Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 February 2016 - 4 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the absence of existing reliable data, the Murine Local Lymph Node Assay (LLNA) was the endorsed method of to assess skin sensitisation potential under REACH, as no EU-OECD in vitro tests for skin sensitisation had been validated. However, the REACH Annex VII information requirements were revised in 2016 to endorse a battery of in vitro assays for skin sensitisation. The LLNA for skin sensitisation (OECD 429) was initiated and completed for the test item prior to changes in regulatory guidance. The reliable and GLP compliant in vivo LLNA was considered sufficient to fulfil the endpoint as the key study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

yes

Remarks:

Relative humidity was at times outside protocol range and Day 4 observations of Set 1 animal were not recorded. The deviations were not considered to affect the outcome of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

yes

Remarks:

Relative humidity was at times outside protocol range and Day 4 observations of Set 1 animal were not recorded. The deviations were not considered to affect the outcome of the study.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Molecular formula:

C17H21NO2

IUPAC Name:

Methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Constituent 2

Reference substance name:

Methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Molecular formula:

C17H21NO2

IUPAC Name:

Methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JcrHSD

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Weight at study initiation: 18.3 to 24.2 g
- Housing: 1 to 5 per cage in polycarbonate box with bedding
- Diet:, PMI Feeds Inc. Formulab #5008, ad libitum
- Water: Municipal water supply, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): 31-94%
- Air changes (per hr): 10+
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

- IN-LIFE DATES: From: 25 December 2015 and 26 February 2016 To: 07 March 2016 and 02 May 2016

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Vehicle control, 2.5, 5, 10, 25 and 50% test item in vehicle, and 100% test item

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS: A range finding test was not conducted.

MAIN STUDY: The study was conducted in two sets, each consisting of a vehicle control and positive control. Test item concentrations 25, 50 and 100% were tested in Set 1 and test item concentrations of 2.5, 5 and 10% were tested in Set 2.

ANIMAL ASSIGNMENT AND TREATMENT: five animals per test item group

- Name of test method: Determination of disintegrations per minute (DPM) by liquid scintillation counting
- Criteria used to consider a positive response: ECETOC Potency Classification: Weak (10% ≤ EC3 ≤ 100%), moderate (1% ≤ EC3 < 10%), strong (0.1% ≤ EC3 < 1%), extreme (EC3 < 0.1%)

TREATMENT PREPARATION AND ADMINISTRATION: On Days 1, 2 and 3, each test animal received an open application of 25 µL of the treatment solution to the dorsum of both ears. Animals were given a two-day rest period on Days 4 and 5. On Day 6, animals were injected in the tail vein with tritiated methyl-thymidine and sacrificed 5 hours after injection by CO2 overdose.

Individual body weights were recorded on Day 1 prior to dosing and on Day 6 prior to injection. Animals were observed daily for clinical signs of toxicity and any excessive test site irritation.

Positive control substance(s):

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

Statistics:

A one-way parametric analysis of variance (ANOVA) with Dunnett's Multiple Comparisons Test was performed on DPM counts at a significance level of p<0.01. The EC3 (estimated concentration required for a 3-fold SI value) were then calculated.

Results and discussion

Positive control results:

SI = 11.8 and 4.9

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

9.5

Results 2

Parameter:

SI

Value:

< 3

Test group / Remarks:

2.5 and 5% test item concentration

Results 3

Parameter:

SI

Value:

>= 3

Test group / Remarks:

10, 25, 50 and 100% test item concentration

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Test item / vehicle control

EC3 CALCULATION
EC3 = c+[(3-d/(b-d)] x (a-c), using SI values closest to 3, one above and one below, where:
a = the dose concentration with higher SI
b = the higher SI value
c = the dose concentration with lower SI
d = the lower SI value

CLINICAL OBSERVATIONS: One mortality was observed in the positive control (Set I) and 100% test item concentration. No clinical signs were observed in animals in Set 1. All animals in Set 2 had matted fur around the ears.

BODY WEIGHTS: All survivors in Set 1 gained weight normally. In Set 2, one animal in the positive control, one animal treated with 2.5% test item concentration and three animals treated with 10% test item concentration lost weight during the study.

Any other information on results incl. tables

Table 1. LLNA results

Test item concentration (%)	Average count per mouse (DPM)	Number of mice in group	Test/Vehicle Control Ratio
Set 1
Vehicle control	1911	5	NA
25	15948	5	8.4
50	15378	5	8.0
100	12846	4	6.7
Positive control	22454	4	11.8
Set 2
Vehicle control	1684	5	NA
2.5	3615	5	2.2
5	3645	5	2.2
10	5224	5	3.1
Positive control	8320	5	4.9

NA: Not Applicable

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test tem produced a stimulation index ≥ 3 at all test item concentrations except 2.5 and 5%. The EC3 value was calculated to be 9.5%.

Executive summary:

The skin sensitisation potential of the test item was determined in a local lymph node assay (LLNA) in female CBA/JcrHSD mice (Murphy 2016). Doses of 2.5, 5, 10, 25, 50 and 100% test item was applied to the dorsum of both ears on Days 1, 2 and 3, alongside a vehicle control (4:1 v/v acetone:oilve oil) and positive control. All animals were given a two-day rest period on Days 4 and 5 and animals were injected with tritiated methyl-thymidine and sacrificed on Day 6. The test item produced a stimulation index ≥ 3 at all test item concentrations except 2.5 and 5%. The EC3 value was calculated to be 9.5%.

The LLNA (EU B.42; OECD 429) is an intentionally accepted in vivo test method to detect skin sensitisation (Category 1, 1A or 1B under CLP) and/or absence of effects requiring classification for skin sensitisation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the LLNA passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).