4''-Butoxy-4-ethyl-2',2'',3''-trifluoro-[1,1':4',1'']-terphenyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-14 to 2017-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-07-22

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-butoxy-4'-(4-ethylphenyl)-2,3,3'-trifluoro-1,1'-biphenyl

EC Number:

812-028-8

Cas Number:

612543-60-9

Molecular formula:

C24H23F3O

IUPAC Name:

4-butoxy-4'-(4-ethylphenyl)-2,3,3'-trifluoro-1,1'-biphenyl

Test material form:

solid: bulk

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
-Source: Envigo RMS B.V., AD Horst, The Netherlands
-Age (beginning of treatment): Pre-test: 8 - 9 weeks, Main study: 9 - 10 weeks
-Weight at study initiation: 17.7 - 21.7 g
-Housing: groupwise in Makrolon Type II/III cages
-Diet: ad libitum
-Water: ad libitum
-Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 22 +/- 2
-Humidity (%): 45 - 65
Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Pre-test for signs of irritation: 5 and 10 % (v/v)
Main study: 0, 2.5, 5 and 10 % (v/v)

No. of animals per dose:

Pre-test: 2 f
Main study: 5 f per group

Details on study design:

RANGE FINDING TEST
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 10% solution in acetone/olive oil (4+1 v/v).
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. The mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5 and 10% 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. From day 2 to 5, the animals showed an erythema of the ear skin (Score 1). Additionally, the animals showed a slight erythema on the scalp on days 3 and 4 and the animal treated with 10% showed scaly ears on days 5 and 6. No signs of exuberant local skin irritation were observed.
Thus, the test item in the main study was assayed at 2.5, 5, and 10%. 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.

MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT
-Name of test method: OECD GL 429
-Criteria used to consider a positive response: 3-fold greater response at one concentration in SI than control animals.

TREATMENT PREPARATION AND ADMINISTRATION
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1 v/v). The application volume (25 μL/ear/day) was spread over the entire dorsal surface 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.
Determination of Ear Thickness: Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer.
Administration of 3H-methyl-thymidine: Five days after the first topical application (day 6), 3H-methyl thymidine was injected into each test and control mouse via the tail vein.
Determination of incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanized and the lymph nodes were harvested. The draining lymph nodes were rapidly excised, pooled per animal (2 nodes per animal) and further processed. The level of 3HTdR incorporation was measured in a β-scintillation counter.

Positive control substance(s):

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

Statistics:

Standard statistical methods were applied.

Results and discussion

Positive control results:

Concentration S.I.
0 % 1.0
5 % 1.5
10 % 3.84
25 % 11.76

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

2.5 %

Results 2

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

5 %

Results 3

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

From the data it can be concluded that the test item is not a skin sensitiser under the conditions of this assay.

Executive summary:

Study Design

In this GLP study performed according to OECD TG 429, three groups each of five female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% 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 technically be achieved. A control group of five 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 animal. 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.

 

Results

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On day 3 (1h after the third application), an erythema of ear skin was observed (score 1) in the mid and high dose group. On day 4, the high dose group continued to show an erythema of ear skin (score 1). The low dose group did not show any signs of skin irritation. A relevant increase in ear thickness was not observed in any dose group.

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 2.4, 1.8, and 1.6 were determined with the test item at concentrations of 2.5, 5, and 10% 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.

 

Conclusion

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