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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
Study period:
2012-03-21 to 2012-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Peroxan BCC; Bis(4-tert-butylcyclohexyl)peroxydicarbonate
- Substance type: organic peroxide
- Physical state: white solid
- Analytical purity: 98.5% (Jodometrie Hauseigene Methode 04Mo114 A)
- Lot/batch No.: 1092801
- Expiration date of the lot/batch: 20 April 2012
- Storage condition of test material: between -15°C and -35 °C, protected from light, 2-8°C during the study

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, 33178 Borchen, Germany
- Age at study initiation: 7 – 8 weeks
- Weight at study initiation: absolute body weights 20 - 21 g
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH value of approx. 2.8
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (w/v)
No. of animals per dose:
20 animals were used for the main test: 5 mice per group; 3 mice in the preliminary test; 3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Due to the solubility properties of the test item the vehicle AOO (4:1 (v/v) acetone / olive oil) was used. The maximum technically applicable concentration of the test item was found to be 25% in AOO (4:1 (v/v) acetone / olive oil)
- Irritation: two animals were treated by topical application with the test item on three consecutive days at a concentration of 25% (diluted with AOO (4:1 (v/v) acetone / olive oil) to the entire dorsal surface of each ear. Neither signs of systemic toxicity nor signs of irritation at the application site could be
detected in any animal.
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay - LLNA
- Criteria used to consider a positive response: A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
- topical application of 25 μL of the selected solution to the entire dorsal surface of each ear
- topical applications were performed once daily over three consecutive days
- Administration of 3H-Methyl Thymidine: Five days after the first topical application all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250μL of 3H-methyl thymidine, diluted to a working concentration of 80μCi/mL
- 5 h after injection all mice were sacrificed
- excision of the auricular lymp nodes and collection in PBS; preparation of single cell suspension
- the cells were pelleted and washed twice with PBS
- resuspension of the pellet in 1 ml 5 % TCA and 7 ml scintillation fluid
- Determination of incorporated 3H -Methyl Thymidine with a ß-counter and expression number of disintegrations per minute (DPM) (see "any other information on materials and methods incl. tables)
Positive control substance(s):
other: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot 060M0186V6) 1%
Statistics:
no data

Results and discussion

Positive control results:
The mean stimulation index of the positive control subtance Phenylenediamine was determined to be 10.9.
DPM (mean): 13469.6

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
All tested concentrations exceeded the stimulation index of 3. The stimulation index at a concentration of 6.25% was 7.8 The stimulation index at a concentration of 12.5% was 10.4 The stimulation index at a concentration of 25% was 9.0 The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were above 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM mean (BCC 6.25 %): 16291.8 DPM mean (BCC 12.5 %): 21821.2 DPM mean (BCC 25 %): 18895.6

Any other information on results incl. tables

All animals survived throughout the test period without showing any clinical signs. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Peroxan BCC is considered to be sensitising. Since the EC3 value could not be calculated (stimulation indices > 3) the test item could not be classified into the sub-categories 1A and 1B.The test item is classified into Category 1.
Executive summary:

In a dermal sensitization study according to OECD guideline 429 (Local lymph node assay) with Peroxan BCC (6.25, 12.5 and 25 %) in 4:1 (v/v) acetone / olive oil, 20 female 7 – 8 weeks old CBA/CaOlaHsD mice were tested using the LLNA. As positive control substance P-Phenylenediamine was used. All animals survived throughout the test period without showing any clinical signs. 

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were above 3. Therefore the test item could not be classified into any of the sub-categories (category 1A or B) according to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System).
The test item is classified into Category 1 and obligatory labelling requirement for skin sensitisation.