(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-07-1994 to 25-08-1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

methods of testing for skin sensitisation described in Document L 383 A (29 December 1992), Annex to Commission Directive 92/69/EEC (31 July 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: October 1992 ; signature: December 1992

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study conducted prior to Regulation (EC) 1907/2006 and/or Regulation (EC) 1272/2008 publication and implementation.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-120-6

EC Name:

-

Cas Number:

166432-53-7

Molecular formula:

C14H22O

IUPAC Name:

2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: in the refrigerator (ca. 4°C) in the dark
- Other: clear colourless liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult (approximately 3 months old).
- Weight at study initiation: 405 – 509 g
- Housing: group housed (5) in stainless steel cages
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test (19 days for main study).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2 °C
- Humidity (%): 30 – 70% (actual 51 – 91% - deviations were not considered to impact the integrity of the study)
- Air changes (per hr): at least 12 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 13-07-1994 to 25-07-1994

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

- Intradermal: 10% test material in light liquid paraffin

Day(s)/duration:

Day 1

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Induction 2

Route:

epicutaneous, occlusive

Concentration / amount:

- Topical: 100% test material (undiluted)

Day(s)/duration:

Day 8

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

- Challenge: 100%

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

- Challenge: 2% and/or 0.5% in acetone

Day(s)/duration:

Day 32

No. of animals per dose:

Test group: 20 ; Control group: 10

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. By intradermal route: tested concentrations: 50%, 25%, 10%, 5%, 1% and 0.5% (w/w). Cutaneous reactions were evaluated approximately 24, 48 hours and daily up to 6 days after the injections. By cutaneous route (occlusive dressing, 24 hours) tested concentrations: 100%, 50%, 25% and 12.5% (w/w). Reactions were evaluated approximately 24 and 48 hours after removal.
At 24 and 48 hours: intradermal and cutaneous/topical route 100%v/v was selected due to absence of irritation. Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 10% test material
- Topical: 100% test material (undiluted)
- Challenge: 1st challenge: 100% (undiluted) ; 2nd challenge: 0.5 and 2% in acetone

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 1 intradermal induction and Day 8 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in water; 2 ID: test item at 10% in liquid paraffin ; 2 ID: a test mixture of the test item at 50% in FCA at 50% and in water to give final concentration of 10.0%.
- Control group: Vehicle and 50:50 FCA in water, and vehicle at 50% in 50:50 FCA/water, only.
- Site: intradermal induction – three pairs of injections in clipped interscapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, clipped and SLS at 10% application. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) ID: Freund's Complete Adjuvant diluted at 50% in water ; B) test item at 10% in liquid paraffin ; C a test mixture of the test item at 50% in FCA at 50% and in water to give final concentration of 10.0%.. Topical induction: The scapular area between the injection sites was clipped and brushed with a solution of SLS at 10%. Then the following day subsequently treated with 0.5 mL of a 100% test item concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered along with injections of (A) and (C) in three sites.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 initial challenge ; 1 further challenge
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 22 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing. The second challenge was performed. The rest period was approximately 5 days.
- Test groups: 1 challenge; test item 100% and 2 challenge: 0.5% and 2%v/v in acetone.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 100% and 2 challenge: 0.5% and 2%v/v in acetone using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24) and/or following re-challenge on day 27.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study

Challenge controls:

(Naive) negative control groups consisting of 10 females were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test item only the vehicle was administered during induction.

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (10%w/w intradermal and 50%w/w topical)

Results and discussion

Positive control results:

A reliability check was performed (presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using Mercaptobenzothiazole (at 10%w/w intradermal induction and 50%w/w topical concentrations) as positive control.
The skin reactions observed in ten experimental animals in response to the > 20 % PC item concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 100% to the 10%w/w induction and 50%w/w challenge concentrations.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Total no. in groups: 20.0 ; Clinical observations: None reported; maximum score = 3 (n=5) to 2 (n=13) (discrete or moderate erythema).

Remarks on result:

positive indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Total no. in groups: 20.0 ; Clinical observations: None reported; maximum score = 3 (n=13) to 2 (n=6) (discrete or moderate erythema).

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 0 (n=10)

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 0 (n=10)

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10% intradermal induction and 50% cutaneous

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

non-concurrent PC conducted within 6 months of the definitive test; % sensitised was based on comparison of challenge sites with test and controls.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is considered to be a contact sensitizer.

Executive summary:

The study was performed using a method equivalent or similar to guideline OECD TG 406 and EU Method B.6 under GLP. The method was consistent with the Magnusson-Kligman Guinea Pig Maximisation test to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 10% in light paraffin oil vehicle and topical application at 100%, on days 1 and 8 respectively) with 10% SLS application on day 7. A treatment group of twenty and a control group of ten, respectively were on day 22, challenged with 100% (undiluted) test item along with parallel control challenged at 100% (undiluted) test item. The left flank was treated with vehicle only. Skin reactions were evaluated 24 to 48 hours after removal of the occlusive dressing. A second challenge was conducted on day 27 with 0.5% and 2% test item in acetone. No clinical signs were reported. Bodyweight gain in the treated group was comparable to controls. In the first challenge, an intense or moderate or discrete erythema (grade 3 or 2 or 1) was noted in 20/20 of the treated group and 0/10 of the control group. The maximum score in control was zero. In the second challenge utilising 2% test item in acetone, a discrete erythema (grade 1) was noted in 6/20 of the treated group and 0/10 of the control group. In the 0.5% test item in acetone and in control, the maximum score was zero. Under the conditions of this study, the test item is considered to be a contact skin sensitizer.