Oligomerisation products of alpha-pinene and beta-pinene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February - 10 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

alpha-pinene and beta-pinene dimers

IUPAC Name:

alpha-pinene and beta-pinene dimers

Constituent 2

Reference substance name:

alpha-pinene and beta-pinene trimers

IUPAC Name:

alpha-pinene and beta-pinene trimers

Constituent 3

Reference substance name:

alpha-pinene and beta-pinene tetramers

IUPAC Name:

alpha-pinene and beta-pinene tetramers

Constituent 4

Reference substance name:

alpha-pinene and beta-pinene pentamers

IUPAC Name:

alpha-pinene and beta-pinene pentamers

Test material form:

liquid: viscous

Details on test material:

Batch No. 17-04 SI
Purity: 100% (UVCB substance)
Name of the test item (as cited in the study report): TERPENIC OLIGOMERS
Physical state: amber viscous liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Reassay date: 01 March 2018

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J (CBA/JRj)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs, Le Genest-Saint-Isle, France.
- Age at study initiation: 8 weeks
- Weight at study initiation: g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood wood flakes.
- Diet: Teklad Global 16% Protein Rodent Diet (Envigo, 2016), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: at least5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 10 changes/h
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary test: 100 % and 50 %
Main test: 10, 25 and 50 %

No. of animals per dose:

Preliminary test: 2 animals
Main test: 4 females/dose

Details on study design:

PRELIMINARY TEST:
- The mice were treated by daily application of 25 μL of the test item undiluted (100 %) or at 50% in AOO to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on Days 1, 3 and 6.
- Cutaneous reactions were noted at the concentration of 100 % but not at 50%. Therefore 50% was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Animals were randomly allocated to the groups.
- Name of test method: Local Lymph Node Assay (LLNA:BrdU)
- Criteria used to consider a positive response: Test item should be classified if at least one concentration of the test item results in a SI≥ 1.6, as sensitiser. Any test item failing to produce a SI> 1.6 was classified as a "non-sensitiser". However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of control and test item were applied to the dorsal surface of both ears on Days 1, 2 and 3. On Day 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. On day 6 (end of the test), the animals were were sacrificed by pentobarbital sodium and the auricular lymph nodes were excised. Then, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No data

Results and discussion

Positive control results:

Based on the EC1.6 of the current positive control, tested with HCA, the positive control is classified as sensitiser, as expected.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.34

Test group / Remarks:

Treated groups at 10%

Results 2

Parameter:

SI

Value:

1.65

Test group / Remarks:

Treated group at 25%

Results 3

Parameter:

SI

Value:

2.08

Test group / Remarks:

Treated group at 50%

Results 4

Key result

Parameter:

other: EC1.6

Remarks:

%

Value:

22.58

Any other information on results incl. tables

Clinical observations and mortality

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Bodyweights

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Local irritation

Dryness of the skin was noted on Day 6 in all animals treated with test item at 25 and 50%.

An increase in ear weight (+3%, +27% +47.8%) was noted in animals treated at 10, 25 and 50 %, respectively, compared with the control group.

An increase in ear thickness (+1.4%, +1.6% and +13.5%) was noted in animals treated at 10, 25 and 50%, respectively, compared with the control group.

 

Table 7.4.1/1: BrdU index & Stimulation index per group and calculation of EC_1.6

Groups	Test item	Brdu-index (Mean*)	Stimulation Index (SI)	Result
1	0 (AOO)	0.455	-	Negative
2	10 %	0.610	1.34	Negative
3	25 %	0.750	1.65	Positive
4	50 %	0.946	2.08	Positive

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item has to be classified as skin sensitiser category 1B (H317) in accordance with CLP Regulation (EC) No 1272/2008.

Executive summary:

In a local lymph node assay (LLNA:BrdU) performed according to OECD Guideline 442B and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of the registered substance at concentrations of 10, 25 and 50% to the dorsal surface of both ears for three consecutive days. Vehicle control group of four females received the vehicle (acetone/olive oil (4/1; v/v)). On Day 5, 0.5 mL of BrdU solution (10 mg/mL) was injected by intra-peritoneal route. On Day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit and Stimulation Indices (SI) were calculated. Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study.

No mortality and no signs of systemic toxicity were noted in the treated and control animals during the test.

Dryness of the skin was noted on Day 6 in all animals treated with the test item at 25 and 50%.

An increase in ear weight (+3%, +27% +47.8%) was noted in animals treated at 10, 25 and 50%, respectively, compared with the control group.

An increase in ear thickness (+1.4%, +1.6% and +13.5%) was noted in animals treated at 10, 25 and 50%, respectively, compared with the control group.

Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Stimulation Index was 1.34, 1.65 and 2.08 for the treated groups at 10, 25 and 50%, respectively. The EC1.6 was estimated to be 22.58% by linear regression.

Based on the EC1.6 of the current positive control, tested with HCA, the positive control is classified as sensitiser, as expected.

Therefore, the test item has to be classified as skin sensitiser category 1B (H317) in accordance with CLP Regulation (EC) No 1272 /2008.