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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2000 to 11 December 2001
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:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
In addition to routine end points for a 90-day study, measurements of serum TSH, T3 and T4 levels, vaginal cytology and sperm assessments, and measurement of test article concentration in adipose tissue were performed.
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)IGS BR
- Source:
Charles River Laboratories, Portage, Michigan.
- Age at study initiation:
36 days old upon receipt, approximately 7 weeks old at the initiation of dosing.
- Weight at study initiation:
Toxicity groups (90-day treatment +/- 28-day recovery period):
Males: 212 to 289 grams
Females: 147 to 205 grams
Satellite groups (control and high dose - up to 90-day treatment; sampling of blood and body fat at scheduled time points):
Males: 204 to 268 grams
Females: 143 to 201 grams
Body weights at randomization were within ± 20% of the mean for all animals.
- Housing:
All animals were housed individually in clean, wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum):
PMI Nutrition International, Inc., Certified Rodent LabDiet® 5002, ad libitum, except during the period of fasting prior to blood collection when food but not water was withheld.
- Water (e.g. ad libitum):
reverse osmosis-treated (on-site) drinking water, ad libitum.
- Acclimation period:
All animals were housed for a 15-day acclimation and pretest period; observations were made twice daily for mortality and general changes in appearance or behavior.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21.1ºC to 24.7ºC
- Humidity (%):
37.7 to 68%
- Air changes (per hr):
Not indicated
- Photoperiod (hrs dark / hrs light):
12 hours dark/12 hours light

IN-LIFE DATES: From: Day 1 To: Day 90 + 28 days recovery period

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
A sufficient volume of the vehicle, Mazola® corn oil, was dispensed into a storage container. The vehicle was stirred continuously throughout use with a magnetic stirrer.
The appropriate amount of test article for each group was weighed into tared, calibrated storage containers. A sufficient amount of the vehicle was added and stirred until a uniform mixture was obtained using a magnetic stirrer. The appropriate amount of vehicle was added to bring the formulation to volume. Preparations were stirred until uniform and continuously throughout use. After preparation, dosing formulations were divided into daily aliquots.
All dosing formulations were prepared weekly and stored at room temperature.

DIET PREPARATION
Not feeding study

- Rate of preparation of diet (frequency):
Not applicable.

- Mixing appropriate amounts with (Type of food):
Not applicable (gavage).

- Storage temperature of food:
Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not reported.

- Concentration in vehicle:
0, 100, 300, 1000 mg/kg/day

- Amount of vehicle (if gavage):
5 mL/kg

- Lot/batch no. (if required):
Not reported.
- Purity: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dosing, sample dosing suspensions were prepared for the lowest and highest concentration groups, and 10 mL aliquots were withdrawn from the top, middle and bottom of each suspension for homogeneity determinations. Samples were also collected from the middle of these preparations and stored under laboratory conditions for 10 days for stability analyses. In addition, the remainer of each preparation was subdivided into seven aliquots and stored at room temperature. Following storage for 10 days and re-suspension using the same procedure to be used for dosing, samples were taken from the top and bottom of the last daily aliquot from each preparation and were analysed to confirm homogeneity in a dosing aliquot. Dosage preparations, including the control group, were verified for test material concentration using a validated HPLC method during study weeks 0, 1, 2, 3, 7 and 12. All analyses were performed at the testing facility. The results of these analyses confirmed that corn oil dosing suspensions were stable for at least 10 days when stored at room temperature. In addition, the dosing formulations were homogeneous and within 100% (+/- 10%) of target dose concentrations.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000
Basis:
actual ingested
No. of animals per sex per dose:
Toxicity groups:
30 animals (15 male/15 female) per dose level

Satellite groups:
40 animals (20 male/20 female) per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not given
- Rationale for animal assignment (if not random): computerized randomization procedure
- Rationale for selecting satellite groups: computerized randomization procedure
- Post-exposure recovery period: 28 days
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. Final fasted body weight recorded for each animal on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not a feeding study
- Food consumption for each animal determined as g food/day: Yes, weekly

FOOD EFFICIENCY:
Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not a drinking water study. No.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weeks -1, 12 and 15 (recovery period)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 month (week 3), end of dosing period (week 13) and end of recovery period (week 17)
- Anaesthetic used for blood collection: no anaesthetic for the 1 month sampling (from lateral tail vein); animals anesthetised with carbon dioxide for terminal sacrifice (samplig from vena cava)
- Animals fasted: Yes
- How many animals: 1 month samplig: 5 sex/group; terminal sacrifice: 10 sex/group; recovery sacrifice: 5 sex per control and high dose groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for haematology
- In addition to standard parameters, THS, total T3 and Total T4 were determined
- Animals fasted: Yes
- How many animals: as for haematology

URINALYSIS: Yes
- Time schedule for collection of urine: overnight, the day prior to blood collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weeks -1, 12 and 16
- Dose groups that were examined: all (control and high dose at week 16 - recovery)
- Battery of functions tested: sensory activity / grip strength / motor activity / other: home cage observations, handling observations, open field observations, physiological observations

OTHER:
- Vaginal smears for determination of stage of estrus were obtained from all females once daily beginning study day 69 through the the last primary necropsy.
- Spermatogenesis evaluations (motility/viability assessment, morphology assessment, enumeration of epididymal and testicular sperm numbers and sperm production rate) were performed for all males at terminal and recovery sacrifice;
- Test article fat concentration was determined from 2 animals/sex/group (satellite groups) on days 2, 6, 9, 13, 20, 27, 55, 89, 104 and 118
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. All animals from the control and high dose groups at primary and recovery necropsies. Liver, lungs, prostate and thyroid glands examined also from all animals of the low and mid dose groups at primary necropsy. The liver from 5 animals/sex for the control and high dose groups were stained with Oil Red O or periodic acid Shiff's reagent for evidence of lipid and glycogen accumulation or depletion, respectively.
Statistics:
All statistical tests were performed using appropriate computer devices or programs. All analyses were two-tailed fro significance levels 5% and 1%. Each mean was presented with the standard deviation (SD) and the number of animals (N) used to calculate the mean. Statistical analyses were not performed if the number of animals was two or less. Body weights, body weight changes, food cunsumption, estous cycle, clinical pathology data, organ weights, epididymal and testicular sperm numbers and sperm production rates were subjected to a one-way analysis of variance (ANOVA). If significant differences (p<0.05) were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups. Clinical laboratory values for leukocytes that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

The percentage motile spermatozoa and the percentage of sperm with normal morphology were analysed by the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences, followed by the Mann-Whitney U-Test comparing the control and treated groups if the ANOVA revealed statistical significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL CHEMISTRY
Statistically significant changes were noted at weeks 3 and/or 13 and included an increase in albumin (all dose levels for males), total protein (all dose levels for females and males treated at 1000 mg/kg bw/day), globulin (300 and 1000 mg/kg bw/day for females), and chloride (all doses both sexes). Gamma glutamyltransferase levels were increased in the 1000 mg/kg bw/day group. Thyroxine (T4) levels were decreased at week 13 in all male groups and in females treated at 300 and 1000 mg/kg bw/day, while no corresponding statistical effects on T3 and TSH were observed. These changes 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 serum albumin and gamma glutamyl transerase increases were probably secondary to the increased liver weight, while the increased serun chloride was probably secondary to the presence of free bromine in the test article formulations which interferred with the chloride determination methodology. The decrease in T4 which was also reversible was considered secondary to increased liver weight due to microsomal induction, known to cause increased metabolism and clearance of T4 in the rat.

ORGAN WEIGHTS
Mean liver weights (absolute and relative) were increased in all treated male and female groups at primary necropsy (week 13), consistent with histopathological chenges. Prostate wieghts (absolute and relative) were also increased at week 13 in males treated at 1000 mg/kg bw/day. Increases in liver weights completely, or neraly completely resolved in all groups by the week 17 recovery necropsy. At that time prostate weights were also comparable to control values.

HISTOPATHOLOGY: NON-NEOPLASTIC
At the 90-day sacrifice, minimal hepatocellular vacuolation was noted in 10% of control males and ca. 50% of the males at 100, 300 and 1000 mg/kg bw/day. Minimal hepatocellular vacuolation was detected in the control and treated females, without a clear dose-relationship (3/10; 6/10; 3/10; 5/10) and with a slight increase in severity noted at 300 and 1000 mg/kg bw/day. Centrilobular hepatocyte hypertrophy (minimal in 20% of the animals, mild in 30% of the animals) was also noted in females at 1000 mg/kg bw/day. Results of the Oil Red O and PAS staining suggested that the content of the vacuoles was lipidic in nature, and that the lipid content was not present in unusually amounts. Minimal thyroid follicular cell hypertrphy was detected in 1/10, 1/10, 5/10 and 7/9 male rats in the control, 100, 300 and 1000 mg/kg bw/day groups, and in 4/10 and 3/10 females treated at 300 and 1000 mg/kg bw/day. Mild follicular cell hypertrophy was noted in 1/10 males and 4/10 females receiving 1000 mg/kg bw/day. At the recovery necropsy, the liver changes had resolved in all treated animals without any delay or long-term toxic effects. The thyroid changes had also resolved in all males and in females previously receiving 300 mg/kg bw/day, and nearly completely resolved in females at 1000 mg/kg bw/day. Despite the increased prostate weights, there were no corroborative findings at histopathology or sperm analysis.

OTHER FINDINGS
The test substance was detected in the adipose tissue of animals of both sexes treated with 1000 mg/kg bw/day fro 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 at achieved by study day 27. Levels in male and female rats were similar at all time points and declined during the recovery period.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Adaptive and reversible changes in the liver and thyroid, considered secondary to enzyme induction.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

The test article, a composite of three lots of commercial hexabromocyclododecane (HBCD), was administered by oral gavage in corn oil once daily to four groups of Crl:CD(SD)IGS BR rats (n=15/sex/group) at dose levels of 0 (control), 100 (low), 300 (mid) and 1000 (high) mg/kg/day seven days per week for 90 days. The dosage volume was 5 mL/kg. The control animals received the vehicle, corn oil, only. At the end of the 90-day treatment period, 10 animals/sex/group were euthanized and necropsied. The remaining rats continued on test untreated for a 28-day recovery period prior to necropsy.

In addition to the main toxicology groups, two satellite groups of 20 animals/sex/group were treated concurrently in an identical manner at dose levels of 0 or 1000 mg HBCD/kg/day for up to 90 days. Body weights were recorded weekly. Two animals/sex/group were euthanized on study days 2, 6, 9, 13, 20, 27, 55, 89, 104 and 118, and blood and body fat (mesenteric and/or omental) were collected. The body fat was analyzed for HBCD content.

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 hematology, 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. Livers from five randomly chosen animals/sex from the control and 1000 mg/kg/day dose groups were 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 glutamyl transferase levels were noted in the 1000 mg/kg/day group (p <0.01). Thyroxine (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; furthermore, these serum albumin and gamma glutamyl transferase increases were probably secondary to the increases in liver weight. The increases in serum chloride were probably secondary to be presence of free bromide in the test article preparation which interfered with the chloride determination methodology. The decrease in T4, which was also reversible, was also probably secondary to increased liver weight (secondary to microsomal enzyme induction, known to cause increased metabolism and clearance of T4 in the rat).

The incidence of observations noted at gross necropsy was low and there was no evidence of frank organ damage. On histopathologic examination of tissues, relatively mild findings occurred in both the control and treated groups. Potential test article-related histologic 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 histologic changes in the liver were accompanied by an increase in liver weight. In contrast there were no statistically significant changes in thyroid weight (absolute, relative to body weight and relative to brain weight). At study week 13, mean liver weights in all dose levels of both sexes (absolute, relative to body weight 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 a result of a microsomal enzyme inducing effect and were not typically considered indicative of toxicity in absence of frank organ damage. 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.

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 were probably not of toxicological significance since the increases did not persist to the recovery period, there were no correlating histologic findings and no change in sperm production.

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, 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, toxicologic 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.