4-sec-butyl-2,6-di-tert-butylphenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2018 - 16 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Weight of Evidence report attached.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay

Version / remarks:

July 2012

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-sec-butyl-2,6-di-tert-butylphenol

EC Number:

241-533-0

EC Name:

4-sec-butyl-2,6-di-tert-butylphenol

Cas Number:

17540-75-9

Molecular formula:

C18H30O

IUPAC Name:

4-(butan-2-yl)-2,6-di-tert-butylphenol

Test material form:

liquid

Details on test material:

Appearance: Colourless to yellowish liquid
Test item storage: At room temperature

Specific details on test material used for the study:

No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 18.2 to 24.0 g
- Housing: Animals were group housed (up to 5 animals) in polycarbonate cages (Makrolon type: MIII) containing sterilized sawdust as bedding material. For psychological/environmental enrichment, animals were provided with paper and paper shelters
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to municipal tap-water
- Acclimation period: At least 5 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 41 - 61
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 19 September 2018 To: 15 October 2018

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5%, 10%, 25% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING STUDY:
In a pre-screen test, two test item concentrations were tested: a 50% and 100% concentration. Based on the results of the initially treated animals, two additional animals were treated in a similar manner with two lower concentrations (10% and 25%). The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation was not performed. At these test item concentrations variation in ear thickness (more than 25% from Day 1 pre-dose values) and/or clinical signs of systemic toxicity were noted. At a 25% and 10% test item concentration, no signs of systemic toxicity were noted and very slight irritation were observed and therefore the 25% concentration was selected as highest concentration for the main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Results were evaluated according to: (see Table 1)
- The recommendations made in the test guidelines (including all amendments).
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments).
- The Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were stirred until and during dosing. The dosing formulations were kept at room temperature until dosing.
Rationale for vehicle: The vehicle was chosen from the vehicles specified in the test guideline and selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7: Performed according to test guidelines.

OBSERVATIONS
Mortality/Viability: Twice daily.
Body weights: On day 1 (pre-dose) and day 6.
Clinical signs: Once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on days 1-6 (on days 1 - 3 within 1 hour after dosing) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Necropsy: No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not performed

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicate that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

The SI values calculated for the item concentrations 5%, 10% and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

4.2

Test group / Remarks:

25%

Any other information on results incl. tables

RANGE FINDING STUDY:

At a test item concentration of 100% and 50%, variation in ear thickness during the observation period were more than 25% from day 1 pre-dose values.

Clinical signs of systemic toxicity: Piloerection was observed until day 3; hunched posture was observed for rats until day 4 at both dose levels. Hyperactivity was observed for rats on day 1 and uncoordinated movements until day 2 for rats dosed 100%.

At a 25% and 10% test item concentration, no clinicial signs or systemic toxicity were noted and a very slight irritation were observed and therefore the 25% concentration was selected as highest concentration for the main study.

MAIN STUDY:

Radioactivity measurements:

Mean DPM/animal values for the experimental groups treated with test item concentrations 5%, 10% and 25% were 456, 881 and 2262 DPM, respectively. 

The mean DPM/animal value for the vehicle control group was 538 DPM.

The SI values calculated for the test item concentrations 5%, 10% and 25% were 0.8, 1.6 and 4.2, respectively

The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 18% was calculated.

OTHER:

- Skin reactions: Very slight erythema of the ear of animals treated at 25% between days 1 and 3 was observed, it was considered not to have a toxicologically significant effect on the activity of the nodes.

- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals

- Bodyweights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

- Macroscopic examination of the lymphnodes: The majority of auricular lymph nodes were considered normal in size, except for the nodes in three animals treated at 10% and the nodes in all animals treated at 25%, which were considered to be enlarged.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on an LLNA performed according to OECD TG 429 and under GLP principles, it is concluded that 2,6-di-tert-butyl-4-sec-butylphenol is a skin sensitizer (Category 1B) with an EC3 value of 18% and should be labeled as H317: May cause an allergic skin reaction.

Executive summary:

An LLNA was performed according to OECD TG 429/EC No 640/2012 Part B and GLP principles with 2,6-di-tert-butyl-4-sec-butylphenol. Based on the results of a pre-screen test, concentrations of 5%, 10% and 25% (w/w) were selected for the main study. In the main study, between Days 1 and 3 very slight erythema of the ear of animals treated at 25% was observed which was considered not to have a toxicologically significant effect on the activity of the nodes. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The majority of auricular lymph nodes were considered normal in size, except for the nodes in three animals treated at 10% and the nodes in all animals treated at 25%, which were considered to be enlarged. Mean DPM/animal values for the experimental groups treated with test item concentrations 5%, 10% and 25% were 456, 881 and 2262 DPM, respectively. The mean DPM/animal value for the vehicle control group was 538 DPM. The SI values calculated for the test item concentrations 5%, 10% and 25% were 0.8, 1.6 and 4.2, respectively. These results show that the test item elicits a SI ≥ 3. The data showed a dose-response and the EC3 value was established to be 18%. Reliable positive and negative controls were included, demonstrating that the test system functioned properply and is an appropriate model for testing for contact hypersensitivity. According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and a according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 2,6-di-tert-butyl-4-sec-butylphenol is classified as skin sensitizer (Category 1B) and  labeled as H317: May cause an allergic skin reaction.