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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ISOPROPYL-1,3-DIOXANE-5,5-DICARBOXYLIC ACID DIETHYL ESTER
EC Number:
609-063-4
Cas Number:
35113-48-5
Molecular formula:
C13H22O6
IUPAC Name:
2-ISOPROPYL-1,3-DIOXANE-5,5-DICARBOXYLIC ACID DIETHYL ESTER
Test material form:
other: LIQUID

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Twenty-six male albino Dunkin Hartley guinea pigs were supplied by David Hall Limited, Burton-on-Trent, Staffordshire, UK. After an acclimatisation period of at least five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen. At the start of the main study the animals were in the weight range of 300 to 450g, and were approximately eight to twelve weeks old.
The animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes. Free access to mains tap water and food (Certified Guinea Pig Diet (Code 5026) supplied by PMI Nutrition International, Nottingham, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Intradermal induction : 5% v/v in arachis oil BP
topical induction: undiluted as supplied
topical challenge: 75% and 50% v/v in arachis oil BP
topical re-challenge: 50% and 25% v/v in arachis oil BP
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Intradermal induction : 5% v/v in arachis oil BP
topical induction: undiluted as supplied
topical challenge: 75% and 50% v/v in arachis oil BP
topical re-challenge: 50% and 25% v/v in arachis oil BP
No. of animals per dose:
10 for test group
5 for control
Details on study design:
Selection of Concentrations for Main Study (Sighting Tests)
for Intradermal Induction : Intradermal injections (0.1 ml/injection site) were made on the clipped shoulder of two guinea pigs, using concentrations of 1% and 5% v/v in arachis oil BP. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours and 7 days after injection. The highest concentration that caused only mild to moderate skin irritation was selected for the intradermal induction stage of the main study.

for Topical Induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant fourteen days earlier) were treated with the undiluted test material and three preparations of the test material (75%,50% and 25% v/v in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

for Topical Challenge
The undiluted test material and three preparations of the test material (75%, 25% and 10% v/v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Induction of the Test Animals:
Shortly before treatment on Day 0 the hair was removed from an area on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 ml each) was made on each side :
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 5% v/v formulation of the test material in arachis oil BP
c) a 5% v/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. injection site b) was evaluated
On Day 7 the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the undiluted test material. This occlusive dressing was kept in place for 48 hours.The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches.

Challenge
Shortly before treatment on Day 21, an area on both flanks of each animal, was clipped free of hair with veterinary clippers.
A square filter paper patch, loaded with the test material formulation at the maximum non-irritant concentration (75% v/v in arachis oil BP) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% v/v in arachis oil BP was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified

Rechallenge
Fourteen days after the original challenge the test group animals were re-challenged on previously untreated areas of skin using the test material at concentrations of 50% and 25% v/v in arachis oil BP. An additional control group of five animals was similarly treated. The bodyweight of each animal was recorded at the start and end of the rechallenge phase of the study. These control animals had not previously been exposed to the test material but had received intradermal injections of Freund’s Complete Adjuvant and had formed part of a previous study on an unrelated test material. Approximately 24 and 48 hours after rechallenge dressing removal, the degree of erythema and oedema was quantified.

Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
no clinical sign
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: no clinical sign.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
no clinical sign
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: no clinical sign.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
no clinical sign
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: no clinical sign.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
no clinical sign
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: no clinical sign.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
no clinical sign
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: no clinical sign.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
no clinical sign
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: no clinical sign.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
severe desquamation prevents evaluation of erythema
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: severe desquamation prevents evaluation of erythema.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
desquamation
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: desquamation.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test material produced a 80% sensitisation rate following a rechallenge and was classified as a strong sensitiser to guinea pig skin.
Executive summary:

The study was performed to assess the contact sensitisation potential of the test material in the albino guinea pig. The method was designed to meet the requirements of the following:

§ OECD Guidelines for the Testing of Chemicals No. 406 “Skin Sensitisation” (adopted 17 July 1992)

§ Commission Directive 96/54/EC Method B6 Acute Toxicity (Skin Sensitisation)

Method. Ten test and five control animals were used for the study. Two phases were involved in the main study; an induction of a response by intradermal injection and topical application and a topical challenge of that response.

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:

Intradermal Induction                      :          5% v/v in arachis oil BP

Topical Induction                  :          undiluted as supplied

Topical Challenge                 :          75% and 50% v/v in arachis oil BP

A topical rechallenge was performed at concentrations of 50% and 25% v/v in arachis oil BP.

Conclusion. Under the conditions of the test, the test material produced a 80% (8/10) sensitisation rate following a rechallenge and was classified as a strong sensitiser to guinea pig skin.

The test material was classified as a sensitiser. The test material requires labelling with the symbol “Xi”, indication of danger ‘irritant’ and the risk phrase R 43 “May Cause Sensitisation by Skin Contact” according to EU labelling regulations Commission Directive 93/21/EEC.