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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:
20 Sep - 30 Oct 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
96/54/EC
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Behörde für Arbeit, Gesundheit und Soziales, Hamburg, Germany
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was done before LLNA as first-choice method for in-vivo testing was set into force.

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate
EC Number:
946-282-7
Molecular formula:
C11H20O2
IUPAC Name:
reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 27 days
- Weight at study initiation: 247 - 315 g
- Housing: in pairs in MAKROLON cages (type IV) on granulated textured wood
- Diet: ssniff Ms-H V2233 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum, analysis was performed
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
10%
Day(s)/duration:
Day 0 / single injections
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
Day 7 / 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: sesame oil
Concentration / amount:
50%
Day(s)/duration:
Day 21 / 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
5 (controls), 10 (test groups)
Details on study design:
RANGE FINDING TESTS:
A range finding study was performed to determine the appropriate dose level of the test substance following intradermal and epicutaneous administrations. 8 animals were used: 6 animals for the topical and 2 for the intracutaneous administration.
For the intradermal administration 3 concentrations of the test item (0.01, 0.1 and 1%) were injected intradermally into one and 3 further concentrations (5, 10 and 50%) into a second animal. Solutions up to 1% did not reveal any skin reactions, a concentration of 5% revealed a discrete or patchy erythema, a concentration of 10% revealed a moderate and confluent erythema, a concentration of 50% revealed an intense erythema and swelling 24, 48 and 72 hours after start of administration.
For the epicutaneous administration 2 concentrations each (1% and 5%, 10% and 25%, 50% and 100%) were applied to the shaved or depilated flanks of 3 animals each.
Administration of a 50% solution to the shaved skin revealed a discrete or patchy erythema, a concentration of 100% revealed a moderate and confluent erythema 48 and 72 hours after start of administration.
Administration of a 50% solution to the depilated skin revealed no skin reaction, a concentration of 100% revealed a discrete or patchy erythema 24, 48 and 72 hours after start of administration.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/0.9% NaCI
Injection 2: test substance in sesame oil
Injection 3: test substance in a 1:1 mixture (v/v) FCA/physiological saline
Epicutaneous: undiluted test substance
- Control group:
Injection 1: a 1:1 mixture (v/v) FCA/0.9% NaCI
Injection 2: sesame oil
Injection 3: sesame oil in a 1:1 mixture (v/v) FCA/physiological saline
Epicutaneous: sesame oil
- Site: shoulder region (intradermal + epicutaneous)
- Duration: Days 0-7
- Concentrations: intradermal 10%, epicutaneous 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day of challenge: 21 (challenge)
- Exposure period: 24 h
- Test groups: test substance in sesame oil and sesame oil
- Control group: test substance in sesame oil and sesame oil
- Site: left flank (test substance in sesame oil) and right flank (sesame oil)
- Concentrations: 50%
- Evaluation: 24 and 48 h after patch removal
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
benzocaine was dissolved in 40% ethanolic 0.9% sodium chloride solution, Induction: intradermal 2% (w/v), epicutaneous 5% (w/v), challenge: 5% (w/v)

Results and discussion

Positive control results:
The positive control substance induced positive reactions in 20/20 animals (100%), thus meeting the reliability criteria for the Guinea Pig Maximisation Test (≥ 30% positive response). The positive control group was not carried out concurrently with this study but is a historical background data group from a study performed during April/May 2004.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction intradermal: 10%, challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Induction intradermal: 2%; challenge: 5%
No. with + reactions:
20
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction intradermal: 10%, challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Induction intradermal: 2%; challenge: 5%
No. with + reactions:
20
Total no. in group:
20

Any other information on results incl. tables

A 10% solution of test substance in sesame oil chosen for the intradermal induction stage revealed a moderate and confluent erythema in 5 animals 25 h after administration, a discrete or patchy erythema in 5 animals 25 h and in 7 animals 48 h after administration.

2 mL of 100% test substance chosen for the epicutaneous induction revealed a moderate and confluent erythema in 6 animals 49 h after start of administration, a discrete or patchy erythema in 4 animals 49 h and in 6 animals 72 h after start of administration.

The challenge with 2 mL of a 50% solution of the test substance in sesame oil revealed no skin irritation in any animal.

The body weight gain of the animals treated with test substance was within the range of the vehicle reference during the experiment. The behaviour remained unchanged.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the guinea pig maximisation test the test substance revealed no sensitising properties.
Executive summary:

The skin sensitising potential of the test substance was investigated in a guinea pig maximisation test (GPMT test) according to OECD Guideline 406 and in compliance with GLP (2004). A range finding study was performed to determine the appropriate dose level of the test substance in sesame oil following intradermal and epicutaneous administrations. For the intradermal administration 3 concentrations of the test item (0.01, 0.1 and 1%) were injected intradermally into one and 3 further concentrations (5, 10 and 50%) into a second animal. For the epicutaneous administration 2 concentrations each (1% and 5%, 10% and 25%, 50% and 100%) were applied to the shaved or depilated flanks of 3 animals each. Based on the results of the preliminary study, a 10% solution of the test substance in sesame oil for the first induction stage (intradermal) on Day 0, the undiluted test substance for the second induction stage (epicutaneous) on Day 7 and a 50% concentration for the challenge on Day 21 were selected.

In the main study, 10 animals were used to investigate the skin sensitising potential of the test substance. In addition, 5 animals treated with sesame oil only served as vehicle reference. The 10% solution of the test substance used for the first induction stage on Day 0 revealed a moderate and confluent erythema in 5 animals 25 hours after administration, a discrete or patchy erythema in 5 animals 25 hours and in 7 animals 48 hours after administration. In the second induction stage on Day 7, 2 mL of the undiluted test substance revealed a moderate and confluent erythema in 6 animals 49 hours after start of administration, a discrete or patchy erythema in 4 animals 49 hours and in 6 animals 72 hours after start of administration. The challenge with 2 mL of the 50% solution of the test substance in sesame oil on Day 21 revealed no skin irritation in any animal. The body weight gain of animals treated with test substance was within the expected range during the experiment. The vehicle reference revealed no skin reactions per se. Based on the results of this GPMT test, the test substance was not regarded as a skin sensitizer under the conditions of the test.