Ethyl 2-methylvalerate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February to 15 March 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

not specified

Principles of method if other than guideline:

The experiment was conducted essentially according to the guinea pig maximization test as described by B. Magnusson and A.M. Kligman (Allergic contact dermatitis in the guinea pig. Publ. Ch.C. Thomas: Springfield, Illinois, USA, 1970).

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study had already been conducted.

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-methylvalerate

EC Number:

254-384-1

EC Name:

Ethyl 2-methylvalerate

Cas Number:

39255-32-8

Molecular formula:

C8H16O2

IUPAC Name:

ethyl 2-methylpentanoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material (as cited in study report): Ethyl 2-methylpentanoate (charge no. A2-16)
- Physical state: colourless liquid
- Storage condition of test material: The test substance was stored at room temperature, until use
- Other: Test substance was received from Naarden International N.V., Naarden, the Netherlands, on February 4, 1986.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: SPF-bred, albino guinea pigs (Bor:DHPW)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding Centre for Laboratory Animals, F. Winkelmann GmbH, Borchen, (Hannover), F.R. - Germany. The animals were received on February 11, 1986 and the completion date of the test was March 15, 1986.
- Age at study initiation: Young guinea pigs
- Weight at study initiation: The main test was carried out with 20 males (body weight 319 - 407 g) and 20 females (body weight 310 - 378 g) without visible skin abnormalities.

- Preliminary tests (to establish the concentrations of the test substance to be used in the main study) were conducted on animals which had already served as controls in previous sensitization tests.

- Housing: During an acclimatization period of 8 days, and throughout the test period, the animals were kept individually under conventional conditions in suspended, stainless steel cages, fitted with wire mesh floor and front in an animal room conditioned with respect to temperature (18 ± 2°C), relative humidity (40 - 70%), ventilation (at least 10 air changes/hour) and lighting (12 hours light, 12 hours dark).
- Diet (e.g. ad libitum): The animals were fed a pelleted, natural ingredient diet (Hope Fans, Woerden, the Netherlands)
- Water (e.g. ad libitum): The animals received unfluoridated tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

unchanged (no vehicle)

Induction 2

Concentration / amount:

a. Intradermal injections
- Three pairs of intradermal injections were made simultaneously in the clipped area. The following preparations were injected:
- Test animals:
- two injections (0.1 ml) with Freund’s Complete Adjuvant (FCA)
- two injections (0.1 ml) with a 25% dilution (v/v) of ethyl 2-methylpentanoate in PG
- two injections (0.1 ml) with a 25% dilution (v/v) of ethyl 2-methylpentanoate in FCA and PG (1:1)

- Control animals:
- two injections (0.1 ml) with FCA
- two injections (0.1 ml) with PG
- two injections (0.1 ml) with FCA and PG (1:1)

- Skin readings were made 24 h after the treatment.

b. Topical application
- One week after the intradermal injections, the induction by topical application was made in the same scapular region. The area was closely shaved again. The test animals were treated as follows:
- A 2 x 4 cm patch of Whatman No. 3 MM filter paper was loaded with a 25% (w/w) mixture of the test substance with vaseline. The patch was placed over the sites of the intradermal injections and was secured. The dressing was left in place for 48 h. The control animals were similarly treated with patches loaded with vaseline only. Skin readings were made directly after removal of the patches.

- Challenge: The topical challenge was carried out two weeks after the topical induction as follows:
- An area of 5 x 5 cm of the right flank of each animal was clipped free of hair. Subsequently patches of 2 x 2 cm of Whatman No. 3 MM filter paper were loaded with a 25% (w/w) mixture of the test substance with vaseline. One loaded patch was placed in the centre of the shaved areas of each of the test and control animals. The patches were covered with PVC foil and Leukopor bandage, and held in place by Tensoplast for 24 h. Skin readings were made 24 and 48 h after removal of the patch.

Challenge

Challenge 1

Route:

epicutaneous, open

Vehicle:

unchanged (no vehicle)

Challenge 2

Concentration / amount:

a. Intradermal injections
- Three pairs of intradermal injections were made simultaneously in the clipped area. The following preparations were injected:
- Test animals:
- two injections (0.1 ml) with Freund’s Complete Adjuvant (FCA)
- two injections (0.1 ml) with a 25% dilution (v/v) of ethyl 2-methylpentanoate in PG
- two injections (0.1 ml) with a 25% dilution (v/v) of ethyl 2-methylpentanoate in FCA and PG (1:1)

- Control animals:
- two injections (0.1 ml) with FCA
- two injections (0.1 ml) with PG
- two injections (0.1 ml) with FCA and PG (1:1)

- Skin readings were made 24 h after the treatment.

b. Topical application
- One week after the intradermal injections, the induction by topical application was made in the same scapular region. The area was closely shaved again. The test animals were treated as follows:
- A 2 x 4 cm patch of Whatman No. 3 MM filter paper was loaded with a 25% (w/w) mixture of the test substance with vaseline. The patch was placed over the sites of the intradermal injections and was secured. The dressing was left in place for 48 h. The control animals were similarly treated with patches loaded with vaseline only. Skin readings were made directly after removal of the patches.

- Challenge: The topical challenge was carried out two weeks after the topical induction as follows:
- An area of 5 x 5 cm of the right flank of each animal was clipped free of hair. Subsequently patches of 2 x 2 cm of Whatman No. 3 MM filter paper were loaded with a 25% (w/w) mixture of the test substance with vaseline. One loaded patch was placed in the centre of the shaved areas of each of the test and control animals. The patches were covered with PVC foil and Leukopor bandage, and held in place by Tensoplast for 24 h. Skin readings were made 24 and 48 h after removal of the patch.

No. of animals per dose:

20 male and 20 female guinea pigs randomly divided into 2 groups: a control group and a test group of 10 males and 10 females each.

Details on study design:

RANGE FINDING TESTS:
- Preliminary tests: The irritation response upon intradennal injection of various concentrations was examined in 2 guinea pigs. A sufficiently large area of the scapular region was clipped free of hair with electric clippers. Subsequently 0.1 ml portions of 5, 12.5 and 25% (v/v) dilutions of the test substance in propylene glycol (PG), were applied to each animal by intra - dermal injection. Skin readings were made after 24 h. A dilution causing slight to moderate irritation is usually taken for intradermal injection of the test substance in the induction phase of the main test.
- The irritation response upon topical treatment was tested in 3 other guinea pigs. Both flanks of each animal were clipped free of hair with electric clippers. Subsequently several 2 x 2 cm patches of filter paper (Whatman No. 3 MM) were loaded with different concentrations of the test substance in vaseline, viz. 5, 12.5 or 25% (w/w). The patches were placed on the clipped skin and covered with a piece of PVC foil and hypoallergenic paper bandage (Leukopor) which was then secured by elastic adhesive bandage (Tensoplast), 7.5 cm in width, wound around the torso of the animal. Two concentrations were tested per animal. The dressing was left in place for 24 h. Then the animals were examined for signs of irritation. A concentration causing slight to moderate irritation is usually chosen for topical induction and a non - irritant concentration for topical challenge.

MAIN STUDY:
- 20 male and 20 female guinea pigs randomly divided into 2 groups: a control group and a test group of 10 males and 10 females each. The animals were weighed just before the test was initiated. The test consisted of two parts:
a) induction by intradermal injection and topical application
b) challenge by topical application

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

2 animals died from rectal prolapse

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Group:

negative control

Remarks on result:

not measured/tested

Results 4

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

Preliminary tests: Intradermal treatment with 25% ethyl 2-methylpentanoate caused severe erythema and abscesses. Slight to moderate erythema and abscesses were induced by the 5 and 12.5% concentrations. Topical application of 5, 12.5 or 25% ethyl 2-methylpentanoate in vaseline induced no erythema. Based on these results a 25% concentration was used for intradennal injections as well as for challenge treatment in the main test.

 

Main test: Four males (two controls and two test animals) and three females (two controls and one test animal) died in the course of the study, because of a rectal prolapse. All other animals remained in good health during the experimental period.

- Induction: The scores of the skin reactions made during the induction phase of the study are given in appendix 2 of attached report. The intradermal injections caused the following reactions:

- with Freund’s Complete Adjuvant (FCA): moderate to severe erythema and slight oedema in all test and control animals

- with the 25% dilution of the test substance in PG: moderate to severe erythema and abscesses in all test animals

- with PG alone: slight to moderate erythema and abscesses in all controls

- with the 25% dilution of the test substance in a mixture of FCA and PG (1:1): moderate to severe erythenia and abscesses in all test animals and slight oedema in nearly all test animals

- with the mixture of FCA and PG alone: moderate to severe erythema and abscesses in all control animals and slight oedema in nearly all animals.

The topical applications of the 25% dilution of the test substance in vaseline, made in the induction phase, did not induce erythema in any of the test animals. Vaseline alone did not induce skin reactions in any of the controls.

- Challenge: The results of the challenge treatment are given in table 1 of attached report. The challenge treatment with the 25% dilution of the test substance in vaseline did not induce skin reactions in any of the test animals or the controls, either after 24 h or after 48 h.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

From the results of the challenge treatment with the 25% dilution it appeared that the test substance did not induced signs of delayed hypersensitivity. Therefore, the test substance is considered to be a non - sensitizer.

Executive summary:

1. The test substance ethyl 2 - methylpentanoate was examined for possible sensitizing properties by a maximization test in guinea pigs using 20 control and 20 test animals.

2. The test comprised:

- induction treatment by intradermal injection of 25% test substance in propylene glycol, followed one week later by a topical treatment with 25% in vaseline,

- challenge treatment, 14 days after the last induction, by topical application of 25% in vaseline,

3. The challenge treatment with the 25% dilution did not induce signs of sensitization in any of the guinea pigs under the conditions of the test. Therefore, it was concluded that the test substance is a non - sensitizer.