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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November to 18 December 1998
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:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl: CD®(SD)IGS BR
- Source: Charles River Laboratories, Raleigh, North Carolina

- Age at study initiation: approximately 12 weeks old at pairing

- Weight at study initiation: from 232 g to 299 g on day 0 of gestation.

- Housing: upon arrival and until pairing, all animals were individually housed in clean, wire-mesh cages suspended above cage-board. The animals were paired for mating in the home cage of the male. Following positive identification of mating, the females were returned to an individual suspended wire mesh cage; nesting material was not required as the females were euthanized prior to the date of expected parturition.

- Diet (e.g. ad libitum): PMI Nutrition International Inc., Certified Rodent LabDiet® 5002, ad libitum

- Water (e.g. ad libitum): ad libitum

- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 to 22.3°F

- Humidity (%): 42.6% to 60.7%

- Air changes (per hr): 10 changes/hr

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

IN-LIFE DATES: From: 24 November 1998 To: 18 December 1998 - Exposure from day 6 to 19 of presumed pregnancy

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of the vehicle, Mazola® corn oil, was dispensed into a properly labeled storage container for administration to the control group. A stir bar was added and the vehicle was stirred continuously throughout the sampling and dosing procedures. A sufficient volume of the vehicle was dispensed daily for administration to the control group. The test article formulations were prepared as follows. An appropriate amount of test article was weighed for each group into a labeled, precalibrated storage container. A stir bar was added and the container was tared a second time. A sufficient amount of the vehicle was then added to bring the volume of each preparation to the calibration mark. Weights were collected for the vehicle for specific gravity determination. The preparations were stirred continuously throughout the sampling and dosing procedures. Preparations for all dose groups were formulated daily during the treatment period and were maintained at room temperature. The dosing preparations were visually inspected for homogeneity by the study director on November 30, 1998, and were found to be acceptable for use.

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

- Concentration in vehicle: 10, 20, 40 mg/ml

- Amount of vehicle (if gavage): 5ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and concentration determinations were performed at WIL Research Laboratories, Inc. using a gravimetric method provided by the sponsor. On the first, tenth and last days of dosing, three 1-ml samples were collected from the top, middle and bottom of each group formulation, then weighed to assess homogeneity. The samples were taken using a positive displacement pipet. These values were compared to the calculated specific gravity of each formulation. On those days in which homogeneity analyses were not performed, three 1-ml samples were collected from the middle of each group formulation, then weighed to assess concentration. Based on the results of the gravimetric analyses, the dosing formulations were homogeneous and contained the amounts of the test article specified in the protocol.
One-gram samples of test article were collected on the first day of dosing and at the termination of the study, frozen at approximately -20°C and shipped to Albemarle Corporation to assess the storage stability of the neat test article. Analysis demonstrated identity and stability of the net test substance used in the study. At the request of the sponsor, a retrospective confirmation of homogeneity, concentration and 24-hour stability of the test article formulations was performed utilizing a validated HPLC method. Representative batches of test article formulations (low and high dose groups only) were prepared after the completion of the in-life phase of the study. All procedures were identical to those utilized to prepare the formulations used to dose the animals during the administration period. To verify homogeneity, 24-hour stability and concentration of the test article in the dosing formulations, duplicate 1-ml samples were taken from the low and high dose group formulations after 24 hours at room temperature. All of the samples collected (both at time 0 and after 24 hours) were frozen at approximately -20°C as soon as possible. The samples and approximately 3 grams of the test article were shipped to Albemarle Corporation for analysis. The results of the analysis demonstrated that HBCD was homogeneous as a suspension in corn oil, and stable in the formulation for at least 24 hours after preparation.
Details on mating procedure:
- Impregnation procedure: cohoused

- If cohoused:
- M/F ratio per cage: 1:1

- Length of cohabitation: until evidence of breeding was observed.

- Verification of same strain and source of both sexes: male were untreated sexually mature rats from the same strain and source mainatind only for breeding.

- Proof of pregnancy: evidence of mating (vaginal plug / sperm in vaginal smear) referred to as day 0 of gestation.
Duration of treatment / exposure:
from Days 6-19 of gestation.
Frequency of treatment:
single daily dose.
Duration of test:
23 days.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25 (female)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not reported. Study preceed by a RF study.

- Rationale for animal assignment (if not random): at the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the study director, animals judged to be in good health and meeting acceptable body weight requirements (a minimum of 220 g) were placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Following the completion of the mating period, the bred females were assigned to groups using a computer program which randomized tha animals based on body weight stratificationin a block design.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: from day 0 through 20 of gestation (prior to test article administration during the dosing period)

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 and 6-20 (daily). A group mean body weight was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for intervals 6-9, 9-12, 12-20, 6-20 and 0-20.
Gravid uterine weight was collected and net body weight (the day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
not feeding study
Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All maternal animals were euthanized by carbon dioxide inhalation on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents examined. In all instances, the post mortem findings were correlated with the ante mortem comments, and any abnormalities were recorded. The uterus and ovaries were excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathological examination only as indicated by the gross findings.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no macroscopic evidence of nidation were excised, opened and placed in 10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- External examinations: Yes:
Each fetus was sexed, weighed and tagged for identification. Fetal tags contained the WIL study number, the maternal animal number and the fetus number. A detailed external examination of each fetus was conducted to include, but was not limited to, an examination of the eyes, palate and external orifices, and each finding was recorded. Crown-rump measurements were recorded for late resorptions, if present, and the tissues were discarded. Each fetus was examined viscerally by a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development by a method described in Woo and Hoar.
Heads from approximately one-half of the fetuses in each litter were placed in Bouin's fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a mid-coronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue by a method similar to that described by Dawson and Inouye. External, visceral and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effects on animal health or conformity representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function or may be incompatible with life).
Statistics:
All analyses were conducted using two-tailed tests for a minimum significance level of 5%, comparing each treated group to the vehicle control group. Means were presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. The following statistical tests were performed by a Digital® MicroVAX® 3400 computer (with appropriate programming) and referenced in the report tables:
STATISTICAL TEST: PARAMETER:
- One-way ANOVA with Corpora Lutea, Total
Dunnett's test Implantations, Fetal Body Weights, Maternal Body Weights and Weight
Changes, Maternal Net Body Weight Changes and Gravid Uterine Weights, Food Consumption, Viable Fetuses

- Kruskal-Wallis test with Litter Proportions ofIntrauterine Data (Considering the Litter, Rather than the Fetus, as the Experimental Unit); Litter ProMann-Whitney U test portions of Fetal Malformations and Developmental Variations.
Indices:
Intrauterine data were summarized using two methods of calculation. An example of each method of calculation follows:
- Group Mean Litter Basis:
Post-implantation Loss / Litter = No. Dead fetuses, Resorptions (early and late)per group / No. Gravid females per group

- Proportional Litter Basis:
Summation per Group (%) = Post-implantation Loss per Litter (%) / No. of litters per group
Post-implantation Loss / Litter (%) = (No. Dead Fetuses, Resorptions (early and late) per litter / No. Implantation sites per litter) x 100

The fetal developmental findings were summarized by 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in this group; and 2) by considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = Viable fetuses affected per litter (%) / No. of litters per group
Viable fetuses affected per litter (%) = (No. viable fetuses affected per litter / No. viable fetuses / litter) x 100
Historical control data:
Historical control data were included in the report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All animals survived to the scheduled necropsy on gestation day 20. No treatment-related clinical signs were observed in any dose level. Clinical findings in all of the treated groups occurred similarly in the control group, in single animals or in a manner that was not suggestive of a relationship to treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
-EXTERNAL MALFORMATIONS AND VARIATIONS:
External malformations were observed in four fetuses in the 500 mg/kg/day group. Fetus nos. 3594-01, 3639-08 and 3653-03 had anophthalmia (bilateral). The proportional incidence (% per litter) was increased (0.8% per litter) in the 500 mg/kg/day group when compared to the control group (0% per litter). The value was not statistically significant and was within the WIL historical data range (0-1.3 % per litter). Fetus no. 3594-01 also had a facial cleft and exencephaly (without open eyelid), and fetus no. 3639-16 had exencephaly (without open eyelid). Although the values were not statistically significant, the incidence of exencephaly (0.6% per litter) exceeded the maximum value in the WIL historical control data (0.5% per litter). The facial cleft malformation was not previously seen in the WIL historical control data. Fetus no. 3639-08 was hydrocephalic with a dome-shaped head. Since there were no malformations observed in the 1000 mg/kg/day group fetuses, the malformations in the 500 mg/kg/day group were not considered to test article-related. No other external malformations were noted. No external developmental variations were observed in fetuses at any dose level.

-VISCERAL MALFORMATIONS AND VARIATIONS:
There were no soft tissue malformations observed in fetuses at any dose group. Soft tissue developmental variations were observed in 0(0), 0(0), 1(1) and 1(1) fetuses (litters) in the control, 250, 500 and 1000 mg/kg/day groups, respectively. One fetus in each of the 500 and 1000 mg/kg/day groups (nos. 3694-12, and 3652-01 respectively) had a retroesophageal right subclavian artery. These single occurrences were not considered to be related to test article administration.

-SKELETAL MALFORMATIONS AND VARIATIONS:
Skeletal malformations were noted in one fetus in the 500 mg/kg/day group. Fetus no. 3653-03 had a vertebral centra anomaly that consisted of fused lumbar centra (nos. 5 and 6). No other skeletal malformations were observed.
Skeletal developmental variations occurred in all dose groups, including the control group, and consisted primarily of unossified sternebrae nos. 5 and/or 6, ossified cervical centrum no. 1 and 14th rudimentary rib(s). Other skeletal variations observed in the treated groups occurred infrequently and/or similarly in the control group or were within the range of the WIL historical control data. No relationship to treatment was evident.

-SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS:
Fetal external, soft tissue and skeletal malformations were observed in 0(0), 0(0), 4(3) and 0(0) fetuses (litters) in the control, 250, 500 and 1000 mg/kg/day groups, respectively. The findings were not considered to be related to the test article since they were not observed in the 1000 mg/kg/day group. There were no statistically significant differences in malformations observed in any of the treated groups. Fetal developmental variations in the treated groups occurred similarly in the control group or occurred infrequently and were not considered to be related to treatment with the test article.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Discussion

All maternal animals survived to the scheduled necropsy on gestation day 20. One female in the 500 mg/kg/day group delivered on gestation day 20 and was examined at the scheduled laparohysterectomy. Based on pup/fetal weights, the delivery was considered to be an error in the detection of mating and not test article-related. No test article-related clinical findings were observed in the 250, 500 and 1000 mg/kg/day groups. Mean maternal body weights, body weight gains, gravid uterine weights, net body weights, net body weight gains and food consumption were unaffected by test article administration at any dose level. At the scheduled necropsy on gestation day 20, no test article-related internal findings were observed at any dose level. Intrauterine growth and survival were unaffected by test article administration at anydose level. Parameters evaluated included post-implantation loss, live litter size, mean fetal body weights, fetal sex ratios and the mean numbers of corpora lutea and implantation sites. Fetuses (litters) available for morphological evaluation numbered 322(22), 331(23), 338(23) and 290(20) in the control, 250, 500 and 1000 mg/kg/day groups, respectively. Malformations were observed in 0(0), 0(0), 4(3) and 0(0) fetuses (litters) in these same respective dose groups and were considered to be spontaneous in origin. No developmental variants were noted in fetuses in the treated groups that were considered to be related to treatment with the test article.

Applicant's summary and conclusion

Conclusions:
no maternal toxicity or developmental toxicity were observed at any dose level. Based on the results of this study, the limit dose level of 1000 mg/kg/day was found to be the NOAEL (no-observed-adverse-effect level) for maternal toxicity and developmental toxicity of HBCD in rats.
Executive summary:

The potential maternal toxicity and prenatal developmental toxicity of the test article, hexabromocyclododecane (HBCD), were evaluated in a fully compliant GLP study. The test article in the vehicle, Mazola®corn oil, was administered to three groups of 25 bred Crl:CD''(SD)IGS BR rats once daily from gestation days 6 through 19. Dosage levels were 250, 500 and 1000 mg/kg/day (homogeneity and content confirmed) administered at a dose volume of 5 ml/kg. A concurrent control group composed of 25 bred females received the vehicle, Mazola®corn oil, on a comparable regimen at 5 ml/kg. The route of administration was oral by gastric intubation. Clinical observations, body weights and food consumption were recorded. On gestation day 20, a laparohysterectomy was performed on all animals. The uteri and ovaries were examined and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Mean gravid uterine weights and net body weight changes were calculated for each group. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal malformations and variations.

All maternal animals survived to the scheduled necropsy on gestation day 20. One female in the 500 mg/kg/day group delivered on gestation day 20 and was examined at thescheduled laparohysterectomy. No treatment-related clinical signs were observed at any dose level. Body weight gain and food consumption were not adversely affected at any dose level. At necropsy, no treatment-related findings were observed. Intrauterine growth and survival were unaffected by test article administration at any dose level. No treatment-related fetal malformations and developmental variations were observed in any of the treated groups.

Based on the results of this study, the NOAEL(no-observed-adverse-effect level) for maternal toxicity and developmental toxicity was found to be 1000 mg/kg/day.