1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-10-2015 to 27-11-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. Information as to the availability of the study is attached by the applicant.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according 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:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147 (2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: April 2015; signature: October 2015

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Scientific justification and information as to the availability of the study is attached by the applicant in 'attached background material'.

Test material

Test material information

Constituent 1

Reference substance name:

1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

EC Number:

807-612-4

Cas Number:

1393645-32-3

Molecular formula:

C14H22O

IUPAC Name:

1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (6 ± 3°C) in the dark
- Other: colourless

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult (approximately 3- 4 weeks old).
- Weight at study initiation: 262 – 286 g (mean weight: 272.2 g).
- Housing: Individually or group housing of 3 per labelled polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid.
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 – 70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 26-10-2015 to 27-11-2015

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

olive oil

Concentration / amount:

- Intradermal: 20% test material in Olive Oil

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Remarks:

olive oil for the intradermal injections and in liquid paraffin for the topical applications

Concentration / amount:

- Challenge: 50% in liquid paraffin

Day(s)/duration:

Day 21

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group: 10; Control group: 5

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. Pre-MNNC: Two received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in olive oil in view to determine the MNNC with macroscopic evaluation at 24 hours. PreMNIC: Two were treated with test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after dressing removal. MNIC: This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase. Three were treated according to the same treatment as animals from GROUP 1 (control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: diluted 50%v/v, 30%v/v, 20%v/v and 10%v/v in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after dressing removal.

Preliminary irritation testing: A series of test item concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%. This determined the Maximal Non Necrotizing Concentration (MNNC); Pre-Maximal Non Irritant Concentration (Pre-MNIC) and the Maximal Non Irritant Concentration (MNIC) respectively.
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 20% test material in Olive Oil
- Topical: 100% test material (undiluted)
- Challenge: 50% in liquid paraffin
% are percent v/v of test substance in vehicle. Vehicle: Olive oil (intradermal induction) and liquid paraffin (topical), was chosen on the basis of the most suitable formulation at the required concentrations and maximising the solubility of the test substance. The preparation of the test item at 50% in physiological solution (v/v) was non-homogenous with two phases, within in pre-testing solubility testing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 0 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 olive oil; 2 ID: test item at 20% in olive oil ; 2 ID: a test mixture in equal volumes v/v of FCA at 50% and the test item at 40% in olive oil
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, shorn 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) A 1:1 w/w mixture of Freunds' Complete Adjuvant with olive oil for injection; B) The test substance at a 20% concentration ; C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant. 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 substance concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 21 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 and 72 hours after removal of the dressing.
- Test groups: 1; test substance 50% in liquid paraffin vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 50% using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 and 72 hours after dressing removal (at Day 23 and 24 and 25).
The control group were treated as described for the experimental group except that, instead of the test substance, 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 5 females were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test substance only the vehicle was administered during induction.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamicaldehyde (20%)

Results and discussion

Positive control results:

A reliability check was performed (three tests are listed at 6, 12 and 18 months of the study and 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 Alpha-Hexylcinnamicaldehyde (at 6.25 and 12.5 concentrations %v/v) as positive control.
The skin reactions observed in seven experimental animals in response to the > 20 % test substance 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 40 - 90% to the 12.5 %w/w concentration.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 3 (intense eryhema and swelling).

Remarks on result:

positive indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 3 (Intense erythema and swelling).

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

50%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 2 (Moderate and confluent erythema).

Remarks on result:

positive indication of skin sensitisation

Results 4

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Total no. in groups: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Total no. in groups: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Results 6

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Total no. in groups: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

12.5%

No. with + reactions:

9

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.

Any other information on results incl. tables

Table 1: Preliminary irritation study

Skin reactions after intradermal injection (24 hours)

Number	100%v/v	50%v/v	20%v/v	10%v/v
1	Ne	SINe	0	0
2	SINe	0	0	0

Ne = Necrosis

SINe = Slight Necrosis

0 = No visible change

MNNC: 20% in olive oil

 

Table 2: Preliminary irritation study

Skin reactions after topical application (24 hours)

Number	100%v/v	50%v/v	20%v/v	10%v/v
1	1	0	0	0
2	0	0	0	0

0 = No visible change

Pre-MNIC: 100%

 

Table 3. Preliminary irritation study

Number	Hours	50%v/v	30%v/v	20%v/v	10%v/v
1	24	0	0	0	0
	48	0	0	0	0
2	24	0	0	0	0
	48	0	0	0	0
3	24	0	0	0	0
	48	0	0	0	0

MNIC: 50% in liquid paraffin

 

Table 4.

		20% MNIC		Liquid Paraffin
	Number	24 hours	48 hours	24 hours	48 hours
Control	1	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
	4	0	0	0	0
	5	0	0	0	0
Test group	1	3	2	0	0
	2	2	0	0	0
	3	0	0	0	0
	4	1	0	0	0
	5	2	3	0	0
	6	3	3	0	0
	7	1	2	0	0
	8	1	0	0	0
	9	1	0*	0	0
	10	0	0	0	0

Where:

0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

* = dryness

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 according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test substance. Test item concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 20% and topical application at 100%) and a ten day rest phase of ten experimental animals were challenged with 50%v/v in liquid paraffin along with parallel control challenged at test item 50%v/v in liquid paraffin in five animals. The treated group (treatment dose of 50%v/v in liquid paraffin), a discrete to intense erythema was recorded in 80% (8/10), 40% (4/10) and 40% (4110) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. In the control group (associated with the treatment dose of 50%), no cutaneous intolerance reaction was recorded at 24, 48 and 72 hours.No cutaneous reaction was recorded from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item). There was no abnormal clinical signs or abnormal bodyweight gain during the test. Under the conditions of this study, the test item is considered to be a contact skin sensitizer.