Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 April 2003 to 14 July 2003
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:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(see below)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
HBCD was not adequately soluble in 4:1 acetone:olive oil (AOO), the preferred vehicle for the LLNA. The highest concentration of HBCD (50% w/v) was based upon maximal solubility of HBCD in DMF; lower concentrations were selected to provide a range of doses (25%, 10%, 5%, or 1%) to evaluate ear swelling potential of HBCD.
Lymphocyte proliferation by DMF, vehicle treated mice (2,015 dpm) was higher than historical laboratory values (Table 3, attached) commonly observed using acetone and olive oil as a vehicle. This is not inconsistent with that reported in the literature for this vehicle. One mouse in particular demonstrated 3481 dpm in its draining lymph nodes and was confirmed to be a statistical outlier. Exclusion of this vehicle treated animal still resulted in a control value of 1,722 dpm and did not ultimately alter the conclusions of this study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: >95%
- Chemical name: 1,2,5,6,9,10-hexabromocyclododecane
The test material was comprised of three diastereomers: 5.8% alpha, 19.3% beta, 74.9% gamma.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
The Jackson Laboratory, Bar Harbor, Maine.

- Age at study initiation:
The mice were 8 weeks of age at the start of the primary irritancy study and 9 weeks of age at the start of the LLNA.

- Weight at study initiation:
Not reported.

- Housing:
The animals (6/group) were housed in plastic "shoebox" cages with filter lids in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately one week prior to the start of the study. Each cage contained corncob bedding and a cardboard enclosure, which were changed at least two times a week.

- Diet (e.g. ad libitum):
Animals were provided LabDiet® Certified Rodent Diet #5002 food pellets, ad libitum

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

- Acclimation period:
approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 + 1 °C (with a maximum permissible excursion range of + 3 °C)

- Humidity (%):
40-70%

- Air changes (per hr):
approximately 12-15 times/hour.

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

IN-LIFE DATES: From: Day 1 To: Day 14

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
50%, 20% and 2%
No. of animals per dose:
6 (female)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
HBCD was not adequately soluble in 4:1 acetone:olive oil (AOO), the preferred vehicle for the LLNA. The highest concentration of HBCD (50% w/v) was based upon maximal solubility of HBCD in DMF; lower concentrations were selected to provide a range of doses (25%, 10%, 5%, or 1%).

- Irritation:
Since mild irritation was noted during the irritation screen, the thickness of each ear was monitored during the LLNA procedure. Prior to the administration of a dosing material and after the final exposure on day 3, the thickness of each ear was measured with a digital micrometer.

- Lymph node proliferation response:
Not investigated in range finding study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Animal assignment:
Animals were stratified using pre-exposure body weights and randomly assigned to treatment groups using a computer program (Provantis created by Instem Corporation, Philadelphia, Pennsylvania).

- Name of test method:
Local Lymph Node Assay (LLNA)

- Criteria used to consider a positive response:
Chemicals that elicit a SI of >3 in the LLNA are considered positive for dermal sensitization potential.

TREATMENT PREPARATION AND ADMINISTRATION:
The administration of the materials (25 µl/ear) was made on the dorsal surface of both ears using an adjustable pipettor fitted with a disposable tip (n=6 mice/group). All mice received one of three concentrations of HBCD (2%, 20%, or 50% in DMF) or DMF alone on days 1-3.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The % ear swelling was calculated for each ear using the following equation:

% Ear swelling = (B-Ax100)/A where:
A = mean of pretreatment measurement (mm x 10E-2)
B = mean of post treatment measurement (mm x 10E-2)

The Stimulation Index (SI) was calculated for each mouse using the following equation:

SI = Disintegration per minute (dpm) of individual mouse/Average dpm of the VH control mice

Descriptive statistics only (means and standard deviations) are reported for ear irritation data. Body weights (absolute and gains) and lymphocyte proliferation data were first evaluated by Bartlett's test (a = 0.01; Winer, 1971) to determine equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steele and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed.
If the ANOVA was significant at a= 0.05, it was followed respectively by Dunnett's test (a= 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (a= 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. Statistical outliers were identified by a sequential test (a= 0.02; Grubbs, 1969). The final interpretation of the biological significance of the responses was based on both statistical outcome and scientific judgement.

Grubbs, F. E. (1969). Procedures for Detecting Outlying Observations in Samples. Technometrics 11(1):1-21.

Hollander, M. and Wolfe, D. A. (1973). Nonparametric Statistical Methods. John Wiley, New York, New York.

Miller, R. G., Jr. (1966). Simultaneous Statistical Inference. McGraw-Hill, New York, New York pp. 67-70, 101-102.

Steele, R. G. D. and Torrie, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York, New York.

Winer, B. J. (1971). Statistical Principles in Experimental Design (2nd Edition). McGraw-Hill, New York, New York.

Results and discussion

Positive control results:
HCA administrations (30% v/v) elicited proliferation that was 3-fold greater than that of vehicle controls. Exclusion of the statistical outlier in the vehicle group increased the HCA SI value to above 3.5. While this SI value for HCA is slightly lower than reported values when diluted in acetone and olive oil, HCA was still positive according to guideline criteria and demonstrated 3H-thymidine uptake (5,970 dpm) that was statistically higher than that of vehicle controls (2,015 dpm). HCA is a useful positive control for the LLNA as it is a moderate contact sensitizer and can serve as a better indication of the assay's sensitivity compared with extreme sensitizers such as DNCB.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Summary of Lymph Node Proliferation Data: Treatment Group DPM (mean ± SD) SI (mean ± SD) Vehicle (DMF) 2015 ± 749 1.0 ± 0.4 HCA (30% v/v) 5970 ± 1909* 3.0 ± 0.9 2% HBCD 1744 ± 181 0.9 ± 0.1 20% HBCD 1670 ± 155 0.8 ± 0.1 50%HBCD 1946±664 1.0±0.3 Mice (n=6/group) were treated topically with vehicle or HBCD preparation for 3 consecutive days. Mice were injected intraveneously (tail) with 3H-thymidine and draining auricular lymph nodes were evaluated for 3H-thymidine incorporation into proliferating lymphocytes. Values represent mean and standard deviations (S.D.). * represents statistical difference from control mean at p < 0.05 using Dunnett's T-test. DMF = N,N-Dimethylformamide. HBCD =hexabromocyclododecane. HCA = a hexyl cinnamic aldehyde. DPM = disintegrations per minute. SI = stimulation index.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Lymphocyte proliferation by DMF, vehicle treated mice (2,015 dpm) was higher than historical laboratory values (Table 3, attached) commonly observed using acetone and olive oil as a vehicle. This is not inconsistent with that reported in the literature for this vehicle. One mouse in particular demonstrated 3481 dpm in its draining lymph nodes and was confirmed to be a statistical outlier. Exclusion of this vehicle treated animal still resulted in a control value of 1,722 dpm and did not ultimately alter the conclusions of this study. HBCD did not demonstrate results that were consistent with dermal sensitization (See below). SI values were consistently around 1.0 at all doses tested.

Any other information on results incl. tables

Percent Ear Swelling (Irritation, range finding test)

Treatment

Percent Ear Swelling

(mean)

Vehicle (DMF)

1.0

1% HBCD

2.0

5% HBCD

6.2

10% HBCD

19.8

25% HBCD

26.5

50% HBCD

19.8

Mice (n=l/group) were treated topically with vehicle or HBCD preparation for two consecutive days. Ear thickness was measured using a digital micrometer. Values represent mean for each mouse (n=2 ears/dose).

DMF = N,N-Dimethylformamide

HBCD = hexabromocyclododecane

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
HBCD did not elicit lymph node proliferation when tested as high as 50% w/v in the mouse LLNA. HBCD has also been tested in the guinea pig using a Magnusson and Kligman (M&K) Maximization test, and in a human patch test with fabric treated with HBCD. The human patch test did not elicit skin sensitization (McDonnell 1972). In the M&K test, a lack of dermal sensitization was reported by Wenk (1996), whereas Momma et al. (1993) and Nakamura et al. (1994) reported positive results. Specifics of the test material used by Momma and Nakamura were not described in detail and may partially explain differing results. Though both the guinea pig M&K and the LLNA are both considered appropriate for evaluating contact sensitization potential, the LLNA has the advantage of a quantitative, dose-response endpoint for sensitization potential. These results suggest that HBCD does not possess contact sensitization potential.

McDonnell, M. (1972). Haskell Laboratory Report No. 185-72. Haskell Laboratory for Toxicology and Industrial Medicine.

Miller, R. G., Jr. (1966). Simultaneous Statistical Inference. McGraw-Hill, New York, New York pp. 67-70, 101-102.

Nakamura, A., Momma, J., Sekiguchi, H., Noda, T., Yamano, T., Kaaiwa, M., Kojima, S., Tsuda, M. and Kurokawa, Y. (1994). A new protocol and criteria for quantitative determination of sensitization potencies of chemicals by guinea pig maximization test. Contact Dermatitis 31: 72-85.

Wenk, M. (1996). Maximization test in guinea pigs. Test Article: Hexabromocyclododecane. Project no. M96AO61.1X64. Microbiological Associates, Inc., Rockville, Maryland.
Executive summary:

The Local Lymph Node Assay (LLNA) assesses the potential of test materials to cause contact sensitization by measuring the lymphocyte proliferative responses from auricular lymph nodes following topical application of the test materials to mouse ears. Test materials that elicit a Stimulation Index (SI) of >3 (i.e., 3-fold greater proliferation than control animals) should be considered positive for dermal sensitization potential.

All mice received one of three concentrations of HBCD (2%, 20% or 50% w/v) or DMF (dimethylformamide) on days 1-3 (n=6 mice/group). HCA (a-hexyl cinnamaldehyde), a moderate contact sensitizer, was evaluated concurrently as a positive dermal sensitization control. The test materials were administered to the dorsal surface of both ears (25 µl/ear). On day 6, all mice received an intravenous tail vein injection of phosphate buffered saline containing 20 µCi of 3H-thymidine. Uptake into the auricular lymph nodes draining the site of chemical application was measured 5 hours later.

Body weight data were unremarkable and minor increases in ear thickness were noted suggesting slight irritation following applications of 20% and 50% HBCD. There were no indications that HBCD possesses dermal sensitization potential. SI values were consistently around 1.0 at all doses tested. Lymphocyte proliferation by DMF, vehicle treated mice (2015 dpm) was higher than historical laboratory values commonly observed using acetone and olive oil as a vehicle. This is not inconsistent with that reported in the literature for this vehicle. HCA administrations (30% v/v) elicited proliferation that was 3-fold greater than that of vehicle controls thus detecting the moderate contact sensitization potential in this study. On the basis of these results, HBCD would not be considered to have contact sensitization potential. HBCD therefore does not meet the criteria for classification as a sensitiser as set out by 67/458/EEC.