Registration Dossier

Administrative data

Description of key information

Skin sensitization: not sensitizing, no signs of systemic toxicity or local skin irritation (LLNA, OECD 429, GLP)

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:
2015-11-02 - 2016-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented OECD GLP guideline study without deviations on the registered substance itself.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation: Local Lymph Node Assay (adopted 22 July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No. 440/2008, B.42: “Skin Sensitisation: Local Lymph Node Assay”, dated 06 July 2012
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: 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
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17.5 - 21.6 g
- Housing: Conventionally, each dose group (5 mice/group) in one cage, Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top, Bedding: granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): approx. 45-65%
- Photoperiod (hrs dark / hrs light): 12/12 (artificial light 6.00 a.m. - 6.00 p.m.)
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Acetone: Purity: 99.98%; Olive oil: highly refined, low acidity
Concentration:
0, 10%, 25%, 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 50% solution in acetone/olive oil (4+1, v/v). Vortexing was used to formulate the test item.
- Irritation: 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 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 25 and 50% 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.
At the tested concentrations the animals did not show any signs of systemic toxicity. On day 2 and 3, the animal treated with 50% test item concentration showed a very slight erythema of the ear skin (Score 1). The animal treated with 25% test item concentration did not show any signs of local skin irritation.
Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w). 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
- 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:
Test Item Preparation
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical Analysis
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 validated program R Script Outlier.Rnw). No outlier value was detected.
Positive control results:
S.I. values per Test item concentration % in acetone:olive oil (4+1, v/v):
0%: 1.00
5%: 1.62
10%: 4.23
25%: 17.56
Parameter:
SI
Remarks on result:
other: Vehicle Control Group (acetone/olive oil (4+1, v/v)): 1.00 10% : 1.32 25% : 1.10 50% : 1.20
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle Control Group (acetone/olive oil (4+1, v/v)): 1458.5 10% : 1928.7 25% : 1611.1 50% : 1744.1
Interpretation of results:
GHS criteria not met
Conclusions:
The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the sensitizing potential of the test item in mice. The test item did not induce signs of general toxicity.
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted 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 concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.32, 1.10, and 1.20 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in acetone/olive oil (4+1, v/v).
So, the test item was identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.
Executive summary:

In the study according to OECD 429 under GLP the test item formulated in acetone/olive oil (4+1, v/v) 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 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 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 1.32, 1.10, and 1.20 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in acetone/olive oil (4+1, v/v), respectively.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

There is a valid GLP OECD 429 guideline study available to cover this endpoint. Hence, the database is of good quality. 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 1.32, 1.10, and 1.20 were determined with 2,2'-methylenebis[6-cyclohexyl-p-cresol] at concentrations of 10, 25, and 50% (w/w) in acetone/olive oil (4+1, v/v), respectively. So the test item was identified as a non-sensitizing agent in the Local Lymph Node Assay.

There is no indication given that the result is not relevant for human risk assessment, even though no human data is available, as all tested concentrations gave a clear negative result. The tonnage-driven data requirements under REACH are fully met, no data gaps were identified.

Justification for selection of skin sensitisation endpoint:
only study available

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In the available valid GLP OECD 429 guideline study, 2,2'-methylenebis[6-cyclohexyl-p-cresol]did not induce signs of general 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 1.32, 1.10, and 1.20 were determined with 2,2'-methylenebis[6-cyclohexyl-p-cresol] item at concentrations of 10, 25, and 50% (w/w) in acetone/olive oil (4+1, v/v), respectively. The threshold value for classification is a SI ≥ 3, which is not met here. So 2,2'-methylenebis[6-cyclohexyl-p-cresol] was identified as a non-sensitizing agent in the Local Lymph Node Assay, so no classification as skin sensitizer is triggered.