[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 10 July 2018 to 08 August 2018. Report Issued: 16 October 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Due to limited availability of mice at the supplier stated in the study plan mice were obtained from an alternative supplier. This deviation was considered to have not affected the integrity or validity of the study.

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, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Molecular formula:

not applicable as UVCB

IUPAC Name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS (UK) Limited, Oxon, UK).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23g
- Housing: Suspended solid floor polypropylene cages furnished with softwood flake bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least fifteen per hour
- Photoperiod (hrs dark / hrs light): 12 hours in light, 12 hours in darkness

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Control = vehicle only (acetone/olive oil (4:1 v/v))
Low dose = 25% v/v
Mid dose = 50% v/v
High dose = 100% v/v

No. of animals per dose:

Four per dose group.

Details on study design:

PRELIMINARY SCRENING 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. 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.

MAIN TEST

Test Item Administration
Groups of four mice were treated with either the undiluted test item (100% v/v) or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. 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 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, giving a total of 20 μCi to each mouse.

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. The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze.

The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

After approximately 18 hours incubation at approximately 4°C, the precipitates were recovered by centrifugation, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting.

Positive control substance(s):

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

Results and discussion

Positive control results:

The postive control (run as a separate study) responded as expected and confirmed the responsiveness of the test system in the laboratory.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

11.08

Test group / Remarks:

25% v/v test substance

Remarks on result:

other: Positive

Results 2

Key result

Parameter:

SI

Value:

11.99

Test group / Remarks:

50% v/v test substance

Remarks on result:

other: positive

Results 3

Key result

Parameter:

SI

Value:

15.6

Test group / Remarks:

100% v/v test substance

Remarks on result:

other: Positive

Any other information on results incl. tables

There were no deaths on the study. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

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

Executive summary:

Introduction

A study was performed to assess the skin sensitisation potential of the test substance 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 guidelines:

 

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)
• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

 

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, 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 item either undiluted (100% v/v), or as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% 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
25	11.08	Positive
50	11.99	Positive
100	15.60	Positive

 

 

Conclusion

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