4-(3-Butenyl)-4''-ethyl-2'-fluor-1,1':4',1''-terphenyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-02-14 to 2022-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 429

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

yes

Remarks:

Please refer to "Principles of method".

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

Please refer to "Principles of method".

Principles of method if other than guideline:

The relative humidity in the animal room was between approximately 20-65% instead of 45-65% for severaours on several days during the first few days of the acclimation period of the animals used in the main experiment.

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-[4-(but-3-en-1-yl)phenyl]-4'-ethyl-3-fluoro-1,1'-biphenyl

EC Number:

619-966-5

Cas Number:

825633-86-1

Molecular formula:

C24H23F

IUPAC Name:

4-[4-(but-3-en-1-yl)phenyl]-4'-ethyl-3-fluoro-1,1'-biphenyl

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

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 nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: No details provided but only animals without any visible signs of illness were used for the study.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16.0 - 20.3 g
- Housing: group, Makrolon Type II (pre-test) / III (main study) with wire mesh top, on granulated soft wood bedding
- Diet: ad libitum, 2018C Teklad Global 18 % protein rodent diet
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: Not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): approx. 45-65 (20-65 for several hours due to a defective humidity sensor)
- Air changes (per hr): at least 8
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50, and 100 %

No. of animals per dose:

5

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 a 50% solution in MEK. Vortexing was used to formulate the test item.
- Irritation: At the tested concentrations the animals did not show any signs of local skin irritation
- Systemic toxicity: Both animals showed transient, mild and unspecific signs of discomfort: The animal treated with 50% of the test item showed partially closed eyes (transiently at the post-dose observation time after the 2nd and 3rd application). Additionally, the animal treated with 50% test item concentration showed piloerection and decreased activity at the post-dose observation time on day 3. Both animals showed a normal gain in body weight over the course of the study.
- Ear thickness measurements: No findings
- Erythema scores: No irritation observed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item was regarded as a sensitiser in the LLNA if the following criteria were 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 were 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 MEK was added (weight per weight). 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 10, 25, and 50% in MEK. 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). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79.3 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in body weight tables.
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.
Within the program, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. One outlier (DPM value determined for animal 19) was detected in the Grubb’s Test but not in the Dean-Dixon-Test, and was therefore not excluded from any calculations. Furthermore, exclusion of the outlier value would not change the overall test result.

Results and discussion

Positive control results:

In the positive control experiment the group S.I. values were determined to be 2.4, 3.4 and 9.8 after treatment with 5, 10 and 25 % alpha-Hexylcinnamaldehyde, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.11

Test group / Remarks:

50 %

Results 2

Parameter:

SI

Value:

1.64

Test group / Remarks:

25 %

Results 3

Parameter:

SI

Value:

1.19

Test group / Remarks:

10 %

Results 4

Parameter:

EC3

Value:

57.1

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Please refer to “Any other information on results” for tables.

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
Please refer to “Any other information on results” for tables.

EC3 CALCULATION
EC3 value were calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c; where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
Please refer to “Any other information on results” for tables.

CLINICAL OBSERVATIONS AND SIGNS OF TOXICITY
No deaths occurred during the study period. No symptoms of local skin irritation at the ears of the animals were observed during the study period. The animals treated with a test item concentration of 50% showed mild and unspecific signs of discomfort, namely, partially closed eyes and piloerection at the post-dose observation time points on days 2 and 3, possibly due to substance residuals on the ears. Animals treated with 10 and 25%% test item concentration did not show any clinical signs.

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.

Any other information on results incl. tables

 Calculation and Results of Individual Data

Vehicle: MEK

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	12	---	---	---	---
---	BG II	26	---	---	---	---
0	1	5480	5461	8	682.6	1.00
10	2	6540	6521	8	815.1	1.19
25	3	8957	8938	8	1117.2	1.64
50	4	6100	6081	8	760.1	1.11

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In the study the test item formulated in MEK (methyl ethyl ketone) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of local skin irritation during the course of the study and no cases of mortality were observed. The animals treated with a test item concentration of 50% showed mild and unspecific signs of discomfort, such as partially closed eyes and piloerection, directly after the 2nd and 3rd application only. Animals treated with 10 and 25%% test item concentration did not show any clinical signs.

In this study Stimulation Indices (S.I.) of 1.19, 1.64, and 1.11 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in MEK, respectively.