Ethyl 2-cyclohexylpropionate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 november to 14 December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to the EEC Method B.6, Part B and it is GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-cyclohexylpropionate

EC Number:

412-280-5

EC Name:

Ethyl 2-cyclohexylpropionate

Cas Number:

2511-00-4

Molecular formula:

C11H20O2

IUPAC Name:

ethyl 2-cyclohexylpropanoate

Details on test material:

- Physical state: clear liquid
- Storage condition of test material: 4ºC in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dutch

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D.Hall, Newchurch, Staffordshire, England
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 299-358g
- Housing: in group of 10 in suspended metal cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

coconut oil

Induction 2

Concentration / amount:

Table 4 and 5

Challenge

Challenge 1

Route:

other: topical induction

Vehicle:

coconut oil

Challenge 2

Concentration / amount:

Table 4 and 5

No. of animals per dose:

30

Details on study design:

POSITIVE CONTROL
-Results in table 6
TREATMENT PROCEDURE
Preliminary study
- Results in table 4
Induction:
- Intradermal injection: 20% v/v in Alembicol D
- Topical application: As supplied
This was the maximum practical concentration and did not give rise to irritating effects
CHALLENGE
Topical applications: as supplied
From preliminary investigations the test substance, as supplied was the maximum practical concentration and did not give rise to irritating effects.
Main study: the procedure may be considered in two parts, induction and challenge.
INDUCTION
Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant* was diluted with an equal volume of water for irrigation (Ph.eur.)
2. The test substance, 20% v/v in Alembicol D.
3. The test substance, 20% v/v in a 50:50 mixture of Freund's complete adjuvant and Alembicol D.
Control animals: Table 1
CHALLENGE
The control and test animals were challenge topically two weeks after the topical induction applications using the test substance, as supplied and 50% v/v in Alembicol D.

Positive control substance(s):

yes

Results and discussion

Positive control results:

Health status:
No signs of ill health or toxic were recorded
Induction:
Dermal reactions seen following the induction applications are summarised in Table 1:
Intradermal injections:
Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals.
Irritation was seen in test animals at sites receiving the test substance, 20% v/v in Alembicol D. Very slight irritation was observed in control animals.
Topical applications:
Moderate erythema was observed in test animals following topical application with the test subtance, as supplied.
Similar signs of irritation were seen in the controls.
Challenge:
The numerical values given to the dermal reactions elicited by the challenge application are shown in Table 2.
The dermal response seen in test and control animals was similar.

Any other information on results incl. tables

Table 1
Dermal reactions observed after each induction
Site	Intradermal injection		Topical applications
	Test animals	Control animals	Test animals	Control animals
1	Necrosis	Necrosis	Moderate erythema	Moderate erythema
2	Irritation	Necrosis
3	Necrosis	Necrosis

Table 2
Dermal reactions observed after the challenge application with the test substance
Guinea-pig number	E=Erythema O=Oedema	Score						Results Positive (+) Negative (-) Inconclusive (±)
		24 hours		48 hours		72 hours
		A	P	A	P	A	P
4900	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4901	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4902	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4903	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4904	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4905	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4906	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4907	E	0	0	0	0	0*	0	-
	O	0	0	0	0	0	0	-
4908	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4909	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4910	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4911	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4912	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4913	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4914	E	0*	0	0*	0	0*	0	-
	O	0	0	0	0	0	0	-
4915	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4916	E	0*	0	0*	0	0*	0	-
	O	0	0	0	0	0	0	-
4917	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4918	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4919	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4920	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4921	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4922	E	0*	0	0*	0	0*	0	-
	O	0	0	0	0	0	0	-
4923	E	0	0	0	0	0*	0	-
	O	0	0	0	0	0	0	-
4924	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4925	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4926	E	0	0	0	0	0*	0	-
	O	0	0	0	0	0	0	-
4927	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-
4928	E	0	0	0	0	0*	0	-
	O	0	0	0	0	0	0	-
4929	E	0	0	0	0	0	0	-
	O	0	0	0	0	0	0	-

*: Dryness and sloughing of the epidermis

a: Anterior site, exposed to the test substance, as supplied

P: Posterior site, exposed to the test substance, 50% v/v in Alembicol D

Table 3
Group	Guinea-pig number	At start of study 19.11.91	at finish of study
Control	4900	475	632
	4901	548	655
	4902	484	689
	4903	472	592
	4904	458	603
	4905	510	642
	4906	468	644
	4907	503	698
	4908	487	610
	4909	487	621
Test	4910	446	552
	4911	424	553
	4912	443	570
	4913	435	562
	4914	401	534
	4915	451	627
	4916	456	604
	4917	480	649
	4918	453	597
	4919	423	585
	4920	442	574
	4921	454	603
	4922	502	609
	4923	429	571
	4924	469	626
	4925	454	614
	4926	453	594
	4927	464	666
	4928	458	586
	4929	474	594

Table 4
Results of preliminary investigations with the test substance
Intradermal injections
Vehicle: Alembicol D
Guinea-pig number	Concentration % v/v	Score
		Hours	24	72
471	30	D	10	10
		E	SN	SN
		O	2	2
	20	D	8	8
		E	2	2
		O	2	1
454	10	D	10	10
		E	2	2
		O	2	2
	7,5	D	10	10
		E	2	2
		O	2	2
	5	D	8	8
		E	2	2
		O	2	2
	2,5	D	8	8
		E	2	2
		O	2	2
	1	D	8	8
		E	2	2
		O	2	2
	0,5	D	6	6
		E	2	2
		O	2	2
	Vehicle control	D	3	3
		E	1	1
		O	1	1
468	30	D	10	10
		E	SN	N
		O	2	2
	20	D	8	8
		E	2	2
		O	2	2
456	10	D	10	10
		E	2	2
		O	2	2
	7,5	D	10	10
		E	2	2
		O	2	2
	5	D	8	8
		E	2	2
		O	2	2
	2,5	D	8	8
		E	2	2
		O	2	2
	1	D	8	8
		E	2	2
		O	2	2
	0,5	D	6	6
		E	2	2
		O	2	2
	Vehicle control	D	3	3
		E	1	1
		O	1	1
D: Diameter (mm)
E: Erythema (0-4 numerical scores)
O:Oedema (0-4 numerical scores)
N: Necrosis
SN: Slight necrosis

Table 5
Topical appliaction
Vehicle: Alembicol D
Guinea-pig number	Concentration % v/v	Score
		0 Hours		24 Hours		48 Hours
		E	O	E	O	E	O
450	As supplied	0	0	0	0	0	0
	75	0	0	0	0	0	0
	60	0	0	0	0	0	0
	50	0	0	0	0	0	0
451	As supplied	0	0	0	0	0	0
	75	0	0	0	0	0	0
	60	0	0	0	0	0	0
	50	0	0	0	0	0	0
448	As supplied	0	0	0	0	0	0
	75	0	0	0	0	0	0
	60	0	0	0	0	0	0
	50	0	0	0	0	0	0
455	As supplied	0	0	0	0	0	0
	75	0	0	0	0	0	0
	60	0	0	0	0	0	0
	50	0	0	0	0	0	0
E: Erythem (0-4 numerical scores)
O: Oedema (0-4 numerical scores)

Table 6
Summary of positive control data Magnusson and Kligman test using formalin
M&K R&D No.	Numerical of female animals		Dates of study		Dates of study of receipt of Formalin	Dose Levels % (aqueous dilutions)			Results
						Induction		Challenge	Positive	Inconclusive	Negative
	Test	Control	Start	Finish		Intradermal	Topical
20	20	20	06.01.86	03.02.86	11.12.85	0,05	10	5 and 2,5	10/20	7/20	3/20
21	20	20	07.04.86	02.05.86	11.12.85	0,1	10	5 and 1	17/20	2/20	1/20
22	20	10	09.06.86	04.07.86	11.12.85	0,1	10	5 and 1	19/19*	0/19*	0/19*
23	10	10	16.02.87	13.03.87	09.12.86	0,1	10	5 and 1	10/10	0/10	0/10
24	10	10	23.03.87	17.04.87	09.12.86	0,1	10	5 and 1	10/10	0/10	0/10
25	10	10	30.06.87	25.07.87	09.12.86	0,1	10	5 and 1	8/10	0/10	2/10
26	10	10	18.01.88	12.02.88	05.08.87	0,1	10	5 and 1	10/10	0/10	0/10
27	10	10	21.07.88	15.08.88	13.05.88	0,1	10	5 and 1	10/10	0/10	0/10
28	10	10	10.01.89	04.02.89	16.12.88	0,1	10	5 and 1	10/10	0/10	0/10
29	10	10	17.07.89	11.08.89	15.05.89	0,1	10	5 and 1	10/10	0/10	0/10
30	I 10	10	31.10.89	25.11.89	15.05.89	0,1	10	5 and 1	10/10	0/10	0/10
31	10	10	16.01.90	10.02.90	29.11.89	0,1	10	5 and 1	10/10	0/10	0/10
32	10	10	23.07.90	24.08.90	04.07.90	0,05	5	3 and 1,5 5 and 3***	5/10 10/10	0/10 0/10	5/10 0/10
33	10	10	30.10.90	24.11.90	04.07.90	0,1	10	5 and 1	10/10	0/10	0/10
34	10	10	15.01.91	09.01.91	09.01.91	0,1	10	5 and 1	8/10	1/10	1/10
35	10	10	06.03.91	31.03.91	09.01.91	0,1	10	5 and 1	10/10	0/10	0/10
36	10	10	12.03.91	06.04.91	09.01.91	0,1	10	5 and 1	10/10	0/10	0/10
37	10	10	15.08.91	09.09.91	19.06.91	0,1	10	5 and 1	10/10	0/10	0/10

I Animals supplied by Interfauna, Huntingdon, Cambridgeshire, England

All other animals supplied by D.Hall, Newchurch, Staffordshire, England

(Fromain obtained from Savory and Moore, Huntingdon, Cambridgeshire, England or from 1991, supplied by Thornton and Ross, Hunddersfield, England)

* One animal excluded due to poor health status

** Second challenge

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this tes, performed in twenty albino guinea-pigs of the test substance did not produce evidence of skin sensitisation (delayed contact hypersensitivity).

Executive summary:

The study was performed to assess the skin sensitisation potential of the test substance in the guinea-pig. The method followed was that described in the EEC Methods for the determianation of toxicity, Directive 84/449/EEC Part B, Method B.6. Acute toxicity (skin sensitization).

Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected:

- Intradermal injection: 20% v/v in Alembicol D.

- Topical application: as supplied.

- Challenge application: as supplied and 50% v/v in Alembicol D.

Twenty test and ten conrol guinea-pigs were used in this study.

The test substance did not produce evidence of skin sensitisation (delayed contact hypersensitivity).

The test substance does not require labelling with the risk pharse R43 "May cause sensitization" by skin contact" in accordance with Council Directive 79/831/EEC Annex V1, Part II (D) as described in Commission Directive 91/325/EEC.