2-ethoxy-4-(hydroxymethyl)phenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 12 August 2013 Experimental completion date: 27 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-4-(hydroxymethyl)phenol

EC Number:

674-192-5

Cas Number:

4912-58-7

Molecular formula:

C9H12O3

IUPAC Name:

2-ethoxy-4-(hydroxymethyl)phenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 2-Ethoxy-4-(hydroxymethyl)phenol
- CAS No.: 4912-58-7

RADIOLABELLING INFORMATION
- Radiochemical purity: [methyl-3H] thymidine (3 HTdR, lot No. 238-106-002-A-20121113-THN, code No. MT-6032, Moravek Biochemicals, concentration 1.0 mCi/mL))
- Specific activity: 2.0 Ci/mmol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Storage period: from July 2, 2013 to September 5, 2013
- Actual temperature range: 22.4 to 25.4 °C
- Stability under test conditions: Chemically stable under normal conditions

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks
- Weight at study initiation: 19.8 - 23.3 g
- Housing: Wire mesh metal cages (W 10.0 x D 19.6 x H 13.0 cm), Feeders were exchanged once a week.
- Diet: ad libitum, commercial pellet diet CRF-1 (lot No. 130307, Oriental Yeast)
- Water: ad libitum
- Acclimation period: 1 week
- Indication of any skin lesions: None of the animals showed any abnormalities

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26 °C
- Humidity (%): 35 - 70 % RH (actual measurement values: 53.0 to 65.4 % RH)
- Air changes (per hr): 12 times per hour
- Photoperiod: 12hrs dark, 12 hrs light (lights on 7 am, off 7 pm):
- IN-LIFE DATES: From: 21 August 2013 To: 26 August 2013

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10 and 20%

No. of animals per dose:

4 animals per dose, as follows:
Group 1: AOO
Group 2: 5 % test substance
Group 3: 10 % test substance
Group 4: 20 % test substance
Group 5: 25 % HCA

Details on study design:

PRE-SCREEN TESTS:
A pre-screening test [Exp No. E927 (432-024), non-GLP] was conducted before this main study to determine the concentrations for dosing. In the pre-screening test, three concentrations of 5, 10 and 20 % were selected using AOO as a solvent, and 25 µL each of the dose formulations were applied on the dorsal skin of both auricles of each animal, 2 mice for each concentration, once a day for 3 consecutive days. The general conditions including the application site were observed from 1 to 3 hours after application. As a result, no animals showed any abnormalities. The body weights and ear thickness were measured before the initial application on Day 6. As a result, none of the animals showed any changes deviating from the criteria of bofy weights.
From the results above, for the main study 20 % was selected as the high concentration because it was expected not to induce any toxic general condition, 25 % or more increase of the ear thickness, dermal erythema with score of 3 or more on the suricles, or more than 5 % of body weight loss, and two lower concentrations of 10 and 5 % (three concentrations in total).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Animals were randomly assigned to groups based on their body weights on the initial day of dosing (at the end of the quarantine and acclimation period) using a computer system package for safety studies (LATOX-F/V5, FFC). The remaining animals were handled as surplus animals.

Identification of animals
On arrival, the animals were numbered serially, and the numbers were written on their tails with an oil ink felt-tip pent. In addition, the animal ID card was attached to each cage for identification. After assignment to groups, another animal ID card showing the name of test substance and animal ID No. was attached to each cage. Each animal was clipped for intra-cage identification on Day 3.

Handling of surplus animals
Animals that were not assigned to groups were transferred to the animal care section.
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
An amount of 0.2 g of the test was weighed with an electronic balance (AT460DR. Mettler Toledo), dissolve in and diluted to 1 mL with acetone and olive oil mixture (AOO, v/v=4:1) to make the concentration of 20%. The solutions of 10 and 5 % concentrations were made by serial dilution from the 20 % solution.
0.2 mL of the positive control substance was dissolved in 0.6 mL of AOO to make the concentration of 25 %. These formulations were prepared just before use.

Rationale for selection of administration route and method of administration
The test substance was applied to the auricles in accordance with the common administration route for LLNA.
Twenty-five microliters each of a dose formulation were applied on the dorsal skin of the auricles of each animal once a day using a MICROMAN (Moel M100, Gilson).

Administration period and schedule
The administration period was 3 consecutive days.

LLNA Day 1 Day2 Day 3 Day 4 Day 5 Day 6 Day 7
protocol T T T - - 3 H C
T: Administration of dose solutions
3 H: Administration of 20 µCi [methyl-3H] thymidine (3 HTdR)
Local lymph nodes were collected and minced at 5 hours after administration of 3 HTdR, and then pooled lymph node cells (LNC) were treated with 5 % TCA overnight (approximately 18 hours).
C: Counting of 3 HTdR incorporation into pooled LNC.

Preparation of 3 HTdR solution
The 3 HTdR was diluted with phosphate buffered saline to a concentration of 80 µCi/mL just before use.

Confirmation of concentration of 3 HTdR solution
eighty microliters of 3 HTdR solution were diluted to 200 mL with water for injection and 1 mL of this solution was mixed with 10 mL of liquid scintillator (Clear-sol II). amounts of radioactivity were counted with the liquid scintillation counter (LSC-610B, Aloka). Because the actual amount of radioactivity of the 3 HTdR solution was within +/- 20 % of the calculated value, it was confirmed that the 3 HTdR solution was appropriately prepared (actual measurement value: 1,140 Bq, calculated value: 1,184 Bq).

Incorporation of 3 HTdR
On Day 6 all animals in the same dosage group were transferred to polycarbonate cages and transported to the RI laboratory.
Twenty µCi of 3 HTdR (250 µL of 80 µCi/mL 3 HTdR solution) were administered intravenously to each mouse via the tail vein with disposable syringes and 27G needles.

Positive control substance(s):

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

Statistics:

No statistical analyses were used.

Results and discussion

Positive control results:

DPM/animal of the positive control group was 5,502.7 and the SI of this group was calculated as 4.4.
The weights of lymph nodes in the positive control group were higher than that in the vehicle control group.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

5 % concentration

Results 2

Parameter:

SI

Value:

0.7

Test group / Remarks:

10 % concentration

Results 3

Parameter:

SI

Value:

0.8

Test group / Remarks:

20 % concentration

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The DPM/animal of the vehicle control group and 3 test substance groups (5, 10 and 20 %) were 1,263.5, 1,467.5, 931.6 and 950.2 respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The SI for the 5, 10 and 20 % NACET00470 groups were calculated to be 1.2, 0.7 and 0.8 respectively. Therefore NACET00470 was judged as negative.

CLINICAL OBSERVATIONS:
No animals showed abnormal clinical signs due to test substance administration during the observation period.
No aninals showed erythema on the auricles during the observation period.
No animals showed increases of ear thickness during the sensitization period.

BODY WEIGHTS
There were no body weight increases or decreases due to the test substance or positive control substance administration during the sensitization period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

A mouse local lymph node assay (LLNA) was conducted to investigate the potential of NACET00470 to induce allergic contact dermatitis.
NACET00470 showed SI lower than 3 at the concentration of 5, 10 and 20 %. Therefore, NACET00470 was judged to be not a sensitizer under the conditions of this study and was not categorized in anywhere of GHS classificaton for skin sensitization.

Executive summary:

A mouse local lymph node assay (LLNA) was conducted to investigate the potential of NACET00470 to induce allergic contact dermatitis. Female CBA/J [SPF] mice were purchased from Charles River Laboratories Japan, Inc., and 20 mice were used for this study. There were 5 dose groups, each consisting of 4 mice, and the animals in each group were exposed to NACET00470 at 1 of the 3 concentration levels, positive control substance or their vehicle (acetone and olive oil mixture: v/v=4:1).

The results are summarized as follows.

The stimulation indices (SI) for the 5, 10 and 20% NACET00470 groups were calculated to be 1.2, 0.7 and 0.8, respectively. The results were judged to be negative. On the other hand, the SI for the 25% α-Hexylcinnamaldehyde (HCA, positive control) was calculated to be 4.4, which was judged to be positive.

No animals showed abnormal clinical signs due to test substance administration during the observation period. No animals showed erythema on the auricles during the observation period. The ear thickness of all animals were not increased during the sensitizing period. There were no body weight increases or decreases due to the test substance administration during the sensitization period. The weights of lymph nodes in the positive control group were higher than that in the vehicle control group.

In conclusion, NACET00470 was judged to be not a sensitizer under the conditions of this study and was not categorized in anywhere of GHS classificaton for skin sensitization.