oligomeric reaction product of 2-hydroxybenzaldehyde with 1-chloro-2,3-epoxypropane and phenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 August 2015- 15 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

EC Number:

942-639-6

IUPAC Name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

Test material form:

solid

Details on test material:

Name: EPICLON EXA-7250
Batch/Lot number: B006
Appearance: Brown solid (in smaller amount colour is yellow)
Purity: >99%
Manufacture date: 02 October 2014
Expiry date: 02 March 2017
Storage condition: Room temperature 15-25°C, below 70 RH%
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face
mask) for unknown materials were applied to assure personnel
health and safety.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Harlan Laboratories S.r.l. San Pietro al Natisone (UD) Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:10 weeks old (age-matched, within one week)
- Weight at study initiation:20.0 – 20.8 g (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):
- Water (e.g. ad libitum):
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18.0-28.4°C
- Humidity (%):30-87 %
- 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: 9 September 2015 To: 15 September 2015

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

5,10,25 and 50% w/V

No. of animals per dose:

4 animals per dose.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone : Olive oil 4:1 (v:v) mixture (AOO), N,N-Dimethylformamide (DMF), Methyl ethyl ketone (MEK) and Dimethyl sulfoxide (DMSO). The 100% (w/v) formulation was achievable using MEK as vehicle, while the use of other vehicles did not result a suitable formulation at this concentration. The best vehicle taking into account the test item characteristics, the formulations homogeneity and the requirements of the relevant OECD guideline was considered to be MEK, so it was selected for vehicle of study.
- Irritation: There was no indication of irritation at the site of application based on the visual assessment (erythema scoring) in this preliminary experiment.
- Systemic toxicity:During the Preliminary Irritation / Toxicity Test I, no mortality was observed. Marked body weight loss (>5%) was detected for one animal in each dose group, but the mean body weight loss in both dose groups was below the acceptable limit (Table 6 of Appendix 4). Excessive alopecia was observed in both dose groups on Days 2-6 suggesting some kind of local toxicity. Test item precipitate / minimal amount of test item precipitate was detected on the ears of the animals in the 100% (w/v) dose group on Days 1-4 and in the 50% (w/v) dose group on Days 1-6.
- Ear thickness measurements: Increased ear thickness values of at least 25% were detected in several cases on Day 3; the observed results on Day 3 were above the limit acceptable swelling in both dose groups, although the test item precipitate on the ear clearly contributed in the ear thickness measurement in case of the 100% (w/v) dose group.
- Erythema scores:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:

The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: Acetone : Olive oil 4:1 (v:v) mixture (AOO), N,N-Dimethylformamide (DMF), Methyl ethyl ketone (MEK) and Dimethyl sulfoxide (DMSO). The 100% (w/v) formulation was achievable using MEK as vehicle, while the use of other vehicles did not result a suitable formulation at this concentration. The best vehicle taking into account the test item characteristics, the formulations homogeneity and the requirements of the relevant OECD guideline was considered to be MEK, so it was selected for vehicle of study.

To prepare the dosing formulations, the test item was freshly diluted with the selected vehicle to obtain appropriate concentrations in the Pharmacy of CiToxLAB Hungary Ltd. The applicable dose levels were based on the results of the Preliminary Irritation / Toxicity Test. Formulations were prepared on weight: volume basis as % (w/v), and were considered to be stable for this short period (no correction for purity of the test item was applied).
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study, although the formulations were checked for visible homogeneity and physical stability. Test item formulations in the 100-5% (w/v) concentration range appeared by visual inspection to be clear yellow solutions.


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.

Positive control substance(s):

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

Results and discussion

Positive control results:

A significant lymphoproliferative response (stimulation index value of 12.2) was noted for the positive control chemical, this result confirmed the validity of the assay.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

< 3

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Larger than normal lymph nodes were observed in the positive control group, while the appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated groups (subjective judgement by analogy with observations of former experiments).

DETAILS ON STIMULATION INDEX CALCULATION: The stimulation index values were 1.9, 1.8, 2.5 and 2.5 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

EC3 CALCULATION

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity were observed during the main test. Alopecia / extensive alopecia were observed in the 50 and 25% (w/v) dose groups on Days 2-6 indicating some kind of local toxicity. There were no visual indications of any irritancy at the site of application.

BODY WEIGHTS: No treatment related effect on body weight was observed in the main test. Marked body weight loss (≥5%) was detected for two negative control animals, furthermore one animal in the 50% (w/v), two animals in the 25% (w/v) and one animal in the 10% (w/v) dose groups, but based on the body weights of the other animals in those groups, these facts were considered as animal variability.

Any other information on results incl. tables

EAR THICKNESS MEASUREMENTS  

Similarly to the preliminary experiment, ear thickness of the animals was measured using by a thickness gauge and by ear punch weight determination. The ear thickness values and the weights of the ear punches (2 per animal) are summarized in Table 13 of Appendix 8. Significantly increased ear thickness values (increase of ≥25%) were recorded for two animals in the 50% (w/v) dose groups on Days 3 and/or Day 6. Furthermore, increased values was detected for one ear of an animal in the 25% (w/v) dose group and another animal in the positive control group, but in those cases the result of the other ear of the animals was below the limit of excessive local irritation, so those cases were considered as acceptable. The revealing ear punch weights were within the acceptable range in all cases.  

RESULTS AND DISCUSSION  

8.1. CLINICAL OBSERVATIONS  

No mortality or signs of systemic toxicity were observed during the main test. Alopecia / extensive alopecia were observed in the 50 and 25% (w/v) dose groups on Days 2-6 indicating some kind of local toxicity. There were no visual indications of any irritancy at the site of application. The results of the observations are summarized in Table 12 of Appendix 7.  

8.2. BODY WEIGHT MEASUREMENT  

No treatment related effect on body weight was observed in the main test. Marked body weight loss (≥5%) was detected for two negative control animals, furthermore one animal in the 50% (w/v), two animals in the 25% (w/v) and one animal in the 10% (w/v) dose groups, but based on the body weights of the other animals in those groups, these facts were considered as animal variability. Individual and mean body weights are given in Table 4.  

8.3. EAR THICKNESS MEASUREMENTS  

Similarly to the preliminary experiment, ear thickness of the animals was measured using by a thickness gauge and by ear punch weight determination. The ear thickness values and the weights of the ear punches (2 per animal) are summarized in Table 13 of Appendix 8. Significantly increased ear thickness values (increase of ≥25%) were recorded for two animals in the 50% (w/v) dose groups on Days 3 and/or Day 6. Furthermore, increased values was detected for one ear of an animal in the 25% (w/v) dose group and another animal in the positive control group, but in those cases the result of the other ear of the animals was below the limit of excessive local irritation, so those cases were considered as acceptable. The revealing ear punch weights were within the acceptable range in all cases.    

8.4. PROLIFERATION ASSAY  

The results of the proliferation assay are summarized in Table 5 and Figure 1.  

Larger than normal lymph nodes were observed in the positive control group, while the appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated groups (subjective judgement by analogy with observations of former experiments).  

The stimulation index values were 1.9, 1.8, 2.5 and 2.5 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, EPICLON EXA-7250, tested in a suitable vehicle, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of EPICLON  EXA-7250 following dermal exposure. The study was performed with vertebrate animals as no regulatory in vitro alternative was available for classification. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.  

Based on the results of the preliminary solubility / compatibility test and on the recommendations of the OECD Guideline [1], the test item was formulated in  Methyl ethyl ketone (abbreviated as MEK). Due to the physical and chemical characteristics of the test item, the highest achievable concentration was 100% (w/v) (i.e. 1 g/mL).  

The Preliminary Irritation / Toxicity Tests were performed in CBA/CaOlaHsd mice using a total of four doses (100, 50, 25 and 10% (w/v) in MEK). Based on the observations recorded in the preliminary tests, 50% (w/v) dose was selected as top dose for the main test.  

In the main assay, twenty four female CBA/CaOlaHsd mice were allocated to six groups of four animals each: - four groups received EPICLON EXA-7250 (formulated in MEK) at 50, 25, 10 and 5% (w/v) concentrations, - the negative control group received the vehicle (MEK), - the positive control group received 25% (w/v) HCA (dissolved in MEK).  

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. On Day 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. No treatment related body weight loss was observed in the test item treated animals. Alopecia / extensive alopecia were observed in the 50 and 25% (w/v) dose groups on Days 2-6 indicating some kind of local toxicity. There was no indication of any irritancy at the site of application by visual examination, but ear thickness measurements indicated some local irritation for two animals in the 50% (w/v) dose group.  

The stimulation index values were 1.9, 1.8, 2.5 and 2.5 at concentrations of 50, 25 10 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 [1]. A significant lymphoproliferative response (stimulation index value of 12.2) was noted for the positive control chemical, this result confirmed the validity of the assay.  

In conclusion, under the conditions of the present assay, EPICLON EXA-7250, tested in a suitable vehicle, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.