4-Ethoxy-2,3-difluoro-4'-methyl-1,1'-biphenyl

Endpoint summary

Description of key information

The read-across source substance showed no skin sensitising potential in the study and is thus considered to be no skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-03 to 2013-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: not specified; Only animals without any visible signs of illness were used for the study.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 21.5 +/- 1.1 g
- Housing: group
- Diet: ad libitum, 2018C Teklad Global 18 % protein rodent diet (certified)
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

10, 25 and 50 % (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50 % once daily each on three consecutive days.
- Compound solubility: The highest test item concentration, which could be technically used was a 50 % solution in acetone/olive oil 4/1 (v/v). Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37 °C).
- Irritation and Erythema scores: Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. The animal treated with the high dose showed a very slight erythema of the ear skin (score: 1 from days 2-4).
- Systemic toxicity: Clinical signs were recorded at least once daily.
- Ear thickness measurements: 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 cm) and were immediately pooled per animal and weighed using an analytical balance. Increases in ear weights vs. historical vehicle values were 0.2 % after treatment with 25 % test item and 14.7% after treatment with 50 % test item.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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. 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 % (w/w) 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)

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation). The Dean-Dixon-Test and the Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2007). An outlier animal was detected in both the Dean-Dixon-Test and the Grubb’s test. However, as exclusion of the outlier value did not have any impact on the outcome of the study, the value in question was not excluded from calculations of group mean DPM and standard deviation. A statistical analysis (one-way ANOVA) was conducted on the ear thickness values to assess whether a statistically significant increase in ear thickness could be observed when comparing the values measured on day 1 prior to application with the values measured on days 3 or 6 in the respective test item groups or within the vehicle control group. However, both biological and statistical significance were considered together.

Positive control results:

5 %: SI=1.6
10 %: SI=2.4
25 %: SI=5.9

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10 %

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

25 %

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

50 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA and DETAILS ON STIMULATION INDEX CALCULATION
Vehicle control: mean DPM = 290.9 (+/- 100.4), SI = 1.0
10 % test item concentration: mean DPM = 508.9 (+/- 235.7), SI = 1.8
25 % test item concentration: mean DPM = 484.9 (+/- 111.6), SI = 1.7
50% test item concentration: mean DPM = 378.5 (+/- 136.0), SI = 1.3

EC3 CALCULATION
No EC3 calculation was performed as all SI values were below 3.

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 and no signs of systemic toxicity were observed during the study period.
The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A relevant increase in ear thickness was not observed.

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.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no skin sensitising potential in the study and is thus considered to be no skin sensitiser.

Executive summary:

The purpose of the Local Lymph Node Assay was to assess the skin sensitizing potential of the test material when administered to the dorsum of both ears of mice. The study was performed according to GLP and the methods applied were fully compliant with OECD TG 429. In order to study a possible skin sensitizing potential of the test material, three groups each of five female mice were treated with different concentrations (10, 25 and 50 %) of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and 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 then determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. 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. A statistically significant or biologically relevant increase in ear thickness values was not observed in any of the treatment groups in comparison to the vehicle group. Stimulation Indices (S.I.) of 1.8, 1.7 and 1.3 were determined with the test item at concentrations of 10, 25, and 50 % (w/w) in acetone/olive oil 4/1 (v/v), respectively. Based on the results, the test material was not a skin sensitiser under the test conditions of this study.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 429. A detailed read across justification is provided in chapter 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

50%

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No study data with the test item is available for this endpoint. Therefore, a read-across to the read-across source substance with a very similar chemical structure and comparable physico-chemical parameters is used to evaluate the endpoint for the test item.

 

The purpose of the Local Lymph Node Assay was to assess the skin sensitizing potential of the test material when administered to the dorsum of both ears of mice. The study was performed according to GLP and the methods applied were fully compliant with OECD TG 429. In order to study a possible skin sensitizing potential of the test material, three groups each of five female mice were treated with different concentrations (10, 25 and 50 %) of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and 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 then determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. 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. A statistically significant or biologically relevant increase in ear thickness values was not observed in any of the treatment groups in comparison to the vehicle group. Stimulation Indices (S.I.) of 1.8, 1.7 and 1.3 were determined with the test item at concentrations of 10, 25, and 50 % (w/w) in acetone/olive oil 4/1 (v/v), respectively. Based on the results, the test material was not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item does not require classification for causing skin sensitisation according to Regulation (EC) No 1272/2008 (CLP).