Isopropylcyclohexane

Endpoint summary

Description of key information

Under the present test conditions, isopropylcyclohexane was not skin sensitising in a guinea pig maximization test according to OECD 406 (LPT, 2012).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-04 to 2011-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

2008

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A skin sensitization test according to OECD 406 has already excisted since 2011 and is sufficient for evaluation of the skin sensitisation potential of the test substance.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS: 
- Strain: Dunkin-Hartley
- Source: Charles River Laboratories, Germany
- Sex: male
- Age: 32 days at arrival + approx. 2 weeks for acclimatization and  pilot study
- Weight at study initiation: 318 - 399 g 
- Housing: in pairs in MAKROLON cages
Environmental conditions: - Feed: Commercial diet, ssniff Ms-H V 2233
- Water: tap water ad libitum
- Room temperature: 20°C (+/- 3°C)
- Humidity: 55% (+/- 15%)
- Illumination: 12 hour light/dark rhythm

Route:

intradermal and epicutaneous

Vehicle:

other: Sesame oil

Concentration / amount:

1st application: Induction 10 % intracutaneous in sesame oil
2nd application: Induction 100 % occlusive epicutaneous
3rd application: Challenge 75 % occlusive epicutaneous in sesame oil

Route:

epicutaneous, occlusive

Vehicle:

other: Sesame oil

Concentration / amount:

1st application: Induction 10 % intracutaneous in sesame oil
2nd application: Induction 100 % occlusive epicutaneous
3rd application: Challenge 75 % occlusive epicutaneous in sesame oil

No. of animals per dose:

10 animals preliminary study
10 animals vehicle control
20 animals test item

Details on study design:

ADMINISTRATION/EXPOSURE 
- Preliminary study: Six concentrations of Isopropylcyclohexane were tested by intracutaneous injection: 0.01, 0.1, 0.5, 1, 5 or 10% solutions in
sesame oil: No skin reactions were observed up to the concentration of 5%. A concentrations of 10% revealed a discrete or patchy erythema 24, 48 and 72 hours after administration.
Seven concentrations of Isopropylcyclohexane were tested by topical application: 1, 5, 10, 25, 50 and 75% solutions in sesame oil and the undiluted test item.
No skin reactions were observed at any concentration to the non-depilated skin
No skin reactions were observed up to the concentration of 75%. The undiluted test item revealed a discrete or patchy erythema 48 and 72 hours
after start of exposure to the depilated skin
Hence, it was decided to use a 10% concentration in sesame oil for the 1st (intracutaneous) stage, the undiluted test item for the 2nd (topical)
induction stage and a 75% solution in sesame oil for the challenge.
MAIN STUDY
- Preparation of test substance for induction: Mixing with vehicle
Stage 1 (induction)
- Induction schedule:    
Day 0: Injections   
 Injection details: 0.1 ml each at 6 positions in clipped scapular  region:   
2 x Freund's Complete Adjuvant (FCA) emulsified with 0.9% NACl (50:50)   
2 x test substance 10 % in vehicle   
2 x test item in a 1:1 mixture (v/v) FCA/physiological saline
pairwise and symmetrical administration of each solution / suspension   
controls: vehicle instead of test substance
Day 6: Clipping of hair in injection sites and treatment with 0.5 ml of  10 % sodium laurylsulfate in vaseline
Stage 2 (induction)
Day 7: 48 hours occlusive patch with 100 % test substance  (controls: vehicle) on injection sites   
Stage 3 (challenge)
- Challenge schedule:    
Day 21: 24 hours occlusive patches on clipped flanks with 2 mL of a 75% solution of Isopropylcyclohexane in sesame oil/animal. The filter paper
containing the test item was applied to the left flank, the filter paper with the vehicle to the right flank of the animal. 21 hours after the filter paper
had been removed, no cleaning of the treated skin was necessary
Days 23 and 24:
- 21 hours after removing the filter paper the challenge area was cleaned and cleared of hair if necessary
- three hours later (at 48 hours from the start of challenge application) the skin reaction was observed and recorded
- 24 hours after this observation a second observation (72 hours) was made and recorded.
- Rechallenge: no
- Positive control: a-hexyl cinnamic aldehyde - positive control group was not tested concurrently with this study but is a historical background
group from a study performed during October/November 2011.
Other observations
Mortality: daily during the observation period
Clinical signs: daily during the observation period
Body weight: at start of study and at study termination

Challenge controls:

filter paper with the vehicle to the right flank of the animal

Positive control substance(s):

yes

Remarks:

a-hexyl cinnamic aldehyde - positive control group was not tested concurrently with this study but is a historical background group from a study performed during October/November 2011.

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0.

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75% Isopropylcyclohexane

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% Isopropylcyclohexane. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

75% Isopropylcyclohexane

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 75% Isopropylcyclohexane. No with. + reactions: 0.0. Total no. in groups: 20.0.

Flow chart of study design:

	Main experiment
	day 0 (induction)	day 7 (induction)	day 21 (challenge)	day 23 +45 h#	day 23 +48 h#	day 24 +72 h#
Shaving	x	x	x
Depilation			x
Application of test formulation	x	x	x
Cleaning		after patch removal x		x
Evaluation of skin	24 h + 48 h after start of exposure x	48 h and 72 h after start of exposure x			x	x
Mortality	x	x	x	x	x	x
Clinical signs	x	x	x	x	x	x
Body weight	x					x

Conclusions:

Under the present test conditions the test item was found to be not sensitising to guinea pigsin a test model according to
MAGNUSSON and KLIGMAN.

Executive summary:

The purpose of this study was to determine the potential of the test item to produce skin sensitisation reactions in guinea pigs in a test model according to MAGNUSSON and KLIGMAN.

A 10% solution of isopropylcyclohexane in sesame oilchosen for the 1st (intracutaneous) induction stage revealed a discrete or patchy erythema in all 20 animals 24 and 48 hours after administration.

2 mL of the undiluted test item/animal chosen for the 2nd (topical) induction stage were not irritating to the shaved skin in the preliminary experiment.

Hence, in the main study the skin was coated with sodium laurylsulfate on the day before stage 2 induction in order to induce a local irritation. This treatment resulted in a moderate and confluent erythema or a discrete or patchy erythema in all test group animals 48 or 72 hours after start of exposure.

The challenge with 2 mL of a 75% solution of isopropylcyclohexane in sesame oil/animal revealed no skin irritation in any animal and, thus, the test item had no sensitis­ing properties.

The vehicle control revealed no skin reactions.

Animals of the same strain treated with a-hexyl cinnamic aldehyde in sesame oil exhibited a sensitising reaction in all animals in form of

a discrete or patchy erythema (grade 1).

Behaviour of the animals remained unchanged.

Under the present test conditions, the test item was found to be not sensitising to guinea pigsin a test model according to MAGNUSSON and KLIGMAN.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The purpose of this study was to determine the potential of isopropylcyclohexane to produce skin sensitisation reactions in guinea pigs in a test model according to MAGNUSSON and KLIGMAN.

A 10% solution of isopropylcyclohexane in sesame oilchosen for the 1st (intracutaneous) induction stage revealed a discrete or patchy erythema in all 20 animals 24 and 48 hours after administration.

2 mL of the undiluted test item/animal chosen for the 2nd (topical) induction stage were not irritating to the shaved skin in the preliminary experiment.

Hence, in the main study the skin was coated with sodium laurylsulfate on the day before stage 2 induction in order to induce a local irritation. This treatment resulted in a moderate and confluent erythema or a discrete or patchy erythema in all test group animals 48 or 72 hours after start of exposure.

The challenge with 2 mL of a 75% solution of isopropylcyclohexane in sesame oil/animal revealed no skin irritation in any animal and, thus, the test item had no skin sensitising properties.

Justification for selection of skin sensitisation endpoint:
Only one study available. This study is a guideline study with Klimisch score 1 (reliable without restrictions)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the skin sensitization test according to MAGNUSSON and KLIGMAN in guinea pigs and according to the EC Regulation 1272/2008 and subsequent regulations on classification, labelling and packaging of substances and mixtures, isopropylcyclohexane has no skin senitizing properties and hence it does not require to be classified.