6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 October 2016 to 4 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

EC Number:

201-814-0

EC Name:

6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol

Cas Number:

88-24-4

Molecular formula:

C25H36O2

IUPAC Name:

2-tert-butyl-6-[(3-tert-butyl-5-ethyl-2-hydroxyphenyl)methyl]-4-ethylphenol

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo - San Pietro al Natisone (UD) - Zona Industriale Azzida, 57, 33049 Italy
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 10 weeks old (age-matched, within one week) 1
- Weight at study initiation: 20.2 – 22.3 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
- Housing: Group caging (Type II. polypropylene / polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 - 25.7°C
- Humidity (%): 23 - 84 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: November 02 2016 (start treatment) To: November 8, 2016

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 5, 10, 25 and 50%

No. of animals per dose:

4 animals per dose

Details on study design:

PRE-SCREEN TESTS:
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50% (w/v) and 25 % (w/v) in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive
proliferation assay was not performed. Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).
No mortality or signs of systemic toxicity were observed. Minimal amount of test item precipitate was observed for both animals of the 50% (w/v) dose group on Days 1-4 or Days 1-2. There were no indications of any irritancy at the site of application. Marked body weight loss (>5% reduction of body weight) was observed for both animals of the 50% (w/v) dose group, the mean body weight loss of the 50% (w/v) group was more than 5%.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on
Day 6. The ear thickness values and ear punch weights were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former
experiments). Based on these results, 50% (w/v) dose was selected as top dose for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Desintegration per minutes (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide and the draining auricular lymph nodes were excised. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL).
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules at 2-8°C overnight.
After a last centrifugation, pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Statistics:

According to the OECD guideline 429, a statistical analysis of the data is not mandatory.

Results and discussion

Positive control results:

The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 7.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

other: DPN

Value:

354.8

Test group / Remarks:

Negative control

Results 2

Parameter:

other: DPN

Value:

169.9

Test group / Remarks:

50 (w/v)%

Results 3

Parameter:

other: DPN

Value:

239.3

Test group / Remarks:

25(w/v)%

Results 4

Parameter:

other: DPN

Value:

273.9

Test group / Remarks:

10 (w/v)%

Results 5

Parameter:

other: DPN

Value:

387

Test group / Remarks:

5 (w/v)%

Results 6

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control

Results 7

Key result

Parameter:

SI

Value:

0.5

Test group / Remarks:

50 (w/v)%

Results 8

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

25 (w/v)%

Results 9

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

10 (w/v)%

Results 10

Parameter:

SI

Value:

1.1

Test group / Remarks:

5 (w/v)%

Cellular proliferation data / Observations:

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in all the test item treated dose groups.
Larger than normal lymph nodes were observed in the positive control group.
The stimulation index values were 0.5, 0.7, 0.8 and 1.1 at concentrations of 50% (w/v), 25% (w/v), 10% (w/v) and 5 % (w/v), respectively.

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. Test item precipitate or minimal amount of test item precipitate was observed for all animals of the
50% (w/v) dose group on Days 2-3. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
No marked body weight loss (>5% reduction of body weight) was observed.

INTERPRETATION OF OBSERVATIONS
The test item was solid, which was formulated in AOO. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test item does not need classification according to the GHS or CLP.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present assay 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The objective of the study was to determine the skin sensitisation potential of 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative was available at time of study initiation. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

Based on the results of the preliminary compatibility test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 50% (w/v).

The preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50% (w/v) and 25% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 50% (w/v) was selected as top dose for the main test. In the main assay, twenty four female CBA/CaOlHsd mice were allocated to six groups of four animals each:

- four groups received 6,6’-di-tert-butyl-4,4’-diethyl-2,2’- methylenediphenol (formulated in AOO) at 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) concentrations

- the negative control group received the vehicle (AOO),

- the positive control group received 25% (w/v) HCA (dissolved in AOO).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application on the experimental animals. No marked body weight loss (≥ 5%) was observed on the mean body weight changes. The stimulation index values were 0.5, 0.7, 0.8 and 1.1 at concentrations of 50% (w/v), 25% (w/v), 10% (w/v) and 5 % (w/v), respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol, tested in a suitable vehicle, was shown to have

no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. No classification is triggered according to Regulation (EC) No 1272/2008 (CLP).