Fatty acids, C18-unsatd., dimers, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 June 2018 - 08 August 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Fatty acids, C18-unsatd., dimers, oligomeric reaction products with 1-chloro-2,3-epoxypropane

EC Number:

500-215-4

EC Name:

Fatty acids, C18-unsatd., dimers, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Cas Number:

68475-94-5

Molecular formula:

Not applicable for a UVCB substance please see remarks section below.

IUPAC Name:

Reaction products of Fatty acids, C18-unsatd., dimers and trimer with 1-chloro-2,3-epoxypropane

Test material form:

liquid

Details on test material:

Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
Batch: 52611021
CAS Number: 68475-94-5
EC Number: 500-215-4
Purity: 95-100%
Physical state / Appearance: Clear yellow liquid
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo Envigo RMS (UK) Limited, Oxon, UK). On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood flake bedding. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

100%, 50% or 25% v/v in acetone/olive oil 4:1

No. of animals per dose:

4 mice per dose.

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 100%, 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (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.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

4.99

Test group / Remarks:

25% v/v dose group

Remarks on result:

other: Positive

Results 2

Key result

Parameter:

SI

Value:

4.29

Test group / Remarks:

50%(v/v) dose group

Remarks on result:

other: Positive

Results 3

Key result

Parameter:

SI

Value:

0

Test group / Remarks:

100% (v/v) dose group

Remarks on result:

not measured/tested

Cellular proliferation data / Observations:

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).


No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

Any other information on results incl. tables

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	8531.97	1066.50	na	na
25	42612.26	5326.53	4.99	Positive
50	36572.03	4571.50	4.29	Positive
100	No data due to termination of group

dpm=Disintegrations per minute

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

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of100%, 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 undiluted or the test item, as asolutioninacetone/olive oil 4:1,at concentrations of50% or25% v/v. A further group offour animals was treated withacetone/olive oil 4:1alone.

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
25	4.99	Positive
50	4.29	Positive
100	No data as animals terminated

 

Conclusion

The test item was considered to be a sensitizer under the conditions of the test.