Di-tert-butyl α,α,α',α'-tetramethyl-(p-phenylenedimethylene) diperoxide

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2009-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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

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)

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.

Parameter:

SI

Remarks on result:

other: Group 2 - test item 2.5% : 0.38 Group 3 - test item 5% : 0.40 Group 4 - test item 10% : 0.78 Group 5 - test item 25% : 1.87 Group 6 - test item 50% : 1.38

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There is no data on 1, (3) 4-bis(tert-butylperoxyisopropyl)benzene. A read across approach is proposed with 1,4-bis(tert-butylperoxyisopropyl)benzene.

1,(3) 4-bis(tert-butylperoxyisopropyl)benzene did not induce delayed contact hypersensitivity in a well conducted murine Local Lymph Node Assay (LLNA) (Klimisch 1 study).

The test item was non-irritant in the preliminary test, therefore the highest concentration retained for the main test was the maximal practicable concentration (50%).Neither mortality nor clinical signs were observed during the study.Dryness of the skin was noted on day 6 in 3/4 and 2/4 females given 25 and 50%, respectively but no notable increase in ear thickness was observed in the animals of the treated groups.

No notable lymphoproliferation was noted at any tested concentration.

Migrated from Short description of key information:
From the read across with 1, (3) 4-bis(tert-butylperoxyisopropyl)benzene: 1,4 bis(tert butylperoxyisopropyl)benzene does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:
Key study, Klimisch 1.

Justification for classification or non-classification

According to regulation (EC) 1272/2008 and according to the directive 67/548/EEC: 1,4-bis(tert-butylperoxyisopropyl)benzene is not classified for skin sensitisation.