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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:
From 11 May to 18 June 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Adopted on 31st July 1992
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study completion (1994), the LLNA OECD test method was not adopted.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Test material identification: ST 02 C 94
Description: colourless liquid
Date received; 25th November 1994
Specific details on test material used for the study:
Storage conditions: refrigerator in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D.Hall, Darley Oaks, Burton-on-Trent, Staffordshire - U.K.
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 250-300 g
- Housing: in groups of up to 5 in stainless steel cages and identified by the number of the cage to which they were allocated and within each cage by ear tattoo
- Diet (e.g. ad libitum): ad libitum (pelleted diet, SQC FDI guinea pig diet with added vitamin C (Special Diets Services, Witham, Essex)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: preliminary study: 5 days, main study: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 34-62
- Air changes (per hr): air conditionned
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
10%
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
Day 8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
Day 15
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 test animals
10 control animals
Details on study design:
RANGE FINDING TEST:
- Topical induction and challenge: was performed using 4 animals that were in the weight range 396-423g and which had been previously treated with 1:1 FCA/water as described above. The concentrations used were undiluted test article and 80%, 40% and 20% v/v concentrations of the test article in acetone. The results of this preliminary study indicated that undiluted test article did not cause irritation and was therefore selected for use as the induction and challenge concentration in the main study.
- Intradermal induction: From the results of a range finding study using one animal, it was considered that a 10% v/v concentration of the test article in light liquid paraffin could be used intradermally without provoking an unacceptable irritant response.

MAIN STUDY
A. INDUCTION EXPOSURE
Thirty healthy animals with the weight range 315 - 354 g were selected for the study and randomly allocated to groups of 20 test and 10 control animals using a stratified bodyweight procedure. The dorsal area between the shoulders of each animal was clipped free of fur and three pairs of intradermal injections made within this area. Each injection was 0.1 mL and each pair of injections consisted of:
Test Group:
1) 50% v/v Freund's Complete Adjuvant (PCA) emulsified in water.
2) 10% v/v concentration of the test article in light liquid paraffin.
3) Test article mixed with FCA and then emulsified 1:1 with water to give a final 10% v/v concentration of test article.
Control Group:
1) 50% v/v Freund's Complete Adjuvant emulsified in water
2) Light liquid paraffin.
3) Light liquid paraffin mixed with FCA and then emulsified 1:1 with water to give a final light liquid paraffin concentration of 50% v/v.

Six days after the intradermal induction, the area surrounding the injection sites of all animals was clipped free of fur. As the results of the topical rangefinder indicated that undiluted test article was not irritant, the test area of all test and control animals was then treated topicaIly with 0.5mL of 10% sodium lauryl sulphate in light liquid paraffin in order to produce local irritation.
The foIlowing day, the injection process was boosted by topical application of undiluted test article over the injection sites of the test animals. Patches of Whatman No.3 filter paper, 4 x 2cm, were saturated with the test article and placed over the injection sites of animals in the test group. A strip of 5cm wide "Blendcrm" surgical tape was placed over each patch to act as an occlusive barrier and the whole assembly held in position by wrapping the animals with a length of 5cm wide "Elastoplast" elastic adhesive bandage.
Animals of the control group were similarly treated, the patch of filter paper being saturated with acetone. The patches and dressings were removed after 48 hours. Twenty four hours after administration of the intradermal induction and again 24 hours after removal of topical induction patches, all animals were examined for signs of irritation in the treated area.

B. CHALLENGE EXPOSURE
Fourteen days after the topical induction application, the fur was clipped from the back and flanks of all animals. Patches of Whatman No.3 filter paper, 2cm x 2cm, were saturated with undiluted test article and placed on the left flank of all animals. Blank 'sham' patches were similarly applied to the right flank as no vehicle had been used. The patches were occluded and secured using the method previously described for topical application. After a contact period of 24 hours the patches and dressings were removed. Twenty four and 48 hours after removing the dressings the animals were examined and any reaction scored
Challenge controls:
See "Details on study design"
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole (MBT)

Results and discussion

Positive control results:
Mercaptobenzothiazole (MBT) was used as a positive control as this is known to be a sensitiser. This study took place in February-March 1994. The material was administered as a 10% concentration in light liquid paraffin for the intradermal injections and following pretreatment with 10% sodium lauryl sulphate, was used at a 50% concentration in acetone for the topical induction. Challenge was conducted at a concentration of 50% mercaptobenzothiazole in acetone. All ten test animals exhibited positive responses to 50% MBL at the 24 and/or 48 hour examination giving a response incidence of 100% . No responses were seen following challenge with the vehicle.
None of the control animals responded to challenge with either 50% MBT or the vehicle.
These results confirm the ability of the Magnusson Kligman method used at Toxicol Laboratories Ltd to detect sensitising agents.the laboratory has the capability to identify positive dermal sensitizers. (For details, see "background attached material").

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10 %
No. with + reactions:
10
Total no. in group:
100
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

none

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material is not classified as skin sensitiser under the test conditions
Executive summary:

Introduction. This study was performed to assess the potential of the test article, ST 02 C 94, to cause a delayed dermal (Type IV) hypersensitivity response in the guinea pig. The methods used were in accordance with Eu Method B.6, as described in Document L 383 A of 29th December 1992, an Annex to Commission Directive 92/69/EEC of 31st July 1992.

 

Methods. Thirty female albino guinea-pigs were divided up into a group of 10 control and a group of 20 test animals and used to determine the sensitization potential of the test article.

From the results of a range finding study using one animal, it was considered that a 10% v/v concentration of the test article in light liquid paraffin could be used intradermally without provoking an unacceptable irritant response.

A range-finding study in four animals, using undiluted test article and 80%, 40% and 20% concentrations of test article in acetone, applied topically, indicated that undiluted test article was non irritant and could be used for the topical induction and at the challenge phase of the main study.

Treatment of the 30 main study animals was as follows:

 

Day and Procedure

Test animals

Control animals

1. Intradermal induction

Injection between the shoulders of :

(i) A 50% emulsion of FCA and water for injection.

(ii) 10% concentration of the test article in light liquid paraffin.

(iii) 10% concentration of the test article inFCA/water.

Injection between the shoulders of :

(i) a 50% emulsion of FCA/water.

(ii) Light liquid paraffin.

(iii) Light liquid paraffin in FCA/water

8. Topical induction

Undiluted test article

Acetone

15. Topical challenge

Undiluted test article applied to the left flank of all test and control animals.

 

Following the application of undiluted test article at challenge, none of the test animals responded at either the 24 or 48 hour observation, giving a response incidence of 0%.

Similarly, no responses were seen in the control animals following challenge with the test article.

 

Conclusions. Under the conditions of this study, the test article ST 02 C 94 showed no evidence of causing a delayed dermal (Type IV) hypersensitivity response and therefore is not classified according to the criteria described in the Regulation (EC) No. 1272/2008 (CLP).