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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4100 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
while LLNA method was already available, OECD 406 was still an acceptable method at the time.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Chemical name: 2,5-dimethyl-2,5-di(t-butylperoxy)hexane
Purity: 84.8%
Lot No.: 0108513259
Physical Description: Clear pale yellow liquid
Storage Conditions: Room temperature in the dark
Expiration Date: 14 November 2003, allocated by NOTOX



In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Source: Charles River Deutschland, Kisslegg, Germany
Age: young adult animals (approx. 4 weeks old)
Identification: ear tattoo

Group housing of maximally 5 animals per labelled cage containing purified sawdust as bedding material. The acclimatisation period was at least 5 days before the start of treatment.

A controlled environment was maintained in the room with optimal conditions considered as being aproximately 15 air changes perhour, a temperature of 21 +/- 3 °C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.

Free access to standard ginea pig diet. Certificates of analysis were examined and retained in the NOTOX archives. Pressed hay was provided twice a week. Free access to tap water. Certificates of quarterly analysis for tap water were examined and retained in the NOTOX archives.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
50%
Challengeopen allclose all
Route:
epicutaneous, semiocclusive
Vehicle:
corn oil
Concentration / amount:
50%
No. of animals per dose:
10 animals per sex in the test group and 5 per sex in the control group.
Details on study design:
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 systemically 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 four animals selected were between 4 and 9 weeks old. No body weights were determined.

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 using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure

Induction - Experimental animals:

Day 1: The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made in this area: (A), a 1:1 w/w mixture of Freunds' complete adjuvant withwater for injection; (B), the test substance at a 50% concentration; (C) a 1:1 w/w mixtureof the undiluted test substance and Freunds' complete adjuvant.

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

Day 8: The area between the injection sites was tereated with 0.5 ml of a 100% test substance concentration. The dressing was removed after 48 hours exposure, the the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

Challenge:
Day 22: one flank of all animals was clipped and treated by epidermal application of a 50% test substance concentration and the vehicle (0.1 ml each) using patch test plasters. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

MAIN STUDY

Initially, five control and ten experimental animals were treated. Since the animals declined in health due to unknown cause during the study, the results obtained so far were considered to be invalid and the study was intermediately terminated. A new main study was initiated with a new set of animals.
INDUCTION - Experimental animals
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 Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 50% concentration.
C) A 1:1 w/w mixture of the undiluted test substance and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8 The area between the injection sites was treated with 0.5 ml of a 100% 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 using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.
INDUCTION - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.
CHALLENGE - All animals
Day 22 One flank of all animals was clipped and treated by epidermal application of a 50% test
substance concentration and the vehicle (0.1 ml each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). 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 using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
Challenge controls:
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.
Positive control substance(s):
yes
Remarks:
Hexylcinnamaldehyde historical control

Results and discussion

Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. The skin reactions observed in five expierimental animals in response to the 20% test substance

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none observed
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. Clinical observations: none observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none observed
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. Clinical observations: none observed.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
There was no evidence that the test article had caused skin hypersensitivity in the guinea pig, since no responses were obsered in the experimental animals in the challenge phase. This result indicates a sensitisation rate of 0 per cent.
Executive summary:

Assessment for Contact Hypersensitivity to CAS# 1068 -27 -5 in the Albino Guinea Pig(Maximisation Test).

The study was carried out based on the guidelines described in: EC Commission Directive 96/54/EC, Part B.6, "Skin Sensitisation", OECD No. 406, "Skin Sensitisation", EPA OPPTS870.2600 "Skin Sensitisation", August 1998 and JMAFF: Japanese Test Guidelines (draft), July 2000 and based on the method described by Magnusson and Kligman, "Allergic ContactDermatitis in the Guinea Pig - Identification of Contact Allergens".

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 50% concentration and epidermally exposed to a 100% concentration. Five control animals weresimilarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were challenged with a 50% testsubstance concentration and the vehicle. No skin reactions were evident after the challenge exposure in the experimental and control animals.

There was no evidence that the test article had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in the challengephase. This result indicates a sensitisation rate of 0 per cent.

Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC),TRIGONOX 145-E85 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.