration that the dose levels during the lactation period considerably exceeded the accepted limit dose of 1000 mg/kg bw. Consequently a NOAEL for postnatal development of 179 mg/kg bw/day can be derived from this study based on the maternal mid-dose level of the F1generation.

The EU RAC committee in the evaluation of this study concluded that in the absence of a cross fostering study it could not unequivocally differentiate if the observed effects in particular on pup mortality in this study were due to exposure during the lactation period or secondary to effects initiated during gestation. However, the applicant considers this conclusion as very precautionary and is of the opinion that the study data support the conclusion of the absence of developmental effects as outlined above. Eriksson et al. (2006) was not conducted according to recognised testing guidelines, information on GLP conditions was not reported in the literature article. The study was conducted on NMRI mouse pups which were given a single oral dose of 0.9, 13.5 mg/kg/bw on post natal day 10. At 3 months of age animals were assessed for learning and memory capability. A NOEL for developmental toxicity was derived to be 0.9 mg/kg/bw as no significant difference from the control group was observed for learning ability under the conditions of the test.The EU RAR concluded that this study is not sufficiently robust to be used in the risk characterization of HBCDD and needs to be confirmed by other laboratories before. The study by Ema et al. (2008) did explicitly not confirm the results of Eriksson et al. (2006) and this was discussed in the publication. Furthermore the risk assessment suggested, as a similar study had been conducted on decabromodiphenylether (Deca-BDE) by the group of Erikksson with the same experimental design, to wait the results of a guideline developmental neurotoxicity study being performed in the framework of the risk assessment of Deca-BDE before requiring further data on HBCDD for this endpoint. The guideline DNT study with Deca-BDE has been completed in the mean time and was submitted to the EU Rapporteur country. In this study no Deca-BDE related indications of developmental toxicity were observed up to the highest dose tested (1000 mg/kg bw/day) and the findings were thus not confirmed.

Furthermore the study design used by Eriksson et al. was subject of further scientific discussions. For example the publication states that for the testing on endpoints related to developmental toxicity a total of 10 mice was randomly selected from 3 or 4 different litters (litter size 10 to 12) from each treatment group at 3 months of age. That means that the selection was not based on litters. However, mice from the same litter are not independent from one another and cannot be treated as such for experimental studies. Utilizing littermates as independent values grossly inflates the rate of false-positives (Holson and Pearce, 1992). A comprehensive critical discussion of the methodology used by Eriksson et al. in different studies has been published by Williams and de Sesso (2010). Therefore the findings reported in this study are questionable and should not be used for classification purposes.

Lilienthal et al. (2009) and van der Ven et al. (2009) were awarded reliability scores of 2 according to the above criteria as the studies were not conducted according to recognised testing guidelines and no details on GLP compliance were reported. Both literature papers were taken from a reputable journal and have been peer reviewed prior to publication ensuring that the papers follow generally accepted scientific principles. For a detailed description of the van der Ven et al. (2009) study see section 5.9.1.

Parental effects

The authors report a reduction in food intake in females during the third week of gestation. Food and therefore also test substance intake increased considerably during lactation. The authors did not determine the test substance intake during lactation separately. From the group data in the supplementary data it can be deduced that it was only observed at the highest dose level. High dose group males were reported to have lower body weights than controls in the high dose only during the first 3 weeks of dosing. The authors derived a BMDL of 88 and 95.4 mg/kg bw/ day for this effect.

Developmental endpoints

No change in sex ratios of F1 litters was observed. Pup mortality during lactation was comparable to controls in all dose groups. The authors reported a significant decrease in body weights during lactation for females on PND 4, 7, and 21 and males on PND 4, 14 and 21, with no significant dose relationship for the entire period of lactation (d 4 to 21), the lowest BMDL reported was 11.6 mg/kg bw/d in males on PND21. When looking at the group data provided in the supplementary information, a clear dose response cannot be distinguished and the claimed effect rather seems to be an artifact of the BMDL method used. Similarly the authors claim significant dose related reduction of body weights in weeks 4 to 11 after weaning in both sexes with the lowest BMDL being 25.9 mg/kg bw/day in week 4 females. Again no dose related differences were reported for the whole period (weeks 4 to 11). The group data in the supplementary information again show considerable non-dose related variation only in the highest dose group there seems to be an indication of lower body weights than controls indicating a tentative NOAEL of 30 mg/kg bw. The body weights seem to go very much in parallel with the food consumption, although for food consumption the authors only report significant reductions in females of week 9 and males in weeks 5 and 6.

The authors report an increase in anogenital distance (AGD) in male pups of the highest dose group on PND 4, but not on PND 7 and 21. This is not regarded of toxicological relevance as the effect disappeared with age. No changes in AGD were reported in female rats. The time to vaginal opening was reported to be delayed in the highest dose group which was concomitant with a decreased body weight and is thought to be secondary to the lower food intake and body weight observed in these animals. Odum et al. (2004) reported that age at vaginal opening in female rats is related to cumulative energy intake prior to puberty onset. In the high-dose group, female pups consumed 24% less diet at 5 weeks of age (earliest time point reported) and weighed 14% less than vehicle controls. In the intervals prior to puberty onset, high-dose female pups weight markedly less than vehicle controls: 15% less on day 14, 20% less on day 21, and 16% less on week 4.It can therefore be concluded that the delay in vaginal opening was not test substance related. No effects on preputial separation were observed in male F1 pups. Reproductive organ weights were not changed compared to controls at the time of weaning.

Lilienthal et al. (2009) investigated the effect of the registered substance on dopamine-dependant behavior and brainstem auditory evoked potentials in 6 males and females of the F1 generation of the van der Ven et al. (2009) study. The animals were transferred from the Netherlands to the German laboratory at an approximate age of 90 days. The investigations were performed after three weeks of accommodation to the new environment. The animals were housed in groups of three. On day 110 5 males and females from each group, but additional females were taken from two litters in the 0.3 mg/kg bw group and from one litter in the 1 mg/kg bw group (no further details given) were tested for cataleptic behavior. The animals received 0.25 mg/kg bw of haloperidol, a dopamine receptor blocker by i.p. injection. Thirty and 60 minutes thereafter the rats were sequentially put in 3 unusual body positions. The latency time for the onset of corrective movements was measured. At 140 days of age brain stem auditory potential (BAEP) was investigated in 4 to 6 rats per sex per group (with additional females from 2 litters in the 0.3 mg/kg bw dose group and from one litter in the 1 and 30 mg/kg dose groups respectively as well as additional males from 2 litters in the 0.3 mg/kg bw dose group and from one litter in the 30 mg/kg bw dose group) after sedation of the rats with ketamine (90 mg/kg bw males, 55 mg/kg bw females, i.p.). The animals were placed on a heating pad to prevent decreases in body temperature due to anesthesia (no details given). Needle electrodes were placed under the skin at the vertex and behind the stimulated right ear. The ground electrode was placed behind the contra lateral ear. Auditory stimuli were applied to one ear (the other ear was occluded by a tissue plug in the outer ear tube) by an earphone inserted in the outer ear tube. BAEPs were evoked by rarefaction clicks with a pulse duration of 50 micro-s and a repetition rate of 15 Hz. Click stimuli were presented at sound pressure levels (SPL) of 80 followed by 60 dB and then lowered by steps of 10 dB until the most prominent wave II could not longer be detected. Thereafter SPL was elevated in 5 dB steps until the wave II signal was reestablished. The lowest SPL at which wave II could be identified was taken as the threshold. A similar procedure was followed for tone pips with stimulus frequencies of 0.5, 1, 2, 4, 8, and 16 kHz. For each BAEP 400 to 1000 sweeps were averaged until the signal size stabilized. Latencies of wave II and IV were determined in potentials evoked by clicks at 80, 70 and 60 dB and tone pips at 80 dB. The statistical evaluation was performed with a benchmark dose approach as described for the van der Venet al., 2009 study. The selection of critical effect size of 5% for BAEP and 20% for catalepsy was chosen, based on human experience rather than data from the same strain of animals.

For catalepsy the authors report decreased latencies in females, but not males in the two highest dose groups BMD-L range 0.6 to 4.4 mg/kg bw/day. Effects on the auditory BAEPs were on the contrary only observed in male rats with reported slightly elevated thresholds at 0.5 to 4 kHz (5 to 9 dB) at the highest dose and increased latencies between stimuli in the lower frequency range (0.5 to 2 kHz) and after clicks. The BMD-L for effects on thresholds was reported in the range of 1 to 6 mg/kg bw/day while the BMD-Ls for the latencies were close to the highest dose tested.

 

A number of issues indicate that this study should not be used as a basis for a conclusion on test substance related effects:

- The sample sizes for the measured endpoints are small (n = 4-6/group). The animals were taken from the study reported by Van der Ven et al. (2009) that reported a sample size of 6/sex/group. Some of the animals must not have contributed to the data of the Lilienthal et. al. (2008) study and the reasons for that are unclear.

- The animals have been transported between laboratories for the study. This may have had an influence on the results.

- Measured catalepsies are latencies, which are censored (i.e. a maximum of 180 sec is used as a measurement). Censored data are limit values rather than real data, and their quantitative analysis can be challenging. Special statistical tests have been developed to handle such data, but the authors do not mention any special statistical tests, thus it can be assumed that the data were not analysed with the appropriate methods. Some of the catalepsy data do not follow a clear dose response and it is unclear how much they influence the data modeling and how much weight should be given to similar values for a different dose. It is to be noted that the error bars in the figures are standard errors of the mean (not standard deviations) and do not represent the raw data variability that was much larger.

- The critical effect dose from which the effects were considered as truly substance related was taken from human clinical experience and not from historical data of the same rat strain. This renders the evaluation difficult in particular as a very low number of animals was investigated, there was a considerable variation in the raw data and no historical control values for the effects observed in the same strain of rats and the same laboratory were provided.

- The study was a dietary study. It is unclear from the publication whether average daily intakes of HBCDD were calculated on the basis of measured concurrent feed intake or estimated from historical control data.

- The discussion addresses the differential effects in males and females for catalepsy in terms of enzyme induction (females being more sensitive than males). On the other hand, evoked potentials are reported to be effected in males but not in females. The authors only stated that sex-related effects are not uncommon. No comments were made about the finding that in one case females were reported to be more affected and in the other case males.

Given the above mentioned inconsistencies in the study, it is impossible to decide from the data provided whether the reported effects were within the variability of this strain of rats, were an artifact of the statistical method used or represent a real effect.

Saegusa et al. (2009) performed a pre- and postnatal study in pregnant Sprague-Dawley rats. Groups of 10 dams were fed with diets containing 100, 1000 or 10000 ppm of HBCD from GD 10 until postnatal day 20 (day of weaning) (dose levels 8 to 21.6, 80.7 to 212.9, 803.2 to 2231.3 mg/kg bw/day) and body weight and food consumption was monitored throughout the experimental period (no details provided). As this was a dietary study it needs to be considered that the offspring also received the test substance directly in the diet during the later lactation period. On PND1 the number, weight and anogenital distance of the pups were determined. The pups were culled to 4 males and 4 females per litter. After weaning the dams were killed and 20 male and female offspring per group (at least 1 male and female per litter) were killed. Organ weight determination and histopathology was performed on 10 pups per sex per group, thyroid hormone determination in 10 males per group. In dams organ weights and the numbers of implantations were determined and thyroid histopathology performed. The remaining animals were housed in groups of 4 (litters were mixed) and fed basal diet and water ad libitum. Female offspring were observed for vaginal opening from PND 26 and males for preputial separation from PND 34. Estrous cycles of investigated were observed by daily investigation of vaginal smears from post natal week (PNW) 8 to PNW 11.At PNW 11 the F1 animals were killed (males on the first day of week 11, females up to 4 days thereafter when they were in diestrous) and histopathology as well as thyroid hormone determination was performed. Brains of the male offspring on PNW 11 were subjected to immunohistochemistry for 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNPase) and neuron-specific nuclear protein (NeuN) to stain oligodentrocytes and neurons. 3,3’-diaminobenzidine/H2O2 was used as the chromogen. All other tissues were stained with hematoxillin-eosin stain. Morphometric measurements were performed on brains of animals killed in PNW 11 in the lateral to pyramidal cell layers of the hippocampal CA1 region in the NeuN stained brain sections. In areas of the white matter the number of CNPase stained oligodentrocytes surrounding myelinated axons distributed in the cerebral cortical area were measured. Data from the offspring during the lactational period were evaluated using the litter as statistical unit, while after weaning the individual animals were used as statistical unit. This could introduce a litter bias that was not accounted for.

Results

Maternal observations: Maternal body weight gain, food consumption and the duration of pregnancy were comparable between dose and control groups. At necropsy on day 20 of gestation relative thyroid weight and the incidence of diffuse follicular cell hypertrophy was statistically significantly increased in the high dose group animals compared to controls. The number of implantation sites was also comparable to controls.

Observations in offspring on PND 1: The number of live offspring, the sex ratio, the body weights at PND1 and from PND1 through weaning and the ano-genital distance were comparable between dosed and control animals.

PND20: Body weights and organ weights were comparable between treated and control animals except for a statistically significant increase in relative liver weights in both male and female animals of the high dose group (27 and 28%). A statistically significant increase in adrenal weight in males of the 1000 ppm group only and a decrease in relative kidney weights in female of the 100 ppm dose group is not considered treatment related as no dose response was observed and from the tables it can be seen that the values were close to controls. The onset of puberty was comparable to controls in both sexes and no irregularity in estrus cycle was observed in females between PNW 8 and 11. Body weights of females at the onset of puberty were reported to be statistically significantly reduced in the high dose group, but this could be due to a litter bias. Serum thyroid levels were only measured in male offspring. The authors report a statistically significant decrease in serum T3 and an increase TSH levels, but not in serum T4-levels compared to control in PND 20 males of the high dose group. Interestingly, a second control that was used for a study conducted in parallel with another substance showed similar TSH levels as the 10000 ppm HBCDD group. It is therefore possible that the changes in thyroid hormone levels are well within historical control levels. The authors report a statistically significant increase in the incidence of diffuse vacuolar degeneration of liver cells in both sexes of the high dose group.

PNW 11 observations: No statistically significant differences in body weights between treated and control animals were observed. A significant increase in thyroid relative thyroid weight was reported in males of the 1000 and 10000 ppm groups. A significant increase in relative liver weight and a decrease in relative epididymys weights of males in the 100 ppm group is not considered treatment related as no changes were observed in the higher dose groups. Thyroid hormone levels in male offspring revealed no changes in TSH and T4 levels compared to controls and a statistically significant decrease in T3 levels in the high dose group compared to controls. However, again in the second control run in the parallel study with another substance the authors report the same T3 levels in the control animals, while the HBCD control group seemed to have the highest level of all groups. The statistical significance is therefore likely due to the high control values and cannot unequivocally be attributed to the test substance. As histopathological finding the authors report a significant increase of animals with adrenocortical vacuolar degeneration in the high dose males. However, as a similar finding was not observed in females and no changes in adrenal weights were observed the authors consider this finding as of low toxicological relevance. Brain morphology revealed no significant differences between treated and control animals in the distribution of hippocampal CA1 neurons, but the authors report a significant reduction in the number of CNPase positive oligodentrocytes in the cingulate deep cortex at the high dose group compared to controls. Interestingly again also for this finding the control animals of the second substance investigated showed a number of CNPase positive oligodentrocytes in the cingulate deep cortex that was comparable to that of the HBCD high dose group. Due to this observation and in the absence of historical data this observation cannot clearly attributed to treatment. The NOAEL for systemic toxicity based mainly on increased thyroid weights in males at PNW 11 in this study was reported to be 100 ppm or 8.1 to 21.3 mg/kg bw. However as only males were affected in this study and no such findings were reported in females and in the absence of single animal data, the relevance of the finding in the mid dose group is doubtful and the NOAEL could also be in the range of 1000 ppm (80.7 to 212.9 mg/kg bw/day) as maternal dose levels which is corresponding to other studies as well. The findings are consistent with direct exposure of the F1 animals and cannot be regarded as developmental toxicity effects.

 

Furthermore the study has a number of deficiencies that make it difficult to attribute any observed effects to developmental toxicity:

- The authors did not control for litter effect in all of their analyses. The offspring were exposed during gestation and lactation; therefore, litter would clearly have had an impact on both the exposures they received as well as their caregiving. This is particularly important for the weanling necropsy endpoints that were assessed. Similarly, during puberty onset, the authors clearly examined more than 1 pup/litter and did not accommodate litter effect.

- As the study was a dietary study and it is well known that rat pups start eating the diet between day 7 to 14 and day 20 of the lactation period, the study design does not allow for a distinction between direct effects of the test substance, effects from exposure in utero or from lactational exposure.

- The authors did not adjust dietary concentrations during the 2nd and 3rd week of lactation when dams were consuming 2-3 fold as much diet as a non-pregnant rat. It is important to note that human females are much more efficient during lactation and do not increase their feed intake to this degree (estimated increase of ~20% in humans, not 200-300% as in rats). Given these high levels of exposure substantial amounts of HBCDD were likely transferred through the milk. Again, the exposure to the pups was likely higher than human exposures as rat milk has a higher lipid content than human milk (13-15% vs. 4% fat content in human milk (Ecobichon, 1984) which would favor partitioning of lipophilic materials. This together with direct intake through the diet is likely to have led to a massive exposure of the pups in that study. Furthermore the authors did not mention whether the offspring were perfused for brain assessment. Per the OECD and EPA guidelines, perfusions are required to maintain the integrity of brain structures for morphometric assessments. It is difficult to determine how well the 3D architecture of the brains was maintained. Therefore, the observations may have resulted from technical variations in the experimental analysis.

It is unclear why there was a decrease in T3 on PND 20 and no significant change in T4. Small sample sizes (10/group) may have contributed to some imprecision in their thyroid hormone measurements. It would have been helpful if the authors had collected thyroid weights on PND 20. Also, there were no corresponding thyroid histopathology changes at PND 20.

There is only a poor-dose response for changes in T3 and TSH at week 11 (greater effect at 1000 than 10,000). Again, small sample sizes (10/group) may have contributed to some imprecision in their thyroid hormone measurements. It is important to consider that there is typically very high variance in TSH levels (e.g., greater than 50% coefficient of variation in TSH is permitted in the new pubertal assay test guideline at 15 animals/dose). Also, thyroid hormone measurements can change rapidly and are subject to alterations by stress (e.g., Döhler, 1979); therefore point measurements of thyroid hormone are best supported by altered thyroid weights and histopathology to support perturbed thyroid function. No changes in thyroid histopathology in the offspring at PNW 11 were observed. While thyroid weights were increased in males on PNW 11, thyroid weights were decreased in female offspring (PNW 11) exposed to HBCDD. The authors do not discuss this point. 

In conclusion, the effects on offspring reported in this study are mild and of questionable significance. Overall the study depicts the typical effects of HBCDD in rodents on liver and the thyroid axis. It would be unreasonable to expect that young rats would not show these effects upon HBCDD exposure.Consequently, these effects are not specific for developmental toxicity but rather reflect typical rodent repeated dose toxicity of HBCDD.

Toxicity to reproduction: other studies

Additional information

All studies show that the substance can be passed from the mother to the infant in breast milk, further developmental effects were not reported or assessed.

Justification for classification or non-classification

In line with the opinion of the EU Committee for Risk Assessment (RAC) dated 8th December 2010, the third adaptation to technical and scientific progress of Regulation 1272/2008 (Regulation 618/2012) has stated that HBCDD shall be classified as follows: Txoci to reproduction Category 2 (H361: Suspected of damaging fertility or the unborn child; H362: May cause harm to breastfed children). The RAC opinion also specified that HBCDD should be classifed as Toxic to reproduction Category 3 (R63: Possible risk of harm to the unborn child; R64: May cause harm to breastfed babies) in line with Directive 67/548/EEC.