1-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]ethanone

Endpoint summary

Description of key information

Local Lymph Node Assay:

The test item tested at concentrations of 5, 10 and 25% in a suitable vehicle (MEK) was found to be a skin sensitizer and an EC3 value of 23.4 % was derived.

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:

2014-11-12 to 2014-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Roßdorf

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L85-76

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Species:

mouse

Strain:

other: CBA/CaOlaHsd (SPF)

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
- Age at study initiation:
1st pre-test: 11-12 weeks
2nd pre-test: 10-11 weeks
Main study: 8-9 weeks
- Weight at study initiation: 19.6 - 22.7 g
- Housing: group, conventional
- Diet: 2018C Teklad Global 18% protein rodent diet (certified); ad libitum
- Water: Tap 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 (artificial light 6.00 a.m. - 6.00 p.m.)

Vehicle:

methyl ethyl ketone

Remarks:

(MEK)

Concentration:

1st pre-test: 50 and 100%
2nd pre-test: 10 and 25%
Main test: 5, 10 and 25% (w/w)

No. of animals per dose:

Pre-tests: 1 animals per dose
Main test: 5 animals 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 MEK as vehicle.
- Pre-tests:
1st pre-test:
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.
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.
From day 2 to 6, both animals showed an erythema of the ear skin (score 1 to 2. Additionally, the animals showed signs of toxicity such as eyelid closure, reduced spontaneous activity, and hunchbacked position.

2nd pre-test:
A second pre-test was performed using test item concentrations of 10 and 25%. From day 2 to 5, the animal treated with 25% test item concentration showed an erythema of the ear skin (score 1). The animal treated with 10% test item concentration did not show any signs of skin irritation and both animals showed no signs of toxicity. The body weight determined for the animal treated with 25% exceeded the cut-off value of 5% loss, but was considered to be incidental, as only very slight erythema without any signs of systemic toxicity were observed

MAIN STUDY
In the main study the test item 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: 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:
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 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 20.2 μCi of 3H-methyl thymidine (equivalent to 80.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

other: α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v)

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
Where appropriate, the EC3 value was 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.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).
However, both biological and statistical significance were considered together.

Positive control results:

The positive control substance induced a statistically significant stimulation compared to the control.

Key result

Parameter:

EC3

Value:

23.4

Parameter:

SI

Value:

1.12

Test group / Remarks:

5%

Parameter:

SI

Value:

1.43

Test group / Remarks:

10%

Parameter:

SI

Value:

3.19

Test group / Remarks:

25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Radioactive labeling is used to measure proliferations

EC3 CALCULATION: The EC3 value was 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 3-fold increase in draining lyph node cell proliferative activity; (a,b) and (c,d) are respectively to coordinates of the two pair of data lying immediatly above and below the S.I. value of 3 on the loval lymph node assay dose response plot.

VIABILITY/ MORTALITY: No deaths occurred during the study period.

CLINICAL SIGNS: Neither signs of systemic toxicity nor local skin effects were observed during the study period.

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.

LYMPH NODE WEIGHTS AND CELL COUNTS: The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weight and lymph node cell count was observed in the high dose group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the high dose group (index of 1.7).

EAR WEIGHTS: The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item was fount to be an skin sensitizer and an EC3 value of 23.4% (w/w) was derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Local Lymph Node Assay:

A Local lymph node assay was conducted according to OECD test guideline 429 in mice to evaluate the sensitizing potential of the test substance in skin.

Three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10 and 25% in MEK by topical application to the dorsum of each ear for three consecutive days. The test item could be dissolved in the vehicle. The appropriateness of the used concentrations was previously assessed by two pre-experiments. A control group of five mice was treated with the vehicle MEK 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, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards single cell suspensions of lymph node cells were prepared from pooled lymph nodes per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were 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. The animals neither showed any signs of systemic toxicity nor local skin effects during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results 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 test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.12, 1.43, and 3.19 were determined with the test item at concentrations of 5, 10 and 25% in MEK, respectively. A dose response was observed. An EC3 value of 23.4% (w/w) was derived. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and cell count was observed in the high dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the high dose group (index of 1.7).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified for skin sensitization (Category 1B) under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.