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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
guinea-pig maximisation test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Oct-24 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Annex of Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Method B.6.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Adopted 17 Jul 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Good Laboratory Practice, The United Kingdom Compliance Programme, Department of Health & Social Security 1986 and subsequent revision, Department of Health 1989.
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was conducted prior to the adoption of the LLNA; TG 429. Quantitative structure-activity relationships and in vitro models were not yet sufficiently developed to play a significant role in the assessment of the skin-sensitisation potential of substances, which therefore had to continue to be based on in vivo models. The original Test Guideline (TG) for the determination of skin sensitization in the mouse, the Local Lymph Node Assay (LLNA; TG 429) was adopted in 2002.

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
EC Number:
212-990-3
EC Name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
Cas Number:
903-19-5
Molecular formula:
C22H38O2
IUPAC Name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, UK
- Females: no
- Age at study initiation: 4-5 wk
- Weight at study initiation: 271-317 g (range)
- Housing: groups of 5, in suspended metal cages with mesh floors
- Diet: vitamin C enriched diet FD2, ad libitum, supplemented with hay weekly
- Water: drinking water, ad libitum
- Acclimation period: 12 days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ~21
- Humidity (%): 30-70
- Air changes (per hr): ~15
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 31 Oct 1995 To: 24 Nov 1995

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
coconut oil
Concentration / amount:
Induction intradermal injection: 7.5% w/v in 5% acetone in coconut oil
Induction topical application: 60% w/v in acetone
Day(s)/duration:
Induction intradermal injections were on Day 1. Induction topical applications were seven days later on Day 8 for 48 h.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
Topical challenge: 60% and 30% w/v in acetone
Day(s)/duration:
Challenge topical applications were on Day 22 for 24 h. Challenge sites were evaluated 24 and 48 h post patch removal on Day 24 and 25.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
5 (control group), 10 (test group)
Details on study design:
RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase. Animals were pre-treated with an intradermal injection of Freund's complete adjuvant, 50:50 with water for irrigation (Ph. Eur.), approximately three weeks prior to the start of the preliminary investigations. For the intradermal injection, the maximum practical concentration that caused irritation but did not adversely affect the animals was 7.5% w/v in 5% acetone in coconut oil. For the topical application, the highest practical concentration that could be prepared and dosed topically and that did not give rise to irritating effects was 60% w/v in acetone.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 8 days later, 48 h (epicutaneous)
- Test groups:
- Intradermal: 3 pairs of injections
injection 1: 0.1 mL Freund's complete adjuvant 50:50 with water for irrigation (Ph. Eur.)
injection 2: 0.1 mL tetramethylbutylhydrochinon, 7.5% w/v in 5% acetone in coconut oil
injection 3: 0.1 mL tetramethylbutylhydrochinon, 7.5% w/v in a 50:50 mixture of 5% acetone in coconut oil, and Freund's complete adjuvant
- Epicutaneous:
tetramethylbutylhydrochinon 60% w/v in acetone
- Control group:
- Intradermal: 3 pairs of injections
injection 1: 0.1 mL Freund's complete adjuvant 50:50 with water for irrigation (Ph. Eur.)
injection 2: 0.1 mL of 5% acetone in coconut oil
injection 3: 0.1 mL of Freund's complete adjuvant 50:50 with 5% acetone in coconut oil
- Epicutaneous:
acetone
- Site: dorsal skin on the scapular region (injection and epicutaneous)
- Frequency of applications: day 1 and 8. On Day 7 the injection site was clipped and shaved and pretreated with 10% sodium laurel sulphate in petrolatum.
- Duration: Days 0-8
- Concentrations: intradermal 7.5% w/v in vehicle, epicutaneous 60% in vehicle

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Test groups: test substance only
- Control group: test substance only
- Site: left flank anterior and posterior sites
- Concentrations: anterior site: tetramethylbutylhydrochinon 60%, posterior site: tetramethylbutylhydrochinon 30%
- Evaluation (hr after challenge): 24 and 48 h
Challenge controls:
test substance
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde historic data to show efficacy

Results and discussion

Positive control results:
In 7 reliabiltiy studies performed December 1992 - January 1995, the positive control substance hexyl cinnamic aldehyde was shown to induce skin sensitisation reactions in 8-10 animals per group of 10 animals.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
60%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
60%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
60%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
60%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% and undiluted
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Summary of 7 reliability studies performed December 1992 - January 1995

Any other information on results incl. tables

Clinical signs: No signs of ill health or toxicity were recorded.

Bodyweight: Increases were recorded for all guinea-pigs over the period of the study.

Induction:

- Intradermal injections: necrosis was recorded at all sites receiving Freund's complete adjuvant, in test and control animals.

- Slight irritation was seen in test animals' sites receiving tetramethylbutylhydrochinon 7.5% w/v in 5% acetone in coconut oil, and slight irritation was seen in control animals' sites receiving 5% acetone in coconut oil.

- Topical application: Slight erythema was seen in test animals' sites following application with tetramethylbutylhydrochinon 60% w/v in acetone, and slight erythema was seen in control animals' sites following application with acetone.

Challenge:

-Challenge application: No dermal reactions were seen in any test or control animals following the challenge topical application with tetramethylbutylhydrochinon 60% w/v or 30% w/v, in acetone.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
In this study, AF-317 did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the test animals.