cis-2-methyl-4-propyl-1,3-oxathiane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-11-2002 to 28-11-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP; minor deviations in this test is judged not to have affected the reliability of the study.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

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:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

see 'any other information on materials and methods incl. tables

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

see 'any other information on materials and methods incl. tables

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In accordance with REACH Regulation (EC) 1907/2006: Annex VII: column 2 as amended by Commission Regulation (EU) 2017/706, a well documented study report conducted before 10 May 2017, following a method equivalent or similar to guideline with acceptable deviations is available. This is sufficient to fulfil the standard information requirement in accordance with REACH Regulation (EC) 1907/2006: Annex XI: section 1.1.2 since adequate and reliable (with restrictions) study information has been provided suitable for classification and labelling and/or risk assessment.

Test material

Test material information

Constituent 1

Reference substance name:

2-Ethyl-4,4-dimethyl-1,3-oxathiane

IUPAC Name:

2-Ethyl-4,4-dimethyl-1,3-oxathiane

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Refrigerated.
- Other: Clear colourless, Solubility: Soluble in water, Soluble in mineral oil

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult
- Weight at study initiation: 300-570g
- 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: 5 or 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 53-70
- 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

Route:

intradermal and epicutaneous

Vehicle:

other: mineral oil

Concentration / amount:

Preliminary irritation testing: intradermal injection: 1%, 3%, 5% test material; topical: 75%, 100%
Induction:
- Intradermal: 5% test material
- Topical: 100% test material (undiluted)
- Challenge: 75% test material

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

Preliminary irritation testing: intradermal injection: 1%, 3%, 5% test material; topical: 75%, 100%
Induction:
- Intradermal: 5% test material
- Topical: 100% test material (undiluted)
- Challenge: 75% test material

No. of animals per dose:

Preliminary irritation testing: 8
Test group: 20
Sham group: 10
Naive control group: 10

Details on study design:

RANGE FINDING TESTS:
Preliminary Intradermal Injection: Prior to the induction phase, two animals were used to determine the minimally irritating concentration (MIC) of test substance 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. The test substance was diluted with mineral oil or 50% Complete Freund's Adjuvant to yield concentrations of 5%, 3% and 1% w/w. Each mixture was injected into one of the six test sites. Twenty-four 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 minimally irritating concentration. 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 concentration of 75% w/w. Each concentration was applied to one of the two test sites using a 2 cm x 4 cm 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 wiped with ethanol then water and a clean towel to remove any residual test substance. Twenty-four after patch removal, readings were made of local reactions (erythema).

Highest Non-irritating Concentration (HNIC): Prior to the challenge phase, a group of animals was 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 concentrations of75%, 50% and 25% w/w. Each concentration was applied to a test site using an occlusive 25 mm Hill Top Chamber®. The sites were wrapped with nonallergenic Durapore adhesive tape. After 24-hours of exposure, the chambers were removed and the test sites were gently wiped with ethanol then water, using a clean towel to remove 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:
On the first day of the induction period, the test animals received six intradermal injections (0.1 ml each) in the shaved anterior shoulder as follows:
- 50% complete Freund's adjuvant solution
- test substance as a 5% w/w mixture in mineral oil
- 5% w/v mixture of the test substance in Freund's adjuvant solution
[Each were intradermally injected once into the left upper back and once in the right upper back].

The sham treated animals received six intradermal injections (0.1 ml each) in the clipped dorsal area as follows:
- 50% complete Freund's adjuvant
- undiluted vehicle (mineral oil)
- 50% w/v mixture of the vehicle in Freund's adjuvant solution
[Each were intradermally injected once into the left upper back and once in the right upper back].

Topical Application: Seven days after intradermal injections, the shoulder area over the injection sites was clipped free of fur. 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 48hour exposure period, the patches were removed and the test sites were wiped gently with ethanol then water, using a clean towel to remove any residual substance. One hour after patch removal, readings were made of local reactions (erythema). Due to a technical error, the test sham group was exposed to the test substance as well. As a result of this error, a new group of ten animals were placed on test to serve as a naive control group for challenge.

B. CHALLENGE EXPOSURE
Twenty-one days after test initiation, a naive site on the right side of each animal in the test group and naive control group was clipped free of fur. Four-tenths of a milliliter of the test substance at its HNIC (75% w/w mixture in mineral oil) was applied to a naive site on each test and naive control animal using an occlusive 25 mm Hilltop Chamber®. The chambers were secured in place and wrapped with non-allergenic Durapore 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 wiped with ethanol then water, using a clean towel to remove any residual test substance. Approximately 24 and 48 hours after patch removal, these sites were evaluated for a sensitization response (erythema).

Challenge controls:

Due to a deviation it was necessary to assign a naive group to the study to replace the sham control animals that were mistakenly dosed with the test substance at topical induction. A group of 10 animals (male and female) were placed on test to serve as the naive control group. Following the same procedures used for the test group, these animals received a single dose of the HNIC (highest non irritating concentration) of the test substance at challenge only. The response in these animals was compared to the response observed in the test group to determine if the test animals exhibited a sensitization reaction.

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde, technical (HCA)

Results and discussion

Positive control results:

Induction:
- Historical Positive Control Animals (100% HCA): Very faint to moderate erythema (0.5-2) was noted at all test sites during the topical induction phase.
- Historical Positive Control Sham Animals (100% mineral oil): Very faint to faint erythema (0.5-1) was noted at all test sites during the topical induction phase.

Challenge:
- Historical Positive Control Animals (50% HCA in mineral oil): Nine of the ten test animals exhibited signs of a sensitization response (faint to moderate erythema [1-2]) 24 hours after patch removal. Faint erythema (1) persisted at seven sites through 48 hours. Desquamation was evident at several sites following the challenge phase.
- Historical Positive Control Sham Animals (50% HCA in mineral oil): Very faint erythema (0.5) was noted at four positive control sham sites 24 hours after challenge. Irritation persisted at one site through 48 hours.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75%

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

4 animals displayed very faint erythema (score: 0.5)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: 4 animals displayed very faint erythema (score: 0.5).

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

75%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Naive control group

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 75% . No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: Naive control group.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Naive control group

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Naive control group.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50%

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 50%. No with. + reactions: 9.0. Total no. in groups: 10.0.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50%

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 50%. No with. + reactions: 7.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Topical induction evaluation:

Faint erythema was noted at all test sites one hour after removal of the topcial induction patch.

Preliminary test:

Based on these findings, the minimally irritating concentration (MIC) (a concentration which produced mild to moderate irritation) concentration selected for the intradermal injections was 5%. The MIC selected for topical induction was 100%. The highest non-irritating concentration (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) was established and used for challenge. The HNIC selected for the challenge phase was 75%.

Main Study:

Table 1. Challenge phase - skin sensitisation scores

Animal	Score- Hours after patch removal
Test material group	24	48
1	0	0
2	0	0
3	0	0
4	0.5	0
5	0	0
6	0.5	0
7	0	0
8	0	0
9	0	0
10	0.5	0
11	0	0
12	0	0
13	0	0
14	0	0
15	0	0
16	0	0
17	0	0
18	0	0
19	0.5	0
20	0	0
Naive control group
1	0	0
2	0	0
3	0.5	0
4	0	0
5	0	0
6	0.5	0
7	0	0
8	0	0
9	0.5	0
10	0	0

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:

Criteria used for interpretation of results: EU

Conclusions:

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

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 item 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 item 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 item for use in the challenge phase of the study. The concentration of the test item selected for the intradermal phase of the study was 5% w/w mixture in mineral oil. The undiluted test item was determined to be the concentration for the topical induction phase, and a 75% 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 item in mineral oil, a 5% w/w mixture of the test item 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 20 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 item onto the shaved dorsal area of the test group animals. As a result of this error, a new group of ten animals were placed on test to serve as a naive control group for challenge. Twenty days after test initiation, a challenge dose of the test item (75% w /w mixture in mineral oil) was applied to a naive site on each test animal. The naive group was also treated with a 75% w/w mixture of the test item 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 item is not considered to be a contact skin sensitizer.