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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guidelline and GLP compliant study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The available guinea pig maximisation test is enough for risk assessment.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): 3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-/VERTOCITRAL
- Physical state: colourless liquid
- Analytical purity:99.8%
- Lot/batch No.: 7110050
- Storage condition of test material: room temperature in darkness

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: albino Dunkin Hartley guinea pig
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: approximately eight to twelve weeks old
- Weight at study initiation: 300 to 345g
- Housing: housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): free access food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study.
- Water (e.g. ad libitum): free access to mains tap water was allowed throughout the study
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22 °C
- Humidity (%): 34 to 60%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Concentration / amount:
The concentrations of test substance in the main study were selected as follows:
In tradermal Induction: 5% w/v in arachis oil BP
Topical Induction: 25% v/v in ethanol/diethylphthalate 1:1
Topical Challenge: 50% and 25% v/v in ethanol/diethylphthalate 1:1
Challengeopen allclose all
Route:
epicutaneous, occlusive
Concentration / amount:
The concentrations of test substance in the main study were selected as follows:
In tradermal Induction: 5% w/v in arachis oil BP
Topical Induction: 25% v/v in ethanol/diethylphthalate 1:1
Topical Challenge: 50% and 25% v/v in ethanol/diethylphthalate 1:1
No. of animals per dose:
Test group: 20 anmials
Control group: 10 animals
Details on study design:
RANGE FINDING TESTS:
The concentrations of of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The concentration for Intraderrnal Induction (1% and 5% w/v in arachis oil BP) was selected through 2 guinea pigs receiving different concentration and the highest concentration that caused only mild to moderate skin Irritation was selected for the intradermal induction stage of the main study. The concentraion for topical induction (50%, 25%, 10% and 5% v/v in ethanol/diethylphthalate 1:1) was selected as well as the intradermal induction. In addition, the concentraiton for topic challenge ((50%, 25%, 10% and 5% v/v in ethanol/diethylphthalate 1:1) was also selected following the highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: On Day 0, three injections (0.1mL each) in test and control animals as indicated below.
- Exposure period: Test and control animals: Approx 24 and 48 hours after intradermal injection, the degree of erythema was evaluated. One week later (Day 7), the same shoulder region was topically treated and secured with an occlusive dressing for 48 hrs. The degree of erythema and odema was quantified 1 and 24 hr following removal of dressing.
- Test groups: 20 animals
- Control group: 10 animals
- Site: the shoulder region
- Concentrations:
test animals:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 5% w/v formulation of the test material in arachis oil BP
c) a 5% w/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
control animals:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) arachis oil BP
c) a 50% w/v formulation of arachis oil BP in Freund's Complete Adjuvant/distilled water 1:1

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: on Day 21
- Exposure period: Test and control animals:
On Day 21, flanks were topically treated and secured with an occlusive dressing for 24 hrs. The dressing was removed and the challenge sites were swabbed with cotton wool with diethyl ether to remove residual material.
- Test groups: 20 animals
- Control group: 10 animals
- Site: on both flanks of each animal
- Concentrations:
a) 50% v/v in ethanol/diethylphthalate 1:1
b) 25% v/v in ethanol/diethylphthalate 1:1
- Evaluation (hr after challenge): Approx 24 and 48 hrs after challenge dressing removal, the degree of erythema and odema was evaluated.

Results and discussion

Positive control results:
There was no positive control study conducted concurrently. However, the reliability of the test system conducted in the laboratory was summarized in a list positive control data for the Mag and Klig max study (Various known positive sensitisers used; July 1995 – November 1997 (See Appendix XI attached).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
20
Clinical observations:
Positive skin responses (well-defined or moderate to severe erythema grades 2 or 3 and very slight or slight oedema) and dark brown-coloured scab below the challenge site were noted at the challenge sites of all test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% v/v in ethanol/diethylphthalate 1:1 . Total no. in groups: 20.0. Clinical observations: Positive skin responses (well-defined or moderate to severe erythema grades 2 or 3 and very slight or slight oedema) and dark brown-coloured scab below the challenge site were noted at the challenge sites of all test group animals. .
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
20
Clinical observations:
8/20 animals:(very slight or well-defined erythema grades 1 or 2; 11/20 anmials: Very slight to slight oedema; other skin reactions: hardened dark brown/black coloured scab, hardened light brown coloured scab, desquamation, crust formation etc.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% v/v in ethanol/diethylphthalate 1:1 . Total no. in groups: 20.0. Clinical observations: 8/20 animals:(very slight or well-defined erythema grades 1 or 2; 11/20 anmials: Very slight to slight oedema; other skin reactions: hardened dark brown/black coloured scab, hardened light brown coloured scab, desquamation, crust formation etc..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
10
Clinical observations:
No skin reactions
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50% v/v in ethanol/diethylphthalate 1:1 . Total no. in groups: 10.0. Clinical observations: No skin reactions.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
10
Clinical observations:
No skin reactions
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50% v/v in ethanol/diethylphthalate 1:1 . Total no. in groups: 10.0. Clinical observations: No skin reactions.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
20
Clinical observations:
Positive skin responses (very slight to moderate to severe erythema grades 1 to 3 and very slight to slight oedema)
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% v/v in ethanol/diethylphthalate 1:1 . Total no. in groups: 20.0. Clinical observations: Positive skin responses (very slight to moderate to severe erythema grades 1 to 3 and very slight to slight oedema).
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
20
Clinical observations:
9/20 anmials: Positive skin responses (very slight or well-defined erythema - grades 1 to 2); 12/20 animals: Very slight or slight oedema; Other skin reactions: hardened dark brown/black coloured scab, hardened light brown coloured scab etc.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% v/v in ethanol/diethylphthalate 1:1. Total no. in groups: 20.0. Clinical observations: 9/20 anmials: Positive skin responses (very slight or well-defined erythema - grades 1 to 2); 12/20 animals: Very slight or slight oedema; Other skin reactions: hardened dark brown/black coloured scab, hardened light brown coloured scab etc..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
10
Clinical observations:
No signs of erythema or oedema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% v/v in ethanol/diethylphthalate 1:1 . Total no. in groups: 10.0. Clinical observations: No signs of erythema or oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% v/v in ethanol/diethylphthalate 1:1
Total no. in group:
10
Clinical observations:
Desquamation was noted at the challenge site of one control group animal
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% v/v in ethanol/diethylphthalate 1:1. Total no. in groups: 10.0. Clinical observations: Desquamation was noted at the challenge site of one control group animal.

Any other information on results incl. tables

The test substance were indicated a 100% sensitisation rate and was classified as a strongly sensitiser to guinea pig skin and the individual skin reactions in test and control animals at challenge were presented in Tables 1 and 2.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance Ligustral in 25% and 50% v/v ethanol/diethylphthalate 1:1, produced a 100% (20/20) sensitisation rate and was classified as an extreme sensitiser to guinea pig skin.
Executive summary:

In a dermal sensitization study (012/251), with Ligustral/Cyclal C/Trigustral (in 25% and 50% v/v ethanol/diethylphthalate (DEP) 1:1), 38 female albino Dunkin Hartley guinea pigs were tested in the in vivo guinea pig maximisation test.

Based on sighting study results, Ligustral/Cyclal C/Trigustral was used at 5% in arachis oil (intradermal induction), 25% in DEP 1:1 (topical induction) and 50% and 25% in DEP 1:1 for challenge. There was no positive control study conducted concurrently but historical laboratory data indicated that known positive sensitisers produced appropriate sensitisation results using the test system.

The results indicated that Ligustral/Cyclal C/Trigustral was a skin sensitiser at both challenge concentrations in 20/20 animals and no reactions were noted in control groups.

Ligustral/Cyclal C/Trigustral is a dermal sensitizer in this OECD 406 (GPMT) study.