Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 02 November 2009 and 02 December 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

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)

Remarks:

Date of inspection: 15/09/2009 Date of signature: 26/11/2009

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

IUPAC Name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

Constituent 2

Reference substance name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

EC Number:

935-853-6

IUPAC Name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Analytical purity: 80 - 95%
- Lot/batch No.: 200901317-17
- Stability under test conditions : stable
- Storage condition of test material : approximately 4°C in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
Harlan UK Limited, Bicester, Oxon, UK.

- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23g.

- Housing:
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet:
Food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.

- Water:
Free access to mains tap water was allowed throughout the study.

- Acclimation period:
At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 to 25 °C.

- Humidity (%): 30 to 70%.

- Air changes (per hr): Approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES:
From: Day 1 To: Day 6 (Day 1= day of dosing, day 6 = day of termination).

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary Test
25%, 10%, 5%, 2.5% and 1% v/v (in acetone/olive oil 4:1)
Main Test
2.5%, 1% or 0.5% v/v (in acetone/olive oil 4:1)

No. of animals per dose:

Preliminary Test
Five mice: one mouse per test material concentration
Main Test
Four mice per dose group

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using five mice, one mouse per test material concentration. The mice were treated by daily application of 25 µl of the test material at concentrations of 25%, 10%, 5%, 2.5% and 1% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Day 1 and pre-dose on Day 2. Surviving mice were observed pre-dose on Day 3 and remaining surviving mice were observed once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mice on Day 6. The bodyweight of the mouse, treated with the test material at a concentration of 5% v/v in acetone/olive oil 4:1, that was humanely killed, was recorded immediately prior to termination. The bodyweight of the mice treated with the test material at a concentration of 10% or 25% v/v in acetone/olive oil 4:1, that were humanely killed, were not recorded prior to termination in error.

- Lymph node proliferation response:
No signs of systemic toxicity were noted in animals treated with the test material at concentrations of 2.5% or 1% v/v in acetone/olive oil 4:1.

Based on this information the dose levels selected for the main test were 2.5%, 1% or 0.5% v/v in acetone/olive oil 4:1.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse.

- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:

Test Material Administration
Groups of four mice were treated with the test material at concentrations of 2.5%, 1% or 0.5% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.


3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

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

Statistics:

None provided.

Results and discussion

Positive control results:

One group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration % v/v in acetone/olive oil 4:1 Stimulation Index (SI) Result
15 3.12 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: A stimulation index of less than 3 was recorded for the undiluted test material and the test material at concentrations of 50% and 25% v/v in acetone/olive oil 4:1. The stimulation index (SI) results are given in Table 1 in the following section.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute (dpm) per lymph node given in Table 1.

Any other information on results incl. tables

Table 1. Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%v/v) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	13825.58	1728.20	na	na
0.5	114785.4	14348.18	8.30	Positive
1	161068.7	20133.59	11.65	Positive
2.5	116783.9	14597.99	8.45	Positive

dpm =  Disintegrations per minut

a =      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b =      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

Current Positive Control Study for the Local Lymph Node Assay

Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The method was designed to meet the requirements of the following:

- OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

- Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Test Material : a-Hexylcinnamaldehyde, Tech, 85%

Safepharm Laboratories Project number :        0039/1032

Study dates :        11 April 2008 to 17 April 2008

Methods. One group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further group of five animals was treated withacetone/olive oil 4:1alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration % v/v in acetone/olive oil 4:1		Stimulation Index		Result
15		10.91		Positive

Conclusion. alpha-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

 

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test.

The test material was classified as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, indication of danger ‘irritant’ and the risk phrase R 43 “May Cause Sensitisation by Skin Contact” are therefore required.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

-OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

-Method B42 Skin Sensitisation (Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 2.5% v/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as asolutioninacetone/olive oil 4:1at concentrations of 2.5% ,1% or 0.5% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
0.5	8.30	Positive
1	11.65	Positive
2.5	8.45	Positive

Conclusion. The test material was considered to be a sensitiser under the conditions of the test.

The test material was classified as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, indication of danger ‘irritant’ and the risk phrase R 43 “May Cause Sensitisation by Skin Contact” are therefore required.