Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2001-01-26 to 2001-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall (UK) Ltd, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 300 to 324g
- Fasting period before study: no
- Housing: singly or in pairs in solid-floor polypropylene cages.
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): 15
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Main study:
-Induction:
intradermal injection: 1% v/v in arachis oil.
topical application: Undiluted.
-Challenge:
100% and 75% v/v in arachis oil. Rechallenge at 25% and 10% v/v in arachis oil.



Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Main study:
-Induction:
intradermal injection: 1% v/v in arachis oil.
topical application: Undiluted.
-Challenge:
100% and 75% v/v in arachis oil. Rechallenge at 25% and 10% v/v in arachis oil.



No. of animals per dose:
In the preliminary study: 6 animals.
In the main study, test group of 10 animals and a control group of 5.
Details on study design:
RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study

-Intradermal exposure:
Hair over the scapulae was removed using veterinary clippers before treatment.
Intradermal administration of 0.1 ml of the test material (TM). 2 concentrations (5% and 1% v/v in arachis oil) were tested in one animal each.
Evalauation of the potential cutaneous reactions: 24, 48, 72 hours and 7 days later.

- Epicutaneous exposure:
Hair over the scapulae was removed using veterinary clippers before treatment.
Each selected concentration applied to a filter paper patch measuring 2x2cm for 24 hours under occlusive dressing . A total of 4 concentrations (100%, 75%, 50% and 25% v/v in arachis oil, 4 concentrations per animal) were tested in duplicate.
Evaluation of the potential cutaneous reactions 1, 24 and 48 hours after patch removal.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. TM at 1 % v/v in vehicle
. TM at 1 % v/v in a 50/50 (v/v) mixture of FCA and sterile water

Epicutaneous exposure
Application of 4 x 2cm patch saturated with the undiluted TM to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. vehicle
. A mixture 50/50 (v/v) FCA diluted to 50% with sterile water, and vehicle

Epicutaneous exposure
Application of 4 x 2cm patch saturated with the vehicule to the scapular region and held in place for 48 hours using an occlusive dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (2x 4cm) in the scapular region

Epicutaneous exposure
4x 2cm area over the scapulae

- Frequency of applications:
One intradermal injection and one epicutaneous application 8 days (Day 7 of the study) after on the same site.

- Duration:
Epicutaneous exposure: 48 hours

B. CHALLENGE EXPOSURE
- No. of exposures: 2 ( the animals were re-challenged at a lower concentration since a response to the first challenge was noted in a number of animals in both the control and tests groups)
- Day(s) of challenge: 22 and 30
- Exposure period: 24 hours

-> TEST GROUPS:
First challenge: undiluted TM on the right flank and TM at 75% v/v in the vehicule on the left flank (occlusive epicutaneous application)
Second challenge: TM at 25% v/v in the vehicle on the right flank and TM at 10% v/v in the vehicule on the left flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group

- Evaluation (hr after challenge): 24 , 48 hours after removal of the dressing according to the method of Draize.
Challenge controls:
Yes - see above.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole and alpha-hexylcinnamalaldehyde

Results and discussion

Positive control results:
Regular checks were performed at the laboratory with the positive control substances benzothiazole and alpha-hexylcinnamalaldehyde.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Desquamation in all animals + erythema grade 1 in 5 animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 %. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Desquamation in all animals + erythema grade 1 in 5 animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Desquamation in all animals + erythema grade 1 in 1 animal
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75 %. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Desquamation in all animals + erythema grade 1 in 1 animal.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Desquamation in all animals + erythema grade 1 in 4 animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100 %. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: Desquamation in all animals + erythema grade 1 in 4 animals.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Desquamation in all animals + erythema grade 1 in 1 animal
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 75 %. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: Desquamation in all animals + erythema grade 1 in 1 animal.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Desquamation and crusts in all animals. Adverse skin reactions prevented evaluation of erythema in 6 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Desquamation and crusts in all animals. Adverse skin reactions prevented evaluation of erythema in 6 animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Desquamation in all animals. Adverse skin reactions prevented evaluation of erythema in 7 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75 %. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Desquamation in all animals. Adverse skin reactions prevented evaluation of erythema in 7 animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Desquamation in all animals. Adverse skin reactions prevented evaluation of erythema in 2 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100 %. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: Desquamation in all animals. Adverse skin reactions prevented evaluation of erythema in 2 animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Desquamation in all animals.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 75 %. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: Desquamation in all animals..
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

The preliminary study indicated that the test material injected at 1% concentration was reasonably well tolerated. A concentration of 100% was selected for the topical induction as it was judged to be well tolerated and non-irritant. A response to treatment was noted in a number of animals in both the control and test groups after a first challenge with concentrations of 100 and 75%. Therefore, a re-challenge at the lower concentrations of 25 and 10% was performed.

Table 7.4.1/1 Individual skin reactions at first challenge and second challenge

Group

Animal number

Skin reactions (Hours after removal of dressings)

24 hours

48 hours

100%

75%

100%

75%

Erythema

Other

Erythema

Other

Erythema

Other

Erythema

Other

TEST

1

2

3

4

5

6

7

8

9

10

1

0

1

1

0

1

0

0

0

1

D

D

D

D

D

D

D

D

D

D

0

0

0

1

0

0

0

0

0

0

D

D

D

D

D

D

D

D

D

D

?e

0

?e

?e

0

?e

0

?e

0

?e

Cf

D

Cf

D+

D

D+

D

D+

D

D+

?e

?e

?e

?e

0

0

0

?e

?e

?e

D+

D+

D+

D+

D

D

D

D+

D+

D+

CONTROL

11

12

13

14

15

1

1

1

0

1

D

D

D

D

D

0

1

0

0

0

D

D

D

D

D

?e

?e

0

0

0

D+

D+

D

D

D

0

0

0

0

0

D

D

D

D

D

RE-CHALLENGE

 

25%

10%

25%

10%

TEST

1

2

3

4

5

6

7

8

9

10

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

-

-

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

-

-

CONTROL

1R

2R

3R

4R

5R

0

0

0

0

0

-

-

-

-

-

0

0

0

0

0

-

-

-

-

-

0

0

0

0

0

-

-

-

-

-

0

0

0

0

0

-

-

-

-

-

 Er = Erythema D = Desquamation Cf = Crust formation D+ = Severe desquamation ?e = adverse skin reactions prevented evaluation of erythema

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No reactions were observed at challenge with concentrations of 25% or less of test material. Therefore, the substance is not classified according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
Executive summary:

The potential of the substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with Good Laboratory practices.

The induction phase was realized both by intradermal route on day 0 (Test material 1% v/v in arachis oil) and by cutaneous route on day 7 (Undiluted Test material ) in 2 groups of guinea pigs: 5 for control group and 10 for treated group. The challenge phase was realized on day 22 by cutaneous application of the undiluted test material on the right flank and Test material at 75% v/v in the vehicule on the left flank. The cutaneous reactions were scored 24 and 48 after the challenge phase. As a response to treatment was noted in a number of animals in both the control and test groups, a re-challenge at the lower concentrations of 25% and 10% v/v was performed on day 30.

Severe desquamation and crust formation were noted in a majority of animals of both test and control groups at the 48 -hour observation after

the first challenge application with Test material at 100% and 75% v/v . since none of the control group animals had been exposed to Test material prior to the topical challenge, the skin reactions observed in the control group animals were attributed to skin irritation caused by the 100 and 75% concentrations of the Test material, and not skin sensitisation. Therefore the challenge was repeated with lower concentrations of 25 and 10% one week later. On this occasion, no response was observed in any animal of the test or control groups at the 24 or 48-hour observation.

It was concluded that the substance does not elicit a sensitisation response in the guinea pig.