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Description of key information

Several skin sensitization tests have been performed with benzoyl peroxide according to standard protocols, with a good concordance (Kimber et al., 1998; Basketter et al., 1996; Haustein et al., 1985).

In a LLNA performed according to OECD guideline N°429 (and chosen as key study), the delayed contact hypersensitivity of dibenzoyl peroxide was evaluated in mice in different laboratories (Kimber et al., 1998). During the induction phase, dibenzoyl peroxide, was applied over the ears 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). Stimulation indices (SI) of 3 (EC3 values) or greater was considered to have skin sensitizing activity. Benzoyl peroxide elicited strong LLNA response in all trials by 5 laboratories. Even at the lowest concentration tested (0.5 %), benzoyl peroxide induced stimulation indices of considerably greater than 3, and for this reason it proved to be impossible to derive EC3 values mathematically. Under these experimental conditions, dibenzoyl peroxide induced delayed contact hypersensitivity in the murine Local Lymph Node Assay and is considered as sensitizing.

This result is supported by results obtained with human maximisation test (Leyden, 1977) and many case of skin sensitisation in humans

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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.
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)
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
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.
Positive control results:
not applicable
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 %).
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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Compilation of historical results of Buehler tests. Original data not available for evaluation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Route:
epicutaneous, occlusive
Vehicle:
no data
Concentration / amount:
10 %
Route:
epicutaneous, occlusive
Vehicle:
no data
Concentration / amount:
10 %
No. of animals per dose:
20 test animals in the test group, 10 naive control animals for challenge and 10 separate naive control animals for rechallenge.
Details on study design:
A. INDUCTION EXPOSURE
- Site: on the dorsal surface of the test animals
- Frequency of applications: one patch per week for three weeks
- Duration: 6 hours
- Concentration: 10 %

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Site: on naive skin site
- Concentration: 10%
- A rechallenge may have been performed but it is not reported

Reactions were graded for erythema 24 hours and 48 hours after pach removal (with depilation 2-3 hours prior to the 24 hours scoring point).

Positive control substance(s):
yes
Remarks:
several substances (because it was an interlaboratory assessment of the Buehler test)
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
8
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 8.0. Total no. in groups: 20.0.
42 percent of the test group was judged as having positive
allergic reactions. It would be classified as skin sensitizer
according to the threshold defined by EC (> 15 %).
Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under these experimental conditions, Dibenzoyl peroxide was considered as sensitizing in the Buehler Test.
Executive summary:

The delayed contact hypersensivity of dibenzoyl peroxide was evaluated in Guinea pigs according to the Buehler test. It was an interlaboratory assessment of the Buehler test method, invloving many chemicals, included dibenzoyl peroxide. The induction phase and the challenge phase were performed with 10 % dibenzoyl peroxide (purity not reported).

42 percent of the test group was judged as having positive allergic reactions.  Under these experimental conditions, Dibenzoyl peroxide was considered as sensitizing in the Buehler Test and  would be classified as skin sensitizer
according to the threshold defined by the CLP regulation and the directive 67/548/EC(> 15 %).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Dibenzoyl peroxide is classified as skin sensitiser cat 1 according to Annex VI of EU Regulation (EC) N0. 1272/2008 (CLP).