[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-07 to 2012-02-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 24 April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis-[2-(isopropoxycarbonyloxy)-benzoyl]-peroxide

Cas Number:

1310672-92-4

Molecular formula:

C22H20O10

IUPAC Name:

Bis-[2-(isopropoxycarbonyloxy)-benzoyl]-peroxide

Test material form:

semi-solid (amorphous): gel

Details on test material:

- CAS-No: 1310672-92-4
- Molar mass: 446.41 g/mol
- Molecular formula: C22H22O10
- Appearance: opaque white gel

Specific details on test material used for the study:

Test item 1: CD08479 2.5% gel
Batch No.: 11.01474 (2.5%)
Density: 1.0270 (2.5%)
Appearance/description: Opaque white gel
Storage conditions: Store below 25 °C, do not Freeze or refrigerate
Expiry date: December 02, 2011
Labeling color code: Yellow
Purity:103.5% I.C.

Test item 2: CD08479 5% gel
Batch No.: 11.01475
Density: 1.0340
Appearance/description: Opaque white gel
Storage conditions: Store below 25 °C, do not Freeze or refrigerate
Expiry date: December 02, 2011
Labeling color code: Blue
Purity: 103.6% LC

Test item 3: CD08479 10% gel
Batch No.: 11.01476
Density: 1.0453
Appearance/description: Opaque white gel
Storage conditions: Store below 25 °C, do not Freeze or refrigerate
Expiry date: December 02, 2011
Labeling color code: Red
Purity: 103.2% LC

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JRj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (Le Genest-St-Isle, France)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks old
- Weight at study initiation: 19.1 to 23.6 g
- Housing: Animals were housed individually in Makrolon Type I cages. The cages were covered by a stainless-steel lid. Each cage contained dust-free sawdust.
- Diet (e.g. ad libitum): Ad libitum, standard pelleted complete diet (Diet reference A04C, SAFE, Augy, France), ad libitum
- Water (e.g. ad libitum): Ad libitum, filtered mains drinking water (0.22 μm)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50 and 100 %
See Table 1 under "Any other information on materials and methods incl. tables"

No. of animals per dose:

8 females per dose group

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Before allocation, all animals were weighed. At the discretion of the Study Director, animals with abnormalities likely to be prejudicial to the study were excluded from allocation and were replaced by spare animals (if feasible). The required number of animals was allocated to the different dosage groups according to a computerized randomization (CSOR module of Provantis® software) that ensures a similar body weight distribution among group.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dilutions and positive control preparations
1. Preparation of the 100%, 50% and 25% concentration-series: The three concentration-series were manufactured (under GMP environment), packaged and supplied by the Pharmaceutical Unit, Galderma R &D, Sophia Antipolis, France, to the Toxicological Team before the beginning of the study.
2. Preparation of the positive control formulations: The LLNA positive control formulations were prepared freshly on each day of treatment (i.e., Days 1 to 3) by the toxicological team using the appropriate amount of vehicle to obtain the concentration of (approximately) HCA 25 % in Acetone/olive oil (4:1, v/v).

Test item administration: Topical administration of 25 μL of vehicle, placebo, drug product dilution or positive control solution on the dorsum of both ears

OBSERVATIONS:
1.Morbidity/ Mortality
All animals were observed at least twice daily during both pre-dosing and the study periods for mortality and morbidity. The circumstances of any deaths or moribund sacrifice were recorded.
2. Clinical signs
The animals were regularly observed at least once during the pre-dosing period and then at least once daily throughout the study to detect any behavioral and physical abnormalities. The nature, onset, severity, reversibility and duration of clinical signs were recorded.
3. Cutaneous reactions
Observation of the treatment-area (ear) were performed daily, 6 hours ± 1 hour after treatment and on Day 6 just before intravenous injection of 3[H] MT.
4. Body weights
The body weights were recorded once during the pre-period period, before allocation and on the first day of dosing (before dosing) and Day 6 (before intravenous injection of 3[H] MT).

For preparation of the concentration series and experimental design see Tables 1 and 2 in box "Any other information on materials & methods incl. tables".

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was performed using the Provantis 7.0 (validated) system. For each group, the mean and standard deviation were calculated.
A statistical evaluation was performed on the incorporation of the 3[H] MT incorporation (DPM values) results. After a log transformation, an ANOVA (homogeneity of means) and a Dunnett test (comparison for each group against the vehicle group) were performed.

Results and discussion

Positive control results:

The positive-control substance exceeded (5.69) the stimulation index of 3 confirming the reliability of the test system. See Table 3 in box "Any other information on results incl. tables".

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

mean of 8 animals

Value:

0.51

Variability:

SD = 0.34

Test group / Remarks:

2.5% test item

Results 2

Key result

Parameter:

SI

Remarks:

mean of 8 animals

Value:

0.57

Variability:

SD = 0.41

Test group / Remarks:

5% test item

Results 3

Key result

Parameter:

SI

Remarks:

mean of 8 animals

Value:

0.52

Variability:

SD = 0.47

Test group / Remarks:

10% test item

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
For groups 2 to 6, an individual Stimulation Index (SI) was calculated for each animal, as the ratio between the individual DPM of treated animals versus the mean DPM of the vehicle control group (group 1). Then for each of the groups 2 to 6, a mean SI was calculated.
Stimulation Index (mean) of 0.51, 0.57 and 0.52 was calculated in response to CD08479 2.5%, 5% and 10% formulated in gel, respectively.
Stimulation Index (mean) of 0.25 was calculated for the placebo group (group 2), statistically significant when compared to group 1 (negative control group) following Dunnett’s test.
Stimulation Index (mean) of 5.69 was calculated for the positive control group (HCA 25%), statistically significant when compared to group 1 (negative control group) following Dunnett’s test, which demonstrated the performance and the reliability of the assay (SI ≥ 3).

CLINICAL OBSERVATIONS:
No abnormal clinical signs were observed in any treated-groups during the in-life part of this study.

CUTANEOUS REACTIONS:
No cutaneous reaction (erythema or edema) was observed in groups 1 to 5.
In group 6, treated with positive control HCA at 25%, discrete erythema was observed on Days 1 to 3 in 5/8 females, on Days 2 and 3 in 2/8 females and discrete to slight erythema was observed on Days 1 to 3 in 1/8 female. On Day 6, these reactions were no longer visible.

EAR SWELLING MEASUREMENT:
There was no increase of ear thickness observed between Day 1 (before treatment) and Day 6 (before intravenous injection of 3[H] MT) for any group

BODY WEIGHTS
No effect on the body weight was observed in any treated-group.

Any other information on results incl. tables

Chemical analyses

Analytical controls of CD08479 2.5%, 5% and 10% formulated in gel indicated that homogeneity and target concentrations were attained, within target ranges (100.00 ±10%; analytical method for active ingredient assay Reference RDS.03.ATP.03616.R01), and that the pharmaceutical dosage forms were stable for at least 3 weeks, covering the study duration.

Table 3: Results on Simulation Index

	Concentration of test item in applied formulation (%)				Positive control HCA
	Placebo	2.5	5	10	25%
Group number	2	3	4	5	6
N=number of date available	7	8	6	7	6
LLNA Concentration series (%)	NA	25	50	100	NA
SI (Mean)	0.25	0.51*	0.57	0.52	5.69*
Standard deviation	0.08	0.34	0.41	0.47	5.10

*: statistically significant when compared to vehicle group (5% significant level).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of this study, the test item CD08479 formulated in gel is not considered to be a skin sensitiser

Executive summary:

In a dermal sensitization study conducted according to OECD 429 with the test item CD08479 formulated in gel and dissolved in AOO (4:1 (v/v) acetone/olive oil, young adult female CBA/JRj mice (8 per dose group) were treated CD08479 2.5%, 5% and 10% gel or Hexyl Cinnamic Aldehyde (HCA, 25% in AOO; positive control) at a constant dosing volume of 25μL on the dorsum of each ear, for three consecutive days. Simulation index along with other parameters including morbidity and mortality, clinical signs, cutaneous reactions, ear swelling and body weight were evaluated. During the study, no mortality or abnormal effects were observed on the female mice. However, discrete to slight erythema were observed on Days 1 to 3 for animals of positive control group (HCA). Stimulation Index of 0.51, 0.57 and 0.52 was calculated in response to CD08479 2.5%, 5% and 10% formulated in gel, respectively.

Based on these results, the test item did not show any potential for skin sensitization up to the concentration of 10% and thus is categorized as a non-skin sensitizer under UN GHS Criteria.