Trans-2-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-5-(trans-4-propylcyclohexyl)-1,3-dioxane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-01 to 2016-07-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

updated 06 July 2012

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:

Trans-2-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-5-(trans-4-propylcyclohexyl)-1,3-dioxane

EC Number:

700-701-8

Cas Number:

798556-07-7

Molecular formula:

C26H27F7O3

IUPAC Name:

Trans-2-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-5-(trans-4-propylcyclohexyl)-1,3-dioxane

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, NL - 5960, AD Horst
- Age at study initiation: 1st pre-test: 9-10 weeks, 2nd pre-test and main study: 10 - 11 weeks
- Weight at study initiation: 1st pre-test: 18.7 & 19.9 g, 2nd pre-test: 18.3 g, main study: 17.4 - 20.9 g
- Housing: grouped per dose
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 40 – 65 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

1st pre-test: 25 and 50 %
2nd pre-test: 50 %
main study: 10, 25, and 50% (w/w)

No. of animals per dose:

1st / 2nd pre-test: 1 (total 3)
main study: 4 (3 treatment groups and on vehicle control group, total 16)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was a 50% solution in MEK.
- Irritation / Systemic toxicity: From day 3 to 6, the animal treated with 50% test item concentration showed a very slight erythema of the ear skin (Score 1) and a swollen face and ruffled fur on day 6. The animal treated with 25% test item concentration did not show any signs of local skin irritation nor systemic toxicity. Therefore, a second pre-test was performed in one animal using test item concentration of 50% to confirm the clinical symptoms observed in the animal treated in the first pre-test with 50%. From day 3 to 6 the animal showed a very slight erythema of the ear skin (Score 1) and a slightly swollen face. However, no other clinical symptoms and no loss in body weight over the course of the study were observed in the animals. Thus, the test item in the main study was assayed at 10, 25, and 50%. 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 STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3

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 10, 25, and 50% in MEK. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter approx. 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).
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 3H-methyl thymidine were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR, all mice were euthanized by using CO2, draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group) and single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared.

Positive control substance(s):

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

Statistics:

All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Positive control results:

Conc. SI
10% 5.51
25% 7.14
50% 7.31

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

5.51

Test group / Remarks:

Test Group: 10 %

Results 2

Key result

Parameter:

SI

Value:

7.14

Test group / Remarks:

Test Group: 25 %

Results 3

Key result

Parameter:

SI

Value:

7.31

Test group / Remarks:

Test Group: 50 %

Any other information on results incl. tables

Table 1 Summary of results

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG	number of lymph nodes	DPM per lymph node	SI
—	BGI	16	—	—	—	—
—	BG II	15	—	—	—	—
0	1	855	839.5	8	104.9	1.00
10	2	4639	4623.5	8	577.9	5.51
25	3	6008	5992.5	8	749.1	7.14
50	4	6150	6134.5	8	766.8	7.31

  

1 = Control Group

2-4 = Test Group

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

An EC3 could not be calculated since all SI values were above SI of 3.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

The purpose of this Local Lymph Node Assay was to assess the skin sensitizing potential of the test item when administered to the dorsum of both ears of mice. This study should provide a rational basis for risk assessment to the sensitizing potential of the test item in man. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% in Methyl ethyl ketone. The highest concentration tested was the highest concentration that could technically be achieved. The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 5.51, 7.14, and 7.31 were determined with the test item at concentrations of 10, 25 and 50% in Methyl ethyl ketone, respectively. The test item was found to be a skin sensitiser under the test conditions of this study.