Methyl 4-[2-[4-(5-methyl-2-benzoxazolyl)phenyl]vinyl]benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 27th to December 3rd, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

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:

Fluorescent Brightener 371

IUPAC Name:

Fluorescent Brightener 371

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop ZRT.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks old at start of the DRF; 8-11 weeks old (at start of the main test)
- Weight at study initiation: 17.8 – 21.1 g
- Housing: 4 animals/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 26 days in DRF, 7 days in main study
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 h daily, from 6 a.m. to 6 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

DRF: 10 %, 5 % w/v
Main test: 10 %, 5 %, 2.5 %, 1 % w/v

No. of animals per dose:

4

Details on study design:

The DRF was conducted in a similar experimental manner to the exposure phase of the main test except that there was no assessment of lymph node proliferation and fewer animals were used. The test item was formulated in DMSO and evaluated at concentrations of 10 % and 5 % (w/v) in the DRF. Both formulations (suspensions) were adequately applicable on the ears of animals. Groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both ears of each mouse were observed for erythema and scored. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (prior to the treatment) and Day 6.
No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed. No sign of significant irritation (indicated by an erythema score >= 3 and/or an increase of Z= 25 % of ear thickness observed on any day of measurement) or any other local effect was observed.
Based on the results the 10 % (w/v) concentration was selected and used as the maximum in the main test. The test item was tested also at three additional, lower concentrations (5 %, 2.5 % and 1 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.
Criteria for erythema scores
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

Main test design
The test item was administered at four different concentrations.
In vivo treatment
Each mouse was topically treated with 25 µl of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. 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.

Proliferation assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test. All animals treated were processed and therefore no treatment group was excluded from the evaluation.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 µl of sterile PBS (1 × PBS, diluted from 10× concentrate) containing approximately 20 µCi* of 3H-methyl-thymidine using a hypodermic needle with 1 ml sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
*: breaking down of [methyl-3H]-thymidine in aqueous solution (about 3 % per month) was taken into account as necessary when 3HTdR solution was prepared.

Removal and preparation of draining auricular lymph nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps.
Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 ml) of PBS to keep the nodes wet before processing.

Preparation of single cell suspension of lymph node cells
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, 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). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 × g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 ml supernatant above each pellet. The pellets were gently agitated before making up to 10 ml with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 ml) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 ml of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 × g for 10 minutes at 4 °C and decanting the supernatants, than the pellets were re-suspended in 1 ml of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 ml of scintillation liquid, gently mixed and loaded into the ß-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The ß-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 ml aliquots of 5 % TCA. Instrument used for the measurement:
Name: Tri-Carb 3100TR, Liquid Scintillation Analyzer
Serial Number: 072971

Observations in the main test
Clinical observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.

Measurement of body weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g. 

Evaluation of the results
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) was not calculated for the test item.

Interpretation of the results
The test item is considered as a skin sensitizer, if: exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

Assay acceptance criteria
Skin sensitizing effect was observed at the applied concentration of the positive control: the stimulation index was 8.6 (greater than 3).
According to this, the assay acceptance criteria given in the Study Plan was fulfilled, hence the test was valid.

Positive control substance(s):

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

Results and discussion

Positive control results:

SI: 8.6

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.5

Test group / Remarks:

10 %

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

5 %

Results 3

Parameter:

SI

Value:

2

Test group / Remarks:

2.5 %

Results 4

Parameter:

SI

Value:

2.2

Test group / Remarks:

1 %

Cellular proliferation data / Observations:

Body weight measurement
No significant effect on the body weights was observed in any group during the test. A body weight decrease by > 5 % was observed in the positive control group only (1 of the 4 animals with 6 % decrease). No body weight decrease by > 5 % was observed in the other groups.

Clinical observations, signs of irritation
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score >= 3) or any other local effect were observed in any treatment group.

Proliferation assay
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. The appearance of the lymph nodes was normal in both vehicle control groups (AOO or DMSO) and in the test item treated groups.
No significant lymphoproliferative response (SI >= 3) compared to the concurrent control (DMSO) was noted for Fluorescent Brightener 371 at the applied test concentrations. The observed stimulation index values were 1.5, 1.5, 2.0 and 2.2 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % w/v, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No significant dose-related response was observed (p = 0.15, r = 0.73).

Reliability of the test
The positive control group animals were treated with 25 % w/v HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
A significant lymphoproliferative response (SI >= 3) was noted for HCA (SI = 8.6). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

Interpretation of observations
The maximum dose selection was based on results of the formulation evaluation and the dose range finding test (DRF). The solubility of the test item in vehicles preferred in the LLNA was evaluated. Limited solubility of the test item has been observed in all vehicles. The maximum soluble concentration was 0.1 % w/v. Adequate and homogeneous formulation (apparently suspension) was achieved with DMSO with a maximum concentration of 10 % w/v. Based on this and in accordance with the OECD guideline suspension formulations of the test item prepared with DMSO were examined in the LLNA.
As no significant adverse effects (systemic toxicity or irritation) were observed in the DRF at 10 % and 5 % w/v concentrations, the test item was examined in the main test as 10 %, 5 %, 2.5 % and 1 % w/v formulations in DMSO.
Since the test was valid and no confounding effects of irritation or systemic toxicity were observed during the main test, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.
No significant lymphoproliferative response (SI >= 3) compared to the relevant vehicle control (DMSO) was noted for test item at the tested concentrations. The observed stimulation index values were 1.5, 1.5, 2.0 and 2.2 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % w/v, respectively. No dose-related response was observed.

Applicant's summary and conclusion

Interpretation of results:

other: not skin sensitising according to the CLP Regulation (EC 1272/2008)

Conclusions:

Under the conditions of the present assay, test item tested at the maximum feasible concentration of 10 % w/v and also at concentrations of 5 %, 2.5 % or 1 % w/v as adequate homogeneous formulations (suspensions, prepared with DMSO as vehicle) was shown to have no skin sensitization potential in the LLNA.

Executive summary:

The skin sensitization potential of test item was assessed in the LLNA, according to OECD guideline 429.

Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration.

Based on the observed poor solubility (the maximum soluble concentration was 0.1 % w/v in LLNA standard vehicles) the test item was examined in the LLNA as suspension formulations in the most adequate vehicle of DMSO. The maximum concentration of an adequate and homogeneous formulation was 10 % w/v in this vehicle.

No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test at the tested concentrations of 10 % and 5 % w/v. According to this, the test item was examined in the main test at concentrations of 10 %, 5 %, 2.5 % and 1 % w/v.

An appropriate positive control (a-hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item, i.e. 25 % w/v HCA in acetone: olive oil 4:1 v/v mixture (AOO) induced significant stimulation over the relevant control (SI = 8.6) thus confirming the validity of the assay.

No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effect on body weights were observed in any test group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.

No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control (DMSO) was noted for test item at the applied test concentrations. The observed stimulation index values were 1.5, 1.5, 2.0 and 2.2 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % w/v, respectively. No significant dose-response relationship was observed (p = 0.15, r = 0.73; evaluated by linear regression using SI values).

According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI >= 3) up to the maximum attainable concentration of 10 % w/v as well as the lack of a significant dose-related response are considered as evidence that test item is not a skin sensitizer.