Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The substance contains structural elements which may lead to false-positive results regarding LLNAs.

Test material

Test material information

Constituent 1

Reference substance name:

methyl 8-(3-octyloxiran-2-yl)octanoate; methyl 8-[3-({3-[(3-ethyloxiran-2-yl)methyl]oxiran-2-yl}methyl)oxiran-2-yl]octanoate; methyl 8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoate

EC Number:

605-143-8

Cas Number:

158318-67-3

Molecular formula:

Unspecified (UVCB)

IUPAC Name:

methyl 8-(3-octyloxiran-2-yl)octanoate; methyl 8-[3-({3-[(3-ethyloxiran-2-yl)methyl]oxiran-2-yl}methyl)oxiran-2-yl]octanoate; methyl 8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoate

Test material form:

liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, NL-5800 AN Venray
- Housing: in macrolone cages, two or three animals to a cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): at least 30%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.5 ml of undiluted test substance

Day(s)/duration:

6h

Adequacy of induction:

highest technically applicable concentration used

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

0.5ml of diluted test substance (75%)

Day(s)/duration:

6h

Adequacy of induction:

other: For the third induction, a reduced concentration (75%) of the test item was applied because of the development of increasing intensity of skin reaction observed in the test group on day 1 and 8 indicating an irritating effect of the undiluted substance

Challenge

Challenge 1

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

0.5ml of 50% test item

Day(s)/duration:

6h

Adequacy of challenge:

not specified

Challenge 2

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

0.5ml of the 75% test item

Day(s)/duration:

6h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20

Details on study design:

RANGE FINDING TESTS:
The test concentrations for the main study were selected based on the results of a preliminary investigation. Since none of the pre-test animals responded with skin reactions to the single treatment with the test item in concentrations of 25%, 50%, 75% and 100% (w/w) in the vehicle polyethylene glycol (PEG), the undiluted test item was determined to be used in both the induction phase and challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 (topical application for three consecutive weeks)
- Exposure period: 6h
- Test groups: 20 animals
- Control group: 10 animals
- Site: left flank
- Frequency of applications: three-times
- Concentrations: 1st and 2nd induction: 100%, 3rd induction: 75%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: Four weeks after the first induction
- Exposure period: 6h
- Test groups: 20 animals
- Control group: 10 animals
- Site: right cranial flank
- Concentrations: 75% (Re-challenge: 50%)
- Evaluation (hr after challenge): 24h and 48h after the challenge application.

Challenge controls:

The last positive control (PC) study with the reference material α-Hexylcinnamaldehyde, technical grade, 85 %, was conducted from Feb-2020 to Mar-2020.

Positive control substance(s):

yes

Results and discussion

Positive control results:

The last positive control (PC) study with the reference material α-Hexylcinnamaldehyde, technical grade, 85 %, was conducted from Feb-2020 to Mar-2020. In the PC test group, 11 of 20 guinea pigs (55 %) reacted with slight to moderate skin reactions (erythema grade 1 to 2).

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

9

Total no. in group:

10

Results 2

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Results 3

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Results 4

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

75%

No. with + reactions:

10

Total no. in group:

10

Results 6

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

75%

No. with + reactions:

10

Total no. in group:

10

Results 7

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

20

Total no. in group:

20

Results 8

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75%

No. with + reactions:

20

Total no. in group:

20

Results 9

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

85%

No. with + reactions:

11

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on these inconclusive results of the non-adjuvant sensitization study, the test item cannot be classified unambiguously as skin sensi­tizer or non-sensitizer.

Executive summary:

The dermal sensitizing potential of the test substance was investigated by means of the Buehler test.

The main study was performed on 30 guinea pigs in total arranged in a control group of 10 and a test group of 20 animals.

The test concentrations for the main study were selected based on the results of a pre­limi­nary investigation. Since none of the pre-test animals responded with skin reactions to the single treatment with the test item in concentrations of 25 %, 50 %, 75 % and 100 % (w/w) in the vehicle polyethylene glycol (PEG), the undiluted test item was determined to be used in both the induction phase and challenge phase.

The main study started with an induction phase including an occlusive patch topical appli­cation for 6 hours once a week for three consecutive weeks. The animals of the test group were induced twice with the 100 % (w/w) test item, whereas the animals of the control group were induced with the vehicle polyethylene glycol.

Due to occurrence of skin reactions with increasing intensity (grade 1 in 3/20 test group animals after 1^st induction; grade 1 in 7/20 and grade 2 in 12/20 test group animals after 2^nd induction), a reduction of test item concentration to 75 % (w/w) was determined for both the third induction treatment as well as the challenge treatment. In addition, for the third induction the application area was moved to untreated skin.

Erythema of grade 1 was observed in 4/20 test group animals after 3^rd induction with the 75 % (w/w) test item, whereas 10/20 test group animals displayed erythema of grade 2 and 6/20 test group animals even grade 3 erythema.

During the induction phase, none of the ten control group animals displayed any skin reactions.

Four weeks after the first induction, the 6-hour challenge procedure was conducted with an occlusive topical application of the test item on the right cranial flank of all animals in a concentration of 75%. A vehicle exposure took place on the right caudal flank. The skin reactions were evaluated 24 hours and 48 hours after the challenge application.

All animals of the test group (20/20; 100 %) and all animals of the control group (10/10; 100 %) responded with skin reactions to the challenge treatment with the test item in differing intensities. Erythema formation was assessed with grade 1 (7/10) and grade 2 (3/10) in the control group. On the contrary, distinct reaction including even grade-3-erythema (15/20) were observed in the test group among mild to moderate erythema ranging between grades 1 and 2 (5/20) 24 and 48 hours after removal of the patches.

Due to these ambiguous findings providing indications for both irritating effects as well as a sensitizing poten­tial of the test item, a re-challenge was performed using the same animals (test item concentration of 50 % w/w).

One week after the challenge, the 6-hour re-challenge procedure was conducted with an occlusive topical application of the test item on the left cranial flank of all animals. A vehicle exposure took place on the left caudal flank. The skin reactions were evaluated 24 hours and 48 hours after re-challenge application.

Again, all animals of the control group and all animals of the test group responded with skin reactions to the re-challenge treatment with the test item. Control animals displayed mild to moderate erythema (grade 1 and 2), whereas moderate to severe erythema (grade 2 and 3) were observed in the half of test group animals.

In both challenge experiments, stronger reactions in the test group than in the control group were observed, which may be considered as a hint for a skin sensitizing potential of the test item. However, since all animals of all groups showed a skin reaction, a delayed contact sensitization in the guinea pig can be neither excluded nor con­cluded based on the results of that study. Furthermore, the observed skin reactions in all animals of the control group give also evidence of an irritation potential of the chosen concentration

Based on these inconclusive results of the non-adjuvant sensitization study described in this final report, the test item cannot be classified unambiguously as skin sensi­tizer or non-sensitizer.