Methyl 2-ethylhexanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP; minor deviation in test environmental conditions is judged not to have affected the reliability of the study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

The protocol requires that the animal room be humidity (30-70%) controlled. The humidity range fell below the range, to 29%, on one day during the study. This deviation had no material impact on the results of this study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

See Humidity comment above. The deviation was considered not to impact the results of this study.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-ethylhexanoate

EC Number:

212-429-2

EC Name:

Methyl 2-ethylhexanoate

Cas Number:

816-19-3

Molecular formula:

C9H18O2

IUPAC Name:

methyl 2-ethylhexanoate

Test material form:

other: liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Room temperature.
- Other: Clear colourless

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult
- Weight at study initiation: Not reported.
- Housing: The animals were group housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet (e.g. ad libitum): Pelleted Purina Guinea Pig Chow #5025
- Water (e.g. ad libitum): filtered tap water ad libitum
- Acclimation period: 8, 12, or 32 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 29-51
- Air changes (per hr):Not reported.
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: mineral oil

Induction 2

Concentration / amount:

Preliminary irritation testing: 1%, 3%, 5% test material
Induction:
- Intradermal: 5% test material
- Topical: 100% test material (undiluted)
- Challenge: 50%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Challenge 2

Concentration / amount:

Preliminary irritation testing: 1%, 3%, 5% test material
Induction:
- Intradermal: 5% test material
- Topical: 100% test material (undiluted)
- Challenge: 50%

No. of animals per dose:

Preliminary irritation testing: 8
Test group: 10
Sham group: 5

Details on study design:

RANGE FINDING TESTS:
Preliminary Intradermal Injection: Prior to the induction phase, two animals were used to determine the highest concentration of the test substance that causes mild to moderate irritation via intradermal injection. The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into six test sites (three sites on each side of the midline) on each animal. Each guinea pig received six intradermal injections (0.1 ml each); three concentrations (1, 3, and 5%) of the test substance in mineral oil and the same three concentrations in an emulsion of Complete Freund's Adjuvant. Approximately 24 hours after the injections, each site was evaluated for local reactions (erythema).

Preliminary Topical Booster. Prior to the topical booster induction, two animals were used to determine the highest concentration of the test substance that causes mild to moderate irritation. The previously clipped dorsal area of each guinea pig was divided into two sites (one site on each side of the midline). The test substance was applied neat (100%) and also diluted with a mineral oil to yield a w/w concentration of 75%. Each concentration was applied to one of the two test sites using a 2 cm x 4 cm, 2-ply gauze patch. The patch was covered with plastic wrap and secured in place with non-allergenic Durapore adhesive tape to avoid dislocation of the patch and to minimize loss of the test substance. After the 48-hour exposure period, the patches were removed and the test sites were gently cleansed of any residual test substance. One hour after patch removal, readings were made of local reactions (erythema).

Highest Non-irritating Concentration (HNIC): Prior to the challenge phase, four animals were used to determine the highest non-irritating concentration. The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal. The test substance was applied neat (100%) and also diluted with mineral oil to yield w/w concentrations of 75%, 50%, and 25%. Each concentration was applied to a test site using an occlusive 25 mm Hill Top Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 24-hours of exposure, the chambers were removed and the test sites were gently cleansed of any residual test substance. Twenty-four hours after patch removal, each site was evaluated for local reactions (erythema).

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal induction: intradermal injections of a 5% w/w mixture of the test substance in mineral oil, a 5% w/w mixture of the test substance in 50% v/v Complete Freund's Adjuvant in distilled water, and 50% aqueous Complete Freund's Adjuvant alone into the dorsal area of each of 10 guinea pigs. A sham group ( 5 animals) was maintained under the same environmental conditions and injected with mineral oil ( 100% ), mineral oil mixed with 50% v/v Complete Freund's Adjuvant in distilled water, and 50% aqueous Complete Freund's Adjuvant alone into the dorsal area.

Topical Application: Seven days after intradermal injections, the topical induction phase was conducted. Prior to the topical induction, the injection sites were re-clipped free of fur, and the shoulder area of each test group animal was pre-treated with sodium lauryl sulfate (SLS) in order to enhance sensitization by provoking a mild inflammatory reaction. The site remained uncovered until the topical induction patch was applied. Approximately 24 hours after SLS application, the test sites were gently cleansed of any residual SLS. Within one hour, readings were made of local reactions (erythema) according to the scoring system described. Following pre-induction scores, the animals were redipped and five-tenths of a milliliter of the test substance (100%) was applied to a single site on the dorsal midline, between the injection sites, of each test animal and covered with a 2 cm x 4 cm, 2-ply gauze patch. The patches were wrapped with plastic film wrap then secured in place with non-allergenic Durapore adhesive tape to avoid dislocation of the patches and to prevent evaporation. After the 48 hour exposure period, the patches were removed and the test sites were gently cleansed of any residual substance. One hour after patch removal, readings were made of local reactions (erythema). The sham group received the same treatment as described above, using 0.5 ml of mineral oil for the topical application.

B. CHALLENGE EXPOSURE
Twenty days after test initiation, a naive site on the right side of each animal in the test and sham groups was clipped free of fur. Four-tenths of a milliliter of the test substance at its HNIC (50% w/w mixture in mineral oil) was applied to a naive site on each test and sham animal using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic adhesive tape to avoid dislocation of the chambers and to prevent evaporation. After the 24 hour exposure period, the chambers were removed and the sites were gently cleansed of any residual test substance. Approximately 24 and 48 hours after patch removal, these sites were evaluated for a sensitization response (erythema).

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde, technical (HCA)

Results and discussion

Positive control results:

Induction:
- Historical Positive Control Animals (HCA technical, applied as received): Very faint to moderate erythema (0.5-2) was noted at all test sites following the topical induction phase.
- Historical Positive Vehicle Control Animals (mineral oil, 100% ): Very faint erythema (0.5) was noted at two of five vehicle control sites following the topical induction phase.
Challenge:
- Historical Positive Control Animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Eight of ten test animals exhibited signs of a sensitization response (faint to severe erythema [ 1- 3]) 24 hours following the challenge patch removal. Similar indications persisted at seven sites through 48 hours. Very faint erythema (0.5) was noted for two sites at the 24-hour and three sites at the 48-hour interval.
- Historical Positive Vehicle Control Animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Very faint erythema (0.5) was noted at three of five vehicle control sites 24 hours after challenge patch removal. All animals were free of irritation by 48 hours.
- Historical Positive Control Body Weights: All animals increased body weight during the course of the control study.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

1 test animal died prior to challenge

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 1 test animal died prior to challenge.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

1 test animal died prior to challenge

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 1 test animal died prior to challenge.

Any other information on results incl. tables

Preliminary test:

Based on these findings of the preliminary test, the concentration which produced mild to moderate irritation selected for the intradermal injections was a 5% w/w mixture in mineral oil. The concentration selected for topical induction was 100% with a pretreatment of SLS. The HNIC (the highest concentration that produced

responses in 4 guinea pigs no more severe than two scores of 0.5 and two scores of zero) established and used for challenge was a 50% w/w mixture in mineral oil.

Main Study:

Table 1. Challenge phase - skin sensitisation scores

Animal	Score - Hours after patch removal
	24	48
1	0.5	0.5
2	0.5	0
3	0	0
4	0	0
5	0	0
6	0.5	0.5
7	0.5	0.5
8	0.5	0.5
9	0	0
10	-*	-*
Sham
1	0	0
2	0	0
3	0.5	0.5
4	0.5	0.5
5	0	0

*Found dead prior to challenge dose.

Score system

0 - no reaction

0.5 - very faint erythema, usually non-confluent

1 - faint erythema usually confluent

2 - moderate erythema

3 - severe erythema with or without edema

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: US EPA pesticides

Conclusions:

Under the conditions of this study used, the test material is not considered to to be a contact sensitizer. The positive response observed in the historical positive control validation study with technical (HCA) alpha-Hexylcinnamaldehyde, validated the test system used in this study.

Executive summary:

The study was performed according to US EPA OPPTS 870.2600 and OECD 406 to assess the skin sensitisation potential of the test substance in accordance with GLP. The study was conducted using a preliminary irritation screen, a two-stage induction phase and a challenge phase. Preliminary irritation testing was performed on eight animals to establish the highest concentration of the test substance that causes mild to moderate irritation for use in the induction phases of the study and the highest non-irritating concentration (HNIC) of the test substance for use in the challenge phase of the study. The concentration of the test substance selected for the intradermal phase of the study was 5% w/w mixture in mineral oil. The undiluted test substance with the use of sodium lauryl sulfate was determined to be the concentration for the topical induction phase, and a 50% w/w mixture in mineral oil was selected as the HNIC for the challenge phase of the study. The first stage of the induction phase involved intradermal injections of a 5% w/w mixture of the test substance in mineral oil, a 5% w/w mixture of the test substance in 50% v/v Complete Freund's Adjuvant in distilled water, and 50% aqueous Complete Freund's Adjuvant alone into the dorsal area of each of 10 guinea pigs. A sham group ( 5 animals) was maintained under the same environmental conditions and injected with mineral oil ( 100% ), mineral oil mixed with 50% v/v Complete Freund's Adjuvant in distilled water, and 50% aqueous Complete Freund's Adjuvant alone into the dorsal area. The second stage of the induction phase entailed the topical application of the undiluted test substance onto the shaved dorsal area of the test group animals. Due to a lack of irritation produced during preliminary testing all animals received a pretreatment of sodium lauryl sulfate prior to test substance application. The sham group was treated with mineral oil for topical induction. Twenty days after test initiation, a challenge dose of the test substance (50% w /w mixture in mineral oil) was applied to a naive site on each test animal. The sham group was also treated with a 50% w/w mixture of the test substance in mineral oil for challenge. Approximately one hour after the topical induction patch removal and 24 and 48 hours after the challenge phase patch removal, the animals were evaluated for the presence of erythema. Under the conditions of this study, the test material is not considered to be a contact skin sensitizer.