2(3H)-Benzofuranone, 5,7-bis(1,1-dimethylethyl)-3-(4-hydroxy-3,5-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.07. - 04.09.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Remarks:

NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A valid GPMT study is available.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-770-1

EC Name:

-

Cas Number:

216698-07-6

Molecular formula:

C32 H44 O4

IUPAC Name:

2-(2-oxo-5-(1,1,3,3-tetramethylbutyl)-2,3-dihydro-1-benzofuran-3-yl)-4-(1,1,3,3-tetramethylbutyl)phenyl acetate

Details on test material:

- Physical state: White solid
- Analytical purity: 98.7%
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland.
- Age at study initiation: approx. 7 weeks old
- Weight at study initiation: individual body weights < 500 g
- Housing: Group housing of 5 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
- Diet: Free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg) (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage, Germany).
- Water: tap water, ad libitum
- Acclimation period: at least 5 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

corn oil

Induction 2

Concentration / amount:

2% test substance for Intradermal injections and 50 % for epicutaneous treatments.

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

2% test substance for Intradermal injections and 50 % for epicutaneous treatments.

No. of animals per dose:

Preliminary irritation study: 4 animals
Main test: Experimental group: 10 females; Control group: 5 females

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freund’s Complete Adjuvant with water for injection.
B) The test substance at a 2% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used In (B) and Freund’s Complete Adjuvant.

Day 3
The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7
The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8
The 10% SDS treated area between the injection sites was cleaned with water to remove residual SDS and subsequently treated with 0.5 ml of a 50 % test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal exposure were assessed for irritation.

- Control group:
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

B. CHALLENGE EXPOSURE
Day 22
One flank of all animals (control and treated group) was clipped and treated by epidermal application of a 50 % test substance concentration and the vehicle (0.15 mL each), using patch test plasters. The patches were held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.


OTHER:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main Study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated by the animals.
- For the Induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.

Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The animals selected were between 4 and 9 weeks old. No body weights were determined at termination.

Intradermal injections:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal application:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape', which were held in place with Micropore tape' and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.


POSITIVE CONTROL
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques. Prior to this study, animals of the Himalayan strain obtained from BRL Ltd, Basel, Switzerland were treated with a 5% intradermal and 10% epidermal concentration of Alpha-hexylcinnamic aldehyde (Tech. 85%) and a sensitization rate of 100 per cent was observed.

Results and discussion

Positive control results:

The skin reactions in the experimental animals observed in response to the 10% positive control substance concentration in the challenge phase were considered indicative of sensitisation, taking into account the intensity and persistence of the response in the control animals. These results lead to a sensitisation rate of 100 per cent to the 10% concentration. From these results, it was concluded that the female guinea pig of the albino Himalayan strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in a Maximisation type of test.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Results of the preliminary irritation study:

For the main study a 2% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure were selected as suitable concentrations. No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction. A 50% test substance concentration was selected for the challenge phase.

Additional Observations:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period:

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of this test, the test material was devoid of any sensitizing potential and is regarded as not sensitizing.

Executive summary:

The contact hypersensitivity of the test substance was assessed in a guinea pig maximization test with ten animals according to OECD guideline 406 and in compliance with GLP. Test substance concentrations selected for the Main study were based on the results of a preliminary irritation study. Induction was a two-stage operation. First, the scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:

A) A 1:1 w/w mixture of Freund’s Complete Adjuvant with water for injection.

B) The test substance at a 2% concentration.

C) A 1:1 w/w mixture of the test substance, at twice the concentration used In (B) and Freund’s Complete Adjuvant.

One week later, the test article at a concentration of 50% was applied to the area between the injection sites using a Metalline patch (2x3 cm) mounted on Medical tape for 48 hours. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle for 24 hours. Skin reactions were assessed 24 and 48 hours after the removal of the challenge patch. No skin reactions were evident after the challenge exposure in the experimental and control animals. There was no evidence that the test substance had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in the challenge phase. This result indicates a sensitization rate of 0 per cent. Based on these results and according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC), ODH 7800 does not have to be classified for sensitization by skin contact