Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 1989 - 25 March 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
See read-across justification in Section 13.
Cross-reference
Reason / purpose:
other: Target substance
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read across material
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
read-across source
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75 % (w/w) in distilled water
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
One animal showed scattered mild redness
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75 % (w/w) in distilled water
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:
2
Total no. in group:
9
Clinical observations:
Two animals showed scattered mild redness
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
9

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Existing data using a non-LLNA method was already available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: White powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 – 10 weeks.
- Weight at study initiation: 312 – 382g.
- Housing: The animals were housed in groups of up to four in solid-floor polypropylene cages furnished with softwood shavings.
- Diet (e.g. ad libitum): ad libitum. Free access to food (Guinea Pig FDI Diet, Special Diet Services LTD., Witham, Essex, UK) was allowed throughout the study.
- Water (e.g. ad libitum): ad libitum. Free access to mains tap water was allowed throughout the study.
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 – 20 °C
On occasions the minimum temperature was outside the limit specified in the protocol (18°C). This deviation did not affect the purpose or the integrity of the study.
- Humidity (%): 55 – 75% relative.
- Air changes (per hr): approximately 15.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
distilled
Concentration / amount:
For the purpose of this study the test material was freshly prepared as follows:
Intradermal Induction: 25% (w/v) in distilled water
25% (w/v) in Freund's Complete Adjuvant plus distilled water in the ratio 1:1
Topical Induction: 75% (w/w) in distilled water
Topical Challenge: 75% (w/w) in distilled water
The identification and stability of the test material and the prepared formulations were not determined.
Challengeopen allclose all
Route:
epicutaneous, semiocclusive
Vehicle:
water
Remarks:
distilled
Concentration / amount:
For the purpose of this study the test material was freshly prepared as follows:
Intradermal Induction: 25% (w/v) in distilled water
25% (w/v) in Freund's Complete Adjuvant plus distilled water in the ratio 1:1
Topical Induction: 75% (w/w) in distilled water
Topical Challenge: 75% (w/w) in distilled water
The identification and stability of the test material and the prepared formulations were not determined.
No. of animals per dose:
20 test and 10 control
Details on study design:
Procedure
The dose levels for each of the three stages of the main study were determined by 'sighting studies' in which groups of one or two guinea pigs were used and up to four concentrations of test material were tested on each animal. The procedure was as follows:
i) Intradermal Injection (Induction):
Suspensions of the test material in distilled water were tested to determine the highest practical level (up to 25%) that could be intradermally injected and well tolerated both locally and systemically.
ii) Topical Application (Induction):
Suspensions of the test material in distilled water were tested to determine the highest practical level which did not produce excessive inflammation on the flank of animals injected with Freund's Complete Adjuvant (Difco Laboratories, Michigan, U.S.A.) at least seven days previously.
iii) Topical Application (Challenge):
Suspensions of the test material in distilled water were tested to determine the highest practical level which did not produce inflammation or irritation on the flank of animals injected with Freund's Complete Adjuvant at least seven days previously.

A group of thirty guinea pigs was used for the main study, twenty test and ten control. The bodyweight of each animal was recorded at the start and end of the study.

Two main procedures were involved in the maximisation test; (a) an induction of a response and (b) a challenge of that response.

a) INDUCTION
Induction of the Test Animals:
The hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers and a row of three injections (0.1 mL each) was made on each side of the mid-line. The injections were:

i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
ii) a 25% (w/v) suspension of test material in distilled water.
iii) a 25% (w/v) suspension of test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.

One week later, the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation (75% w/w in distilled water). The test material formulation (0.2 - 0.3 mL) was applied on filter paper (WHATMAN No. 4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

Erythematous reactions were quantified one and twenty-four hours following removal of the patches using the 0 - 3 scale shown in Section b.

Induction of the Control Animals:
Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.

ii) Distilled water.
iii) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.

The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

b) CHALLENGE
Two weeks after the topical inductions, an area, approximately 50 - 70 mm x 50 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A quantity of 0.1 - 0.2 mL of the test material formulation (75% w/w in distilled water) was applied to the shorn right flank of each animal on a 20 mm x 20 mm square of filter paper (WHATMAN No. 4) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The position of the treatment sites was identified by using a black indelible marker-pen. After a further 24 and 48 hours, any erythematous reactions were quantified using the four-point scale shown and the number of positive responses recorded.
Scale :
0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

To classify the sensitisation response, the percentage of test animals that showed a more severe reaction at the test material challenge site than the most severe reaction seen in the control animals, was compared with the following scale:
% of animals sensitised Classification of sensitisation potential
0 non-sensitiser
>0 - 8 weak sensitiser
>8 - 28 mild sensitiser
>28 – 64 moderate sensitiser
>64 – 80 strong sensitiser
>80 - 100 extreme sensitiser
Positive control substance(s):
yes
Remarks:
2,4-Dinitrochlorobenzene

Results and discussion

Positive control results:
PROJECT NUMBER SF9:
Date Started: 13 July 1988
Date Completed: 15 August 1988

Experimental Procedures:

1. The sensitivity of the strain of guinea pigs used in these laboratories for skin sensitisation testing is checked using a known positive sensitiser as required by regulatory authorities. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 - "Skin Sensitisation" - Magnusson & Kligman Maximisation Test.
2. Twenty test and ten control animals were used for the main study. Based on results of sighting studies the following concentrations of 2,4-DINITROCHLOROBENZENE (DNCB) were used for the induction and challenge phases.
Intradermal Induction: 0.05% (w/v)
Topical Induction (day 7): 0.25% (w/v)
Topical Challenge (day 21): 0.1% (w/v)

3. The incidence and severity of the skin reactions observed at challenge were:

Group Concentration Observation Time Erythema (Scale 0 – 3) Sensitisation
Identification (% w/v) (Hours) 0 1 2 3 Response

Test 0.1 24 2 6 12 0 18/20
48* 2 8 3 0
Control 0.1 24 10 0 0 0 0/10
48 10 0 0 0
*presence of other adverse skin reactions precluded evaluation of erythema at seven treatment sites.
4. The positive test material 2,4-DINITROCHLOROBENZENE (DNCB), produced a 90% (18/20) sensitisation rate and was classified as an extreme sensitiser to guinea pig skin.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% (w/w) in distilled water
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
One animal showed scattered mild redness
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75% (w/w) in distilled water. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: One animal showed scattered mild redness.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% (w/w) in distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% (w/w) in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
2
Total no. in group:
9
Clinical observations:
Two animals showed scattered mild redness
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 2.0. Total no. in groups: 9.0. Clinical observations: Two animals showed scattered mild redness.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 9.0.

Any other information on results incl. tables

RESULTS

1. Intradermal and Topical Sighting Studies

Based on the results, the following concentrations were selected for the main study:

Intradermal Induction: 25% (w/v) in distilled water

Topical Induction: 75% (w/w) in distilled water

Topical Challenge: 75% (w/w) in distilled water

 

2. Main Study

a) Induction

Scattered mild redness (grade 1) was noted at five test group skin sites one hour after removal of the patches.

No other adverse reactions were noted during the induction period.

 

b) Challenqe

Individual reactions at the challenge sites of test and control animals are summarised as follows:

Incidence of sensitisation Responses in Test Animals

% Sensitisation Response

Classification

0/20

0

Non-Sensitiser

 

One control group animal was found dead on day twenty-one. The absence of this animal did not affect the purpose or integrity of the study.

Scattered mild redness (grade 1) was noted at the test material site of one test group animal at the 24-hour observation. Similar skin reactions were also noted at the test material site of two control group animals at this time. These reactions were, therefore, considered to be skin irritation and were not indicative of skin sensitisation. No adverse skin reactions were noted at the 48-hour observation.

No adverse reactions were noted at the vehicle control sites of the test or control animals at the 24 and 48-hour observations.

The test material, therefore, produced a 0% (0/20) sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin.

3. Bodyweight

Bodyweight gains of guinea pigs in the test group, between day 0 and day 24, were comparable to those observed in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was found to be a non-sensitiser to guinea pig skin.
Executive summary:

The test material, was found to be a non-sensitiser to guinea pig skin. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation" - Magnusson and Kligman Maximisation Test, referenced as method B6 in Annex V of EEC Commission Directive 84/449/EEC.