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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
Study period:
22 Oct 2001 to 19 Nov 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
29 December 1992
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of the test item (as cited in report): Axol C62
- Batch No.: 14 EG 13
- Expiration date of the lot/batch: March 2002
- Purity test date: 100%
- Appearance: Fine ivory-coloured powder
- Storage condition of test material: Ambient, protected from light

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Pirbright White, HsdPoc:DH
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Postfach 11 61, 33178 Borchen, Germany
- Weight at study initiation: 597 - 701 g (males) and 468 - 592 (females)
- Housing: Before animals arrived, the study room and cages were cleaned and disinfected. During the study, the room and cages were cleaned at regular intervals. The guinea pigs were kept in collective housing up to a maximum of 5 animals per cage in a battery of cages, equipped with a paper disposal system.
- Diet: The animals received "2040 Teklad Global Guinea Pig Diet" (pelleted diet, batch no. 1-1169) produced by Harlan Teklad, Shaw's farm, Blackthorn Bicester, Oxon, OX6 OTP, England, offered ad libitum
- Water: Tap water as for human consumption was continuously available ad libitum via drinking nipples.
- Acclimation period: 26 days (range finding test), 30 days (main test)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70%
- Air changes (per hr): 16 times per hour
- Photoperiod: 12/12 with light on at 07:00 a.m.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
50% FCA
Route:
intradermal
Vehicle:
other: water with Tween 80
Concentration / amount:
5% test substance
Route:
intradermal
Vehicle:
other: water with Tween 80/FCA
Concentration / amount:
5% test substance
Route:
intradermal
Vehicle:
water
Concentration / amount:
50% FCA (Control)
Route:
intradermal
Vehicle:
other: water with Tween 80
Concentration / amount:
0% (Control)
Route:
intradermal
Vehicle:
other: water with 50% Tween 80 (v/v) in FCA
Concentration / amount:
0% (Control)
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
50% test substance
Day(s)/duration:
48 h
Adequacy of induction:
other: non-irritant substance, but pretreated with 10% SLS
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Remarks:
Control
Concentration / amount:
0% (Control)
Day(s)/duration:
48 h
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 g
Day(s)/duration:
24 h
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
(Control)
Concentration / amount:
0.5 g
Day(s)/duration:
24 h
No. of animals per dose:
10 test group and 5 control group
Details on study design:
RANGE FINDING TEST
Two animals (1 male, 1 female) were used in the range finding test. The range finding test was performed to determine the concentrations of the test article to be used in the main test. For the intradermal injection, the test article was diluted with aqua ad iniectabilia (containing 1 drop of Tween 80) and Freund's complete adjuvant (FCA; batch no. 20K8933; SIGMA, Deisenhofen) to a final concentration of 5.0 %. Two animals were employed, skin reactions being recorded 48 h after treatment.
For the dermal application, the test article was used 50 % in vaselin (batch no. 419496/144700, Fluka AG, CH-Buchs). A closed patch exposure was effected by means of an occlusive bandage using Hill-Top Chambers (Hill Top, Cincinnati, USA) and nonirritating tape Elastoplast@ (Beiersdorf AG, Hamburg), which enveloped the whole animal's trunk. Two animals were employed and skin reactions were recorded 48 h post application.

MAIN STUDY
On the basis of the results of the range finding test, the concentration of 5 % of the test article was considered to be suitable for intradermal injection and the concentration of 50 % of the test article for dermal application.

A. INDUCTION EXPOSURE
In each test animal, three pairs of intradermal injections (0.1 ml) were made in the clipped intrascapular region on either side of the spine. Seven days later, the previous injection sites were covered occlusively for 48 h with a patch carrying the test article or, in control animals, the control article. Since the 50 % test article formulation was non-irritating after dermal application in the range finding test, the skin area was reclipped and pretreated with 10 % sodium lauryl sulfate (SLS) in vaseline 24 h before application of the test article to induce a mild inflammation.

B. CHALLENGE
Both control and test animals were subjected to a challenge exposure 14 days after the second stage of the induction. The challenge test was performed on a 5 x 5 cm clipped skin area on each flank. The test article was applied to the left flank and the control article petrolatum to the right in a volume of 0.5 g using the patch technique. In each case, the duration of the exposure was 24 h under an occlusive dressing.

OBSERVATIONS
24 and 48 h after patch removal the appearance of the challenge skin sites was observed and skin responses were graded on the basis of the classification system according to draize. The animals were weighed before treatment and at the end of the study. All findings were recorded with respect to duration and severity on special report forms.
Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester (benzocaine)

Results and discussion

Positive control results:
The sensitivity of the test system and the reliability of the experimental technique is assessed at least every six months by use of "4-aminobenzoic acid ethyl ester (benzocaine)" which is known to induce skin sensitisation in guinea pigs. This test was conducted as a Magnusson & Kligman test accorcling to the OECD Guideline for Testing of Chemicals, OECD 406, 17 July 1992 and to the EEC Directive 92/69/EEC, 31 July 1992. The latest test before this study was with acceptable levels of responses to this 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
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50 %
No. with + reactions:
4
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 %
No. with + reactions:
2
Total no. in group:
10

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met