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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:
from 06 November 2009 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

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
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:
- Name of test material (as cited in study report): 1,3(4)-bis(tert-butylperoxyisopropyl)benzene
- Physical state: yellowish solid
- Peroxide content: 97.6%
- Purity test date: 28 July 2009
- Lot/batch No.: 307090713
- Expiration date of the lot/batch: July 2010
- Storage conditions of test material: at room temperature (below 30°C) and protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 22.4 +/- 1.2 g
- Housing: housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust.
- Diet (e.g. ad libitum): conventional laboratory diet (SSNIFF R/M-H pelleted maintenance diet)
- Water (e.g. ad libitum): tap water filtered using a 0.22 micron filter
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: From 18 November 2009 to 30 November 2009

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5, 10, 25 and 50%
No. of animals per dose:
4 animals.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%, after 15 minutes stirring.
- Irritation: non-irritant, whatever the concentration
- Lymph node proliferation response: incorporation of tritiated methyl thymidine

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: SI >= 3 with exclusion of excessive irritation

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
HCA at the concentration of 25%: a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 3.62) were noted. The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.38
Test group / Remarks:
Group 2 (test item 2.5%)
Parameter:
SI
Value:
0.4
Test group / Remarks:
Group 3 (test item 5%)
Parameter:
SI
Value:
0.78
Test group / Remarks:
Group 4 (test item 10%)
Parameter:
SI
Value:
1.87
Test group / Remarks:
Group 5 (test item 25%)
Parameter:
SI
Value:
1.38
Test group / Remarks:
Group 6 (test item 50%)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group 1 - vehicle : 1763.28 (DPM per group) and 220.41 (DPM per node) Group 2 - test item 2.5% : 678.37 (DPM per group) and 84.80 (DPM per node) Group 3 - test item 5% : 705.51 (DPM per group) and 88.19 (DPM per node) Group 4 - test item 10% : 1370.06 (DPM per group) and 171.26 (DPM per node) Group 5 - test item 25% : 3293.61 (DPM per group) and 411.70 (DPM per node) Group 6 - test item 50% : 2438.58 (DPM per group) and 304.82 (DPM per node)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item 1,3(4) bis(tert butylperoxyisopropyl)benzene (batch No. 307090713) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The potential of the test item, 1,3(4)-bis(tert-butylperoxyisopropyl)benzene, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA) was evaluated. Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

. five treated groups of four animals receiving the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene at the concentration of 2.5, 5, 10, 25 or 50% in a mixture acetone/olive oil (4/1, v/v) (AOO),

. one negative control group of four animals receiving the vehicle (AOO),

. one positivie control group of four animals receiving the reference item, alpha-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in AOO.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). 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). 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.

Results

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v).

A solution was obtained at the maximum tested concentration of 50%, after 15 minutes stirring.

Consequently, the concentrations selected for the preliminary test were 5, 10, 25 and 50%.

Since the test item was non-irritant in the preiminary test, the highest concentration retained for the main test was the maximal practicable concentration (50%).

Systemic clinical signs and mortality

Neither mortality nor clinical signs were observed during the study.

Local irritation

Dryness of the skin was noted on day 6 in 3/4 and 2/4 females given 25 and 50%, respectively.

No notable increase in ear thickness was observed in the animals of the treated groups.

Proliferation assay

A significant lymphoproliferation was noted with the positive control group given HCA at 25%. The study was therefore considered valid.

No notable lymphoproliferation was noted at any tested concentration.

The results are presented in the following table:

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

2.5

non-irritant

0.38

Test item

5

non-irritant

0.40

Test item

10

non-irritant

0.78

Test item

25

non-irritant

1.87

Test item

50

slightly irritant

1.38

HCA

25

-

3.62

Conclusion

Under the experimental conditions of this study, the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene (batch No. 307090713) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.