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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This substance was evaluated in each of five independent international laboratories using either the standard LLNA protocol (Kimber & Basketter, 1992) or minor modifications of it. There is no data on GLP but the study is comparable to a GLP guideline study.

Data source

Reference
Reference Type:
publication
Title:
Assessment of the Skin Sensitization Potential of Topical Medicaments Using the Local Lymph Node Assay: An Interlaboratory Evaluation
Author:
Kimber I, Hilton J, Dearman RJ, Gerberick GF, Ryan CA, Basketter DA, Lea L, House RV, Ladics GS, Loveless SE and Hastings KL
Year:
1998
Bibliographic source:
J. Toxicol. Environ. Hlth., Part A, 53 (7), 563-579

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibenzoyl peroxide
EC Number:
202-327-6
EC Name:
Dibenzoyl peroxide
Cas Number:
94-36-0
Molecular formula:
C14H10O4
IUPAC Name:
benzoyl benzenecarboperoxoate
Details on test material:
Purity: 70 %

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca strain or CBA/JHsd strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Five different laboratories (because it was an interlaboratory evaluation)
- Age of animal at study initiation : 6 - 12 weeks
- Housing: under standard conditions (no more details)
- Diet and water: ad libitum

Study design: in vivo (LLNA)

Vehicle:
other: acetone
Concentration:
Concentrations: 0.5, 1.0, 2.5, 5.0 and 10.0 % benzoyl peroxide.
Dosing solution were prepared volumetrically and immediately prior to each treatment.
No. of animals per dose:
5 animals/dose
Details on study design:
Route of administration : topical application on the dorsum of both ears.
Volume of material dosed : 25 µl Duration of exposure for induction : daily for 3 consecutive days. Length of rest period : 2 days prior to analysis
Laboratory A and B used standard LLNA protocol.Five hours after the injection of PBS containing 3Hmethylthymidine, mice were sacrificed and the draining auricular lymph nodes were excised and pooled for each experimental group. Modified LLNA Protocol 1 Laboratories C and D employed the standard protocol for the LLNA, with the exception that lymph nodes pooled for individual mice, rather than from experimental groups, were analyzed. Modified LLNA Protocol 2. Laboratory E employed a further modification of the standard protocol that comprised not only analysis of Iymph nodes pooled for individual mice (as per modified protocol 1 just given) but also the use of[125l]iododeoxyuridine ('251-UdR; specific activity2000Ci/mmol) in place of3H-TdR
Grading system used : in vivo [3H]methylthymidine(or [125I]iododeoxyuridine) : incorporation into lymph node cell DNA associated with proliferation induced by application of benzoyl peroxide was an objective and quantifiable response.

Stimulation indices (SI) were determined as increase in 3H-TdR incorporation relative to vehicle-treated controls. SI of 3 or greater was considered to have skin sensitizing activity. Based on fitted quadratic regression analyses, estimates of the applied concentration of benzoyl peroxide required to elicit a SI of 3 (EC 3 value) were calculated.


For more details:
Laboratories A and B: a standard LLNA protocol was employed; groups of mice (n = 5) were exposed on the dorsum of both ears to 25 pl of 1 of 5 concentrations of the test chemical, or to an equal volume of the relevant vehicle atone. Treatment was performed daily for 3 consecutive days. Five days following the initiation of treatment all mice were injected intravenously via the tait vein with 250 pl of phosphate-buffered saline (PBS) containing 20 pCi of [3H]methylthymidine (3H-TdR; specific activity 2 Ci/mmol; five hours later mice were sacrificed and the draining auricular lymph nodes were excised and pooled for each experimental group. A single-cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. Pooled LNC were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. Approximately 12 h later pellets were resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid. Incorporation of 3H-TdR was measured by 5-scintillation counting and expressed as mean disintegrations per minute (dpm) per node for each experimental group. Increases in 3H-TdR incorporation relative to vehicle-treated controls were derived for each experimental group and recorded as stimulation indices.

Laboratories C and D: standard protocol for the LLNA described already, with the exception that lymph nodes pooled for individual mice, rather than from experimental groups, were analyzed. Five days following the initiation of treatment all mice were injected intravenously via the tait vein with 250 pl of PBS containing 20 pCi of 3H-TdR (specific activity 5.0 or 6.7 Ci/mmol. Five hours later mice were sacrificed and the draining auricular lymph nodes were excised and pooled for each individual mouse. Single-cell suspensions of LNC were prepared by gentle mechanical disaggregation through a nylon mesh filter (100 pm pore size). Cell suspensions were washed twice with an excess of PBS and precipitated with 5% TCA at 4°C. Further processing was conducted as described earlier. Results were recorded as dpm per mouse. Stimulation indices for each experimental group were calculated as the mean increase in 3H-TdR incorporation relative to the mean value obtained with vehicle-treated controls.

Laboratory E employed a further modification of the standard protocol that comprised not only analysis of Iymph nodes pooled for individual mice (as per modified protocol 1 just given), but also the use of [125l]iododeoxyuridine ('251-UdR; specific activity 2000 Ci/mmol) in place of 3H-TdR. Exposure of groups of mice (n 5) was performed as already described. Five days following the initiation of treatment all mice were injected intravenously via the tait vein with 250 pl of PBS containing 2 pCi of 1251-UdR and 10-5 M fluorodeoxyuridine. Five hours later mice were sacrificed and the draining auricular Iymph nodes were excised and pooled for each individual mouse. Single-cell suspensions of LNC were prepared by gentle mechanical disaggregation using the frosted ends of two glass slides. The cells were washed once with Hanks balanced sait solution, once with PBS, and precipitated with 5% TCA at 4°C for 18 h. Samples were centrifuged, resuspended in 1 ml of 5% TCA, and transferred to a gamma counting tube. Sample tubes were counted using an LKB-Wallac 1282 universal gamma counter. Incorporation of 1251-UdR was measured as dpm per mouse. Stimulation indices for each experimental group were calculated as the mean increase in 1251-UdR incorporation relative to the mean value obtained with vehicle-treated controls.
Positive control substance(s):
other: It was an interlaboratory assessment for LLNA with several substances, tehrefore no positive control was used since the substances tested were chosen because of their sensitising potential
Statistics:
Yes (different tests were used included Dunnett's t-test.

Results and discussion

Positive control results:
not applicable

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Value:
< 0.5
Remarks on result:
other:
Remarks:
Stimulation indices were greater than 3, and for this reason it is impossible to derive mathematically EC3 values.
Parameter:
SI
Value:
14.6 - 24.4
Test group / Remarks:
0.5%
Parameter:
SI
Value:
17.2 - 22.8
Test group / Remarks:
1%
Parameter:
SI
Value:
18.1 - 33.7
Test group / Remarks:
2.5%
Parameter:
SI
Value:
20.2 - 31.4
Test group / Remarks:
5%
Parameter:
SI
Value:
18.6 - 26.5
Test group / Remarks:
10%
Cellular proliferation data / Observations:
All concentrations elicited a stat. significant increase in SI in the 3 laboratories (lab C, D, E). Even at lowest concentration tested (0.5 %).

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Benzoyl peroxide provoked very vigorous sensitising responses at all test concentration.
Executive summary:

The delayed contact hypersensitivity of dibenzoyl peroxide was evaluated in mice according to a method similar to the OECD N° 429 Guideline (Local Lymph Node Assay) with or without minor modifications. The study consisted into an interlaboratory envaluation of the LLNA with several substances, included dibenzoyl peroxide. Five laboratories were involved.

During the induction phase, dibenzoyl peroxide, was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3) in groups of 5 female CBA/Ca mice. After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6) or 125 Iododesoxyuridine (depending on the laboratories). The obtained values were used to calculate stimulation indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

A dose-related increase in the SI was recorded for all the concentrations tested and in all laboratories.

Under these experimental conditions, dibenzoyl peroxide induced delayed contact hypersensitivity in the murine Local Lymph Node Assay and is considered as sensitizing.