2-Cyclopentene-1-acetic acid, ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-20 -2012-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study reliable without restrictions Deviations from the EU method B.6 (2008) and OECD 406 (1992), which do not have an effect on the results: - positive control substance is not one of the preferred control substances by the guidelines. No justification was given. - only 0.3 mL instead of 0.5 mL of 10% sodium lauryl sulfate was used during the induction phase - a modified Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions was used

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 1992-07-17

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

, 2008

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

EC Number:

605-150-6

Cas Number:

15848-49-4

Molecular formula:

C9H14O2

IUPAC Name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Molecular formula: C9H14O2
- Molecular weight: 154.21 g/mol

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elm Hill Breeding Laboratories, Chelmsford, MA 01824
- Age at study initiation: 4 - 6 weeks (dose range-finding); 6 weeks (main study)
- Weight at study initiation: 300 - 462 (dose range-finding); 347 - 521 g (main assay)
- Housing: animals were housed (individually) in compliance with USDA guidelines.
- Diet (ad libitum): all animals had access to Harlan Teklad Guinea Pig diet (certified)
- Water (ad libitum): water
- Acclimation period: minimum of 7 days (main study animals); minimum of 5 days (dose range animals)

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 29°C
- Relative humidity: 10 to 70%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: mineral oil

Induction 2

Concentration / amount:

Intradermal induction:5% of test item
Topical induction: 100% of test item
Challenge: 100% of test item

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Challenge 2

Concentration / amount:

Intradermal induction:5% of test item
Topical induction: 100% of test item
Challenge: 100% of test item

No. of animals per dose:

Range finding test: 10 animals
Main study:
- Test article group: 10 males / 10 females
- Vehicle group: 5 males / 5 females
- Positive control group: 3 males / 3 females

Details on study design:

RANGE FINDING TESTS:
1) Intradermal dose range: prior to induction, the irritation potential of the test article was determined. Four naive animals were exposed to four different test article concentrations by the intradermal technique described in "Main study" below. In this test, both sides of the animal were clipped and exposed to concentrations of the test article (0.5, 1.0, 2.5 and 5.0%). For grading of the response, the procedure described below for primary challenge was used, except that only 24-hour grades were obtained.
2) Topical dose range: Four naive guinea pigs were exposed to four test article concentrations (1, 5, 15 and 25%). Based on these results two additional animals were exposed to three test article concentrations (50, 75, and 100%). The procedures described in the topical induction period was employed except wrapping was removed at 18 hours and 54 minutes (first topical dose range finding screening) and 20 hours (additional topical dose range finding screening) after dosing. Twenty-three hours and 49 minutes after unwrapping (first topical dose range finding screening) and twenty-one hours (± 2 hours)(additional topical dose range finding screening) after unwrapping the sites were depilated as described in the challenge section. three hours later (± 2 hours)(first topical dose range finding screening) and 5 hours and 38 minutes later (additional topical dose range finding screening) the sites were examined for skin reactions (24 hour grade). The location of each of the concentrations of test article differed in each of the four animals to compensate for any site-to-site variations.

Results:
Sligthly patch mild redness was observed in 3 of 4 animals at 5% and 2.5% intradermally and in two of four at both 0.5% and 1.0% intradermally. All other intradermal scores were zero. Grades of zero were observed in all the topical administrations of the test article during the dose ranging finding study. It was decided to proceed to the main study using a test article concentration of 5% intradermally and 100% topically.

MAIN STUDY
A. INDUCTION EXPOSURE
1) Intradermal induction stage:
The hair from an area over the shoulders of the guinea pigs was removed 24 to 48 hours prior to dosing using a clipper. The test article, positive and vehicle control groups with and without FCA were injected in the shoulder/intrascapular region of each animal. Respective animal groups received test article, control animals received vehicle and positive control materials were used in the positive control group.
Three pairs of 0.1 mL intradermal injections of each of the following , into each animal, were made in the clipped intrascapular/shoulder region.

Injection A = all animals - FCA (50:50 dilution with distilled water)
Injection B = test article concentration or vehicle for vehicle groups or positive control at 0.1% in saline for the positive group.
Injection C = test article concentration or vehicle emulsified with FCA and distilled water (50:50) or the positive control article at 0.1% in FCA 50:50.

The injection sites were just within the boundaries of a 2 x 4 cm patch, which were applied one week later for the topical induction.

2) Topical induction stage:
On Day 6, the test sites were reclipped free of hair. Since the test article was non-irritating, test and vehicle control sites were pretreated on Day 7 with 10% sodium lauryl sulfate (Sigma Aldrich, lot# 069K0344, exp. 26 Oct 2012) in petrolatum (0.3 mL). One week after intradermal injections, (Day 8) the day after the sodium lauryl sulfate pretreatment, the test article (at 100% - as received) was spread over a 2 x 4 cm filter paper, saturated (0.3 mL) and applied to the injection site area and occluded using Blenderm® tape.
The Blenderm® tape was held in place with an appropriate overlay bandage (Elastoplast). A minimum of forty-eight hours later the dressings were removed. This same procedure was employed with the vehicle control and the positive control materials in the vehicle and positive control animals, respectively.

B. CHALLENGE EXPOSURE
Fourteen days after the topical induction, the test article treated and vehicle and positive control animals were challenged with occluded patches for 24 hours on the right and left flanks. The hair was removed from these areas using a clipper 27-28 hours before application of the patch.
For the test groups a 2 x 2 cm filter paper was saturated (0.2 mL) with the test article (100%) and applied to the left flank. Another 2 x 2 cm filter paper was saturated (0.2 mL) with respective vehicle control (mineral oil) and applied to the right flank. For the vehicle groups, a 2 x 2 cm filter paper was saturated with the respective test article (100%) and applied to the right flanks. Another 2 x 2 filter paper was saturated with respective vehicle control (mineral oil) and applied to the left flank. The positive control animals were similarly treated with the positive control article (0.05% in petrolatum) on the left flank and the appropriate vehicle (petrolatum) on the right flank. The same occlusive technique as for topical induction was employed.
Approximately 24 hours later, the wrapping were removed and the sites wiped clean with gauze and water. Twenty-one± one hours after unwrapping, the sites were depilated with Nair® Lotion Hair Remover (Church & Dwight, lot#LL0050). The depilatory was placed on the test sites and surrounding area and left on for no more than 30 minutes. The depilatory was then thoroughly washed off with water and animals patted dry and returned to their cages. Three to five hours later the sites were graded for elicited skin reactions (24 hour grade). Approximately 24 (± 2 hours) hours later the sites were graded a second time (48-hour grade).

OTHER EXAMINATIONS:
- Mortality: once daily
- Clinical observations: once daily
- Dermal observations: animals were examined for signs of erythema, oedema and the responses scored twenty-four and forty-eight hours after unwrapping following the challenge. Dermal irritation was scored and manually recorded according to the grading system show in Table 1 (please refer to the field "Any other information on materials and methods incl. tables" below.
- Body weight: prior to dose administration and at final skin grading of the challenge

Challenge controls:

Vehicle control group: 5 males / 5 females
Challenge dose: 100% of test item

Positive control substance(s):

yes

Remarks:

1-chloro-2,4-dinitrobenzene (DNCB)(Supplier: Aldrich; Lot/batch#: 86996KJ) (Induction exposures of 0.1% DNCB in saline (intradermal exposure) and petrolatum (topical exposure). A topical challenge exposure of 0.05% DNCB in petrolatum and petrolatum alone.

Results and discussion

Positive control results:

The positive control group exhibited the anticipated positive reaction to validate the study. Moderate and diffuse redness and intense redness and swelling were observed (grade 2 to 3) at the 24 and 48 hour observations.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% of the test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0).

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% of the test item. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0). .

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% of the test item

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

Slight patchy mild redness (grade 1) was observed in one animal at the 48 hour observation. All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0).

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% of the test item. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Slight patchy mild redness (grade 1) was observed in one animal at the 48 hour observation. All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0). .

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100 % of the test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0).

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100 % of the test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0). .

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% of the test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0).

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100% of the test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0). .

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.05% of 1-chloro-2,4-dinitrobenzene

No. with + reactions:

6

Total no. in group:

6

Clinical observations:

Moderate and diffuse redness and intense redness and swelling were observed (grade 2 to 3). All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0).

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.05% of 1-chloro-2,4-dinitrobenzene. No with. + reactions: 6.0. Total no. in groups: 6.0. Clinical observations: Moderate and diffuse redness and intense redness and swelling were observed (grade 2 to 3). All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0). .

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.05% of 1-chloro-2,4-dinitrobenzene

No. with + reactions:

6

Total no. in group:

6

Clinical observations:

Moderate and diffuse redness and intense redness and swelling were observed (grade 2 to 3). All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0).

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.05% of 1-chloro-2,4-dinitrobenzene. No with. + reactions: 6.0. Total no. in groups: 6.0. Clinical observations: Moderate and diffuse redness and intense redness and swelling were observed (grade 2 to 3). All animals appeared normal throughout the course of the study. Vehicle control site showed no skin reaction (grade 0). .

Any other information on results incl. tables

EXAMINATIONS:

- Mortality and clinical signs: no mortality was observed and all animals appeared normal thorughout the course of the study.

- Body weights: no biologically relevant differences in the final body weights occurred.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is not considered to be a skin sensitiser.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not a skin sensitizer.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not a skin sensitizer.