Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26, 2010 to February 16, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol

EC Number:

911-254-5

Molecular formula:

Not applicable - Multiconstituent substance

IUPAC Name:

Reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.8-22.4 g
- Housing: single
- Diet: pelleted standard 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 dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 20-65%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

2.5, 5, and 10%.

No. of animals per dose:

16 females:
3 groups each of 4 female mice and a control group of 4 mice

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 100 % of the undiluted test item. The solutions were formulated in acetone:olive oil (4+1).
- Irritation: to determine the highest non-irritant test concentration, two pre-tests were performed in two animals each. In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. The test item in the main study was assayed at 2.5, 5, and 10%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
- Lymph node proliferation response: the proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: the sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. The validation- / positive control experiment was performed with α-Hexylcinnamaldehyde in acetone:olive oil (4+1) using CBA/CaOlaHsd mice in November 2009, see Annex 2 of the study.

TREATMENT PREPARATION AND ADMINISTRATION: the test item was placed into a volumetric flask on a tared balance and acetone:olive oil (4+1) was quantitatively added. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 2.5, 5, and 10% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface (Ø ~ 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, all mice were administered with 250 µl of 80.9 µCi/ml 3HTdR (corresponds to 20.2 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

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.

Results and discussion

Positive control results:

Test item concentration % (w/v) Stimulation Index
0 1.00
5 1.21
10 2.09
25 6.22

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

6.61

Test group / Remarks:

2.5%

Results 2

Key result

Parameter:

SI

Value:

10.21

Test group / Remarks:

5%

Results 3

Key result

Parameter:

SI

Value:

7.67

Test group / Remarks:

10%

Cellular proliferation data / Observations:

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 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 6.61, 10.21, and 7.67 were determined with the test item at concentrations of 2.5, 5, and 10% in acetone:olive oil (4+1). In the group treated with 10% test item concentration, severe signs of systemic toxicity were observed. Therefore, this group was excluded from the assessment of the test result. The decrease in S.I. at this concentration as compared to the lower doses might be related to the systemic toxicity observed for this group. However, the S.I.'s derived from the mid and low dose were also well above the threshold of 3.
The EC3 value could not be calculated, since all obtained SI's were above 3.

Any other information on results incl. tables

One animal belonging to the group treated with 10% test item concentration was found dead on study day 6.

 

The animals did not show any signs of local irritation during the course of the study. On day 6, three animals of the group treated with a test item concentration of 10% showed reduced spontaneous activity and one animal of the same group was found dead. At the mid and low dose signs of systemic toxicity were not observed.

 

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.

 

Results with test substance

Test item concentration % (w/v)	Group	Measurement DPM	Stimulation Index
---	BG I	15	--
---	BG II	20	--
---	1	4976
2.5	2	32774	6.61
5	3	50640	10.21
10	4	28536	7.67

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitiser (Cat 1) (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item was found to be a skin sensitiser.

Executive summary:

In the study the test item dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10%. The animals did not show any signs of local irritation during the course of the study. On day 6, three animals of the group treated with a test item concentration of 10% showed reduced spontaneous activity and one animal of the same group was found dead. At the mid and low dose signs of systemic toxicity were not observed. In this study Stimulation Indices (S.I.) of 6.61,10.21,and 7.67 were determined with the test item at concentrations of 2.5, 5, and 10% in acetone:olive oil (4+1), respectively. The test item was found to be a skin sensitiser.