Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Endpoint summary

Description of key information

The test item H-32 was not a skin sensitiser under the test conditions of this study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 2017 -12 April 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:Main study: 8 - 9 weeks
- Weight at study initiation:18-20g
- Housing:group
- Diet (e.g. ad libitum):2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period:At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2°C
- Humidity (%):45-65%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light):artificial light 6.00 a.m. - 6.00 p.m.
- IN-LIFE DATES: From: To:

Vehicle:

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

Concentration:

The test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

No. of animals per dose:

4 animal per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (4+1, v/v) as vehicle.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of systemic toxicity. The animals showed an erythema of the ear skin on several days, slight erythema of the scalp, and slight eschar formation on day 6. Additionally, the animal treated with 50% test item concentration showed an increase in ear weight above the threshold of 25%, and the animal treated with the undiluted test item showed an increase in ear weight just below the threshold of 25% (i.e., 24% increase). Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. At the test concentrations, the animals showed an erythema of the ear skin on several days, but no eschar formation or other signs of excessive skin irritation.


MAIN STUDY

Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Skin Sensitisation Local Lymph Node Assay.
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:

The test item was placed into an appropriate container on a tared balance and acetone/olive oil (4+1, v/v) was added (w/w). The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in acetone/olive oil (4+1, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Positive control substance(s):

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

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2016

Key result

Parameter:

SI

Value:

2.44

Test group / Remarks:

Test item concentration 25%

Remarks on result:

other:

Remarks:

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test item concentration 5%

Key result

Parameter:

SI

Value:

1.59

Test group / Remarks:

Test item concentration 10%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: no Data

DETAILS ON STIMULATION INDEX CALCULATION: In this study Stimulation Indices of 1.30, 1.59, 2.44 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone/olive oil (4+1, v/v).

EC3 CALCULATION: The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:No signs of systemic toxicity were observed during the study period. On day 3 (1h hour after the third application), all animals showed an erythema of the ear skin (Score 1).

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

In order to study a possible skin sensitising potential of H-32, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. A control group of four mice was treated with the vehicle (acetone/olive oil (4+1, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On day 3 (1h hour after the third application), all animals showed an erythema of the ear skin (Score 1).

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.30, 1.59, 2.44 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone/olive oil (4+1, v/v). The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item H-32 was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item H-32 formulated in acetone/olive oil (4+1, v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3 (1h hour after the third application), all animals showed an erythema of the ear skin (Score 1).

In this study Stimulation Indices (S.I.) of 1.30, 1.59, and 2.44 were determined with the test item at concentrations of 5, 10, and 25% in acetone/olive oil (4+1, v/v), respectively.

The test item H-32 was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test item H-32 was not a skin sensitiser under the test conditions of this study. The EC3 value could not be calculated due to the low S.I values therfore according to the CLP regulation the substance is not classified as a a skin sensitiser.