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Description of key information

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

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-03-14 to 2012-06-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst /, The Netherlands
- Age at study initiation: 11 - 12 weeks
- Housing: Makrolon Type II / III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: Rodent 2019C Teklad Global, certified diet, ad libitum (Harlan Laboratories B.V. 5960 AD Horst / Netherlands)
- Water: tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: at least 5 days prior to the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
methyl ethyl ketone
Concentration:
5, 10 and 20 %
No. of animals per dose:
5 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: was performed. The highest test item concentration, which can be technically used was 20 %. 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. sonicating).
- Irritation: To determine the highest non-irritant test concentration that does at the same time not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 20 % once daily on three consecutive days. Prior to the first application of the test item and before sacrifice (animal 1 only) the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible reddening 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 (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, 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 reddening 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 4 to 6, the both animals treated showed an erythema of ear skin (animal treated with 10%: days 4 and 5 Score 1, animal treated with 20 %: days 4 and 6 Score 1, day 5 Score 2).
Thus, the test item in the main study was assayed at 5, 10, and 20 %. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.

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:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 5, 10, and 20 % (w/w) in methyl ethyl ketone. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice 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 (PBS) containing 19.8 µCi of 3HTdR (equivalent to 3HTdR 79.2 µCi/mL) were injected into each test and control mouse via the tail vein.
Prior to the first application of the test item and prior to treatment with 3HTdR, the ear thickness was determined using a micrometre (S0247 Kroeplin, 36381 Schlüchtern, Germany).
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. The values obtained were taken down manually.
After the lymph nodes have been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Stiefel, diameter 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.
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 lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; SI). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
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 extrapolated EC3 value was calculated according to the equation:

extrapolated EC3 = 2(log2 (c) + (3-d)/(b-d) *(log2 (a)- log2 (c)))

where extrapolated EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; the point with the higher S.I. is denoted (a, b) and the point with the lower S.I. is denoted (c, d) on the local lymph node assay dose response plot.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb's test were used for identification of possible outliers (performed with Microsoft Excel 2003).
A statistical analysis was conducted on the DPM values, the ear weights and the lymph node weights to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.
Key result
Parameter:
EC3
Value:
2.7
Remarks on result:
other: extrapolated values
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control
Parameter:
SI
Value:
4
Test group / Remarks:
5 % test item
Parameter:
SI
Value:
5.03
Test group / Remarks:
10 % test item
Parameter:
SI
Value:
7.48
Test group / Remarks:
20 % test item
Parameter:
other: disintegrations per minute (DPM) in %
Value:
429.1
Test group / Remarks:
Vehicle control
Parameter:
other: disintegrations per minute (DPM) in %
Value:
1 718.3
Test group / Remarks:
5 % test item
Parameter:
other: disintegrations per minute (DPM) in %
Value:
2 158.1
Test group / Remarks:
10 % test item
Parameter:
other: disintegrations per minute (DPM) in %
Value:
3 210.7
Test group / Remarks:
20 % test item

The mean DPM per animal (two lymph nodes) was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals). The following mean DPM values were determined:

Vehicle Control: 429.1

5 % test item: 1718.3

10 % test item: 2158.1

20 % test item: 3210.7

The following SI values were determined:

Vehicle control: 1.00

5 % test item: 4.00

10 % test item: 5.03

20 % test item: 7.48

EC3 extrapolation

Since all SI values are above the threshold of 3, the EC3 value could not be calculated. Instead it was extrapolated using the higher SI and the lower SI from the dose response plot (refer to table 1).

Table 1 Extrapolated EC3 = Estimated concentration for a S.I. of 3.

 

Test item concentration %

S.I.

Test group 4

10

5.03

Test group 3

5

4.00

Extrapolated EC3 = 2(log2 (c) + (3-d)/(b-d) *(log2 (a)- log2 (c))) = 2.7 % (w/v)

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was found to be a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item 1,1'-(1,1,2,2-tetramethylethylene)dibenzene dissolved in methyl ethyl ketone was assessed for its possible skin sensitising potential according to OECD guideline 429 and EU method B.42.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 20 %. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 to 6, the animals treated with a test item concentration of 20 % showed an erythema of the ear skin (Score 1). Animals treated with the vehicle alone or 5 and 10 % test item concentration did not show any signs of local skin irritation. A statistically significant relevant increase in ear weights and ear thickness was not observed in any treated group in comparison to the vehicle control group.

In this study Stimulation Indices (SI) of 4.00, 5.03, and 7.48 were determined with the test item at concentrations of 5, 10, and 20 % in methyl ethyl ketone, respectively, all exceeding the trigger value of 3 for a biologically relevant S.I. increase. An outlier was identified in the low dose group (DPM value determined for animal number 6). However, as exclusion of the outlier did not change the overall test result, the value in question was not excluded from the calculation. A statistically significant increase in DPM value was observed in all dose groups in comparison to the vehicle control group. A statistically significant and biologically relevant increase in lymph node weight was observed in the mid and high dose groups in comparison to the vehicle control group. The EC3 value could not be calculated, since all obtained SI's were above the threshold value of 3. However, due to the clear dose response, an extrapolated EC3 of 2.7 % could be calculated.

The test item 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was found to be a skin sensitiser.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

OECD 429

In the study the test item 1,1'-(1,1,2,2-tetramethylethylene)dibenzene dissolved in methyl ethyl ketone was assessed for its possible skin sensitising potential according to OECD guideline 429 and EU method B.42.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 20 %. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 to 6, the animals treated with a test item concentration of 20 % showed an erythema of the ear skin (Score 1). Animals treated with the vehicle alone or 5 and 10 % test item concentration did not show any signs of local skin irritation. A statistically significant relevant increase in ear weights and ear thickness was not observed in any treated group in comparison to the vehicle control group.

In this study Stimulation Indices (SI) of 4.00, 5.03, and 7.48 were determined with the test item at concentrations of 5, 10, and 20 % in methyl ethyl ketone, respectively, all exceeding the trigger value of 3 for a biologically relevant S.I. increase. An outlier was identified in the low dose group (DPM value determined for animal number 6). However, as exclusion of the outlier did not change the overall test result, the value in question was not excluded from the calculation. A statistically significant increase in DPM value was observed in all dose groups in comparison to the vehicle control group. A statistically significant and biologically relevant increase in lymph node weight was observed in the mid and high dose groups in comparison to the vehicle control group. The EC3 value could not be calculated, since all obtained SI's were above the threshold value of 3. However, due to the clear dose response, an extrapolated EC3 of 2.7 % could be calculated.

The test item 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was found to be a skin sensitiser.

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 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 as a skin sensitiser in Cat. 1B (H317) under Regulation (EC) No. 1272/2008, as amended for the twelfth time in Regulation (EU) No. 2019/521.