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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:
Between 31 August 2001 and 04 October 2001.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
Study was conducted in 2001 before REACH went into force. An LLNA test does not have to be conducted because adequate information from an OECD 406 study are available.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was conducted in 2001 before REACH went into force. An LLNA test does not have to be conducted because adequate information from an OECD 406 study are available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): R-2581
- Subtsance type and physical state: White crystalline solid
- Analytical purity: Not stated in report
- Impurities (identity and concentrations): Not stated in report
- Batch number: 200-3769
- Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Albino Dunkin Hartley Guinea Pigs from David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 300 - 450g
- Housing: Singly or in pairs in solid floor polypropylene cages furnished with woodflakes
- Diet and water: Free access to mains tap water and food (Guinea Pig FD1 Diet or Certified Guinea Pig Diet)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Controlled by a time switch to give 12 hours darkness and 12 hours light

IN-LIFE DATES: Study was performed between 31 August 2001 and 04 October 2001

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Main study:

Intradermal induction: The injections were:

a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) A 5% w/w formulation of the test material in distilled water
c) A 5% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.

Topical induction:
75% w/w in distilled water

Challenge:
50% and 75% w/w in distilled water was applied on Day 21.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Main study:

Intradermal induction: The injections were:

a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) A 5% w/w formulation of the test material in distilled water
c) A 5% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.

Topical induction:
75% w/w in distilled water

Challenge:
50% and 75% w/w in distilled water was applied on Day 21.
No. of animals per dose:
SIGHTING TESTS
Intradermal induction: 2 guinea pigs
Topical induction: 2 guinea pigs
Topical Challenge: 2 guinea pigs

MAIN STUDY
15 guinea pigs (10 test guinea pigs and 5 control guinea pigs)
Details on study design:
VEHICLE:

Distilled water.

AUXILIARY COMPOUNDS:
Freund's Complete Adjuvant (FCA)

PREPARATION OF TEST MATERIAL:
The test material was freshly prepared in distilled water. The concentrations used are discussed below.

SELECTION OF CONCENTRATION FOR INTRADERMAL INDUCTION:
Intradermal injections were made on the clipped shoulder of two guinea pigs at concentrations of 1% and 5% w/w
in distilled water. The highest concentration that caused only mild to moderate skin irritation was selected
for the intradermal induction stage of the main study. Based on the results, 5% w/w in distilled water was
selected for the intradermal induction stage of the main study.

SELECTION OF CONCENTRATION FOR TOPICAL INDUCTION:
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant 21 days earlier) were treated with test material:
75%, 50%, 25% and 10% w/w in distilled water. Applications were made under occlusive dressings for an exposure period
of 48 hours. The highest concentration producing only mild to moderate dermal irritation was selected for the topical
induction stage of the main study. Based on the results, 75% w/w in distilled water was selected for the main topical
induction study.

SELECTION OF CONCENTRATION FOR TOPICAL CHALLENGE:
Two guinea pigs were treated with four preparations of the test material (75%, 50%, 25% and 10% w/w in distilled water)
which were applied under occlusive dressings for an exposure period of 24 hours. The highest non-irritant concentration
of the test material and one lower concentration were selected for the topical challenge stage of the main study.
The concentrations of material selected for the main study topical challenge were 75% w/w and 50% w/w in distilled water.

MAIN STUDY

Fifteen guinea pigs were used in the main study - ten test and five control.

INDUCTION

Shortly before treatment on Day 0, the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region
of each animal. A row of three injections (0.1 ml each) was made on each side of the mid-line into a 20 mm x 40 mm area.
The injections were:

a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) A 5% w/w formulation of the test material in distilled water
c) A 5% w/w 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
(i.e. 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 test material formulation. A filter paper patch (WHATMAN No. 4: approximate size 40 mm
x 20 mm) loaded with the test material formulation (75% w/w in distilled water) as a thick even layer was applied to the
prepared skin and held in place with surgical adhesive tape covered with a length of aluminium foil. The patch and foil were
further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. The
occlusive dressing was kept in place for 48 hours. The degree of erythema and oedema was quantified 1 and 24 hours
following removal of the patches.

Induction of the control animals: The intradermal induction was performed using an identical procedure to that used
for the test animals except that the test material was omitted from the intradermal injections. Injection (b) was therefore
the vehicle alone, injection (c) was a 50% formulation of the vehicle in a 1:1 preparation of FCA plus distilled water.
Similarly, the topical induction procedure was identical to that used for the test animals except that the the test material
was omitted.

CHALLENGE

Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal was clipped free of hair. A filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm) loaded with the test material formulation (75% w/w in distilled water) as a thick even layer was applied to
shorn right flank of each animal and held in place with surgical adhesive tape. To ensure the maximum non-irritant concentration was used at challenge the
test material at a concentration of 50% w/w in distilled water was applied to the skin site of 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 removed and discarded and the challenge sites were swabbed with cotton wool soaked in distilled water in order to remove
residual material. The treatment sites were identified by using a black indelible marker pen.

Approximately 24 and 48 hours after challenge dressing removal. the degree of erythema and oedema was quantified. Any other reactions were also recorded. One challange animal was killed for humane reasons, due to weight loss, on day 22 which was not considered to affect the integrity of the study.

Challenge controls:
The five control animals at the induction phase also received an application of test substance at challenge (75% w/w and 50% w/w in distilled water).

Note: This is to compare the extent and degree of skin reaction to the challenge exposure in the test animals vs the controls and is in accordance with OECD Guideline 406.

Positive control substance(s):
yes
Remarks:
Positive control data is summarised in Appendix 8 of the study for 2-Mercaptobenzothiazole and α-Hexylcinnamaldehyde.

Results and discussion

Positive control results:
Positive control data is summarised in Appendix 8 of the study for 2-Mercaptobenzothiazole and α-Hexylcinnamaldehyde. In two recent studies for
α-Hexylcinnamaldehyde, the incidence of sensitisation was 50% (5/10 animals) and 40% (4/10 animals). In four recent studies for 2-Mercaptobenzothiazole, the incidence of sensitisation was 90% (9/10 animals), 100% (10/10 animals), 100% (10/10 animals) and 100% (9/9 animals).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% in distilled water
No. with + reactions:
8
Total no. in group:
9
Clinical observations:
Reactions included discrete or patchy to moderate and confluent erythema with or without very slight oedema, desquamation, a hardened light brown-coloured scab and crust formation.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75% in distilled water. No with. + reactions: 8.0. Total no. in groups: 9.0. Clinical observations: Reactions included discrete or patchy to moderate and confluent erythema with or without very slight oedema, desquamation, a hardened light brown-coloured scab and crust formation. .
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% w/w in distilled water
No. with + reactions:
8
Total no. in group:
9
Clinical observations:
Reactions included discrete or patchy to moderate and confluent erythema with or without very slight oedema, desquamation, a hardened light brown-coloured scab and crust formation.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% w/w in distilled water. No with. + reactions: 8.0. Total no. in groups: 9.0. Clinical observations: Reactions included discrete or patchy to moderate and confluent erythema with or without very slight oedema, desquamation, a hardened light brown-coloured scab and crust formation. .
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% w/w in distilled water
No. with + reactions:
7
Total no. in group:
9
Clinical observations:
Positive skin responses (discrete or patchy to moderate and confluent erythema with or without very slight oedema) were noted in the challenge sites of seven test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% w/w in distilled water. No with. + reactions: 7.0. Total no. in groups: 9.0. Clinical observations: Positive skin responses (discrete or patchy to moderate and confluent erythema with or without very slight oedema) were noted in the challenge sites of seven test group animals. .
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% w/w in distilled water
No. with + reactions:
7
Total no. in group:
9
Clinical observations:
Positive skin responses (discrete or patchy to moderate and confluent erythema with or without very slight oedema) were noted in the challenge sites of seven test group animals. Desquamation was noted at the sites of five test group animals at 48 hours.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% w/w in distilled water. No with. + reactions: 7.0. Total no. in groups: 9.0. Clinical observations: Positive skin responses (discrete or patchy to moderate and confluent erythema with or without very slight oedema) were noted in the challenge sites of seven test group animals. Desquamation was noted at the sites of five test group animals at 48 hours..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75% in distilled water (at challenge)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reactions were observed at the challenge sites.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 75% in distilled water (at challenge). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were observed at the challenge sites. .
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% in distilled water (at challenge)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reactions were observed at the challenge sites.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50% in distilled water (at challenge). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were observed at the challenge sites. .

Any other information on results incl. tables

Skin reactions observed after intradermal induction

Individual skin reactions at the intradermal induction sites of the test and control group animals are given in Appendix 4 of the study.

Discrete or patchy erythema was noted at the intradermal induction sites of test group animals.

No skin reactions were noted at the intradermal sites of control group animals.

Skin Reactions Observed after Topical Induction

Individual skin reactions at the topical induction sites of the test and control animals are presented in Appendix 5 of the study.

Discrete or patchy to moderate and confluent erythema with or without very slight oedema was noted at the topical induction sites of test group animals. Discrete or patchy erythema was noted at the topical induction sites of control group animals.

Bleeding from the intradermal injection sites was noted in all test group animals and three control group animals. A hardened dark brown/black-coloured scab was noted in one test group animal at the 24 hour observation and prevented the evaluation of erythema and oedema.

Skin Reactions Observed After Topical Challenge

Individual skin reactions at the challenge sites of the test and control group animals are stated in Table 1 of the study.

75% w/w in distilled water

Positive skin responses were noted at the challenge sites of eight test group animals. Reactions included discrete or patchy to moderate and confluent erythema with or without very slight oedema, desquamation, a hardened light brown-coloured scab and crust formation. Adverse reactions prevented evaluation of erythema and oedema at one treated skin site at the 48 hour observation. A transient challenge reaction was noted at the challenge site of one test group animal at the 24 hour observation. No skin reactions were observed at the challenge sites of the control group animals at the 24 or 48 hour observations.

50% in distilled water

Positive skin responses (discrete or patchy to moderate and confluent erythema with or without very slight oedema) were noted at the challenge sites of five test group animals at the 48 hour observation. Transient challenge reactions were noted at the challenge sites of two test group animals at the 24 hour observation. No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48 hour observations.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance is considered to be a
skin sensitiser to guinea pigs. The study concluded that as the test material produced a sensitisation rate of more than 30%, that the substance requires labelling with the symbol "Xi" and the indication of danger "irritant" and the risk phrase R43 "May cause Sensitisation by Skin Contact".

Based on the study findings, the test substance will be classified under Regulation (EC) No 1272/2008 (CLP) for Skin Sensitisation (Category 1) and is considered to be a moderate skin sensitiser in accordance with the ECHA document "Guidance on the application of the CLP Criteria (Section 3.4.2.3.4.2)".

Executive summary:

Methods

The test substance (R-2581) (Carbohydrazide) was tested for skin sensitisation in guinea pigs according to the Magnusson and Kligman Maximisation Method (OECD Guideline 406; EU Method B.6). The doses used at challenge were 75% and 50% test substance in distilled water and reactions were observed after 24 and 48 hours after challenge.

Results

Based on the dose of 75% w/w in distilled water, the test material produced an 89% sensitisation rate.

Conclusion

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance is considered to be a skin sensitiser to guinea pigs (described as an extreme sensitiser in the study). The study concluded that as the test material produced a sensitisation rate of more than 30%, that the substance requires labelling with the symbol "Xi" and the indication of danger "irritant" and the risk phrase R43 "May cause Sensitisation by Skin Contact".

Based on the study findings, the test substance will be classified under Regulation (EC) No 1272/2008 (CLP) for Skin Sensitisation (Category 1 (sub-category 1B)) and is considered to be a moderate skin sensitiser in accordance with the ECHA document "Guidance on the application of the CLP Criteria (Section 3.4.2.3.4.2)".