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

Endpoint summary

Description of key information

In a GLP compliant OECD 429 LLNA, the test item produced a stimulation index > 3 at concentrations in excess of 10% (stimulation index ≥ 3 for 2.5 and 5% test item concentrations). The EC3 value was calculated to be 9.5%, suggesting the test item is a moderate skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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.

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.

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)

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

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.

Positive control results:

SI = 11.8 and 4.9

Key result

Parameter:

EC3

Value:

9.5

Parameter:

SI

Value:

< 3

Test group / Remarks:

2.5 and 5% test item concentration

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.

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

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).

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 February 1984 - 23 March 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The experimental design and reporting are adequate for risk assessment, however the results may be confounded by the pre-condition of test animals (i.e. lower threshold level for skin irritancy).

Justification for type of information:

The REACH information requirements were revised in 2016 to endorse a battery of in vitro assays for skin sensitisation. Whilst Annex VII does not require in vivo testing for skin sensitisation, all existing available information should be evaluated, including any available animal data, in accordance with Annex VI.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Guinea pigs were induced with the test item on Days 1, 4 and 7. Thirty minutes after treatment application, the test sites were ultraviolet irradiated for 30 minutes. On Day 28, a challenge test was performed on a different test site, following the same irradiation procedure. The degree of erythema was assessed 24 hours after application.
- Short description of test conditions: Guinea pigs were closely clipped and depilated prior to treatment, and were treated with the maximum test item concentration (30%) which did not cause erythema when exposed to untreated guinea pig skin.
- Parameters analysed / observed: Degree of erythema, bodyweight

GLP compliance:

yes

Type of study:

other: Photosensitisation

Justification for non-LLNA method:

The standard information requirements for REACH Annex VII substances for skin sensitisation can be satisfied by an assessment of the available in vivo data, physicochemical properties or in vitro skin sensitisation studies. Conducted prior to the validation and regulatory acceptance of alternative methods, the reliable (Klimisch 1) and GLP compliant in vivo photosensitisation test was available for the test substance and considered sufficient to be used as supporting data for the Annex VII information requirement.

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

ablino

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 5 to 8 weeks
- Weight at study initiation: 268-376 g
- Housing: Solid-floor polypropylene cages with softwood shavings
- Diet: Standard laboratory guinea pig diet, ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: Minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 45-65% RH
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route:

epicutaneous, open

Vehicle:

other: ethanol

Concentration / amount:

30% test item

Day(s)/duration:

Days 1, 4 and 7 / 30 minute irradiation

Adequacy of induction:

other: The highest concentration of the test material which did not cause primary irritation. Test site was depilated before exposure.

No.:

#1

Route:

other: epicutaneous, occlusive and open

Vehicle:

other: ethanol

Concentration / amount:

30% test item concentration

Day(s)/duration:

Day 28 / 30 minute irradiation

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

15

Details on study design:

RANGE FINDING TESTS: A primary irritation test was conducted in a preliminary test. The dorsal surface of two guinea pigs were closely clipped and depilated. Approximately two hours following depilation, 0.05 mL aliquots of the test material at concentrations of 10, 30, 50 and 100% test item were applied on the test sites. The degree of erythema was assessed 24 hours after application. The highest concentration of the test material which did not cause primary irritation in either animal (30%) was selected for the photosensitisation study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 (Days 1, 4 and 7)
- Exposure period: 30 minutes after test item application, animals were ultraviolet irradiated for 30 minutes
- Test groups: 1 test item concentration
- Control group: None
- Site: Nuchal region
- Frequency of applications: Single application for each exposure
- Concentrations: 30% test item, 0.1 mL aliquot

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 28
- Exposure period: 30 minutes after test item application, animals were ultraviolet irradiated for 30 minutes
- Test groups: 1 test item concentration
- Control group: Vehicle
- Site: Dorsal surface (previously untreated)
- Concentrations: 30% test item, 0.05 mL aliquot
- Evaluation (hr after challenge): 24 hrs after application

OTHER: Animals were ultraviolet irradiated at a distance of 35 cm using a Philips MLU 300W Irradiation Lamp. The MLU 300W Irradiation Lamp is a tungsten mercury lamp which emits strong ultra-violet radiation as well as infra-red radiation. The bulb is made of hard glass which filters out radiation below 280 nm. An internal reflector ensures a homogeneous beam of radiant energy. The maximum spectral energy emission was at 365 nm.

Degree of erythema scored as follows:
0: No erythema
1: Scattered mild redness
2: Moderate and diffuse redness
3: Intense redness and swelling

Challenge controls:

Vehicle (absolute ethanol) control

Positive control substance(s):

no

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

30% test item, irradiated

No. with + reactions:

11

Total no. in group:

15

Clinical observations:

Scattered mild redness was observed. Bodyweight gains were within normal limits

Remarks on result:

not determinable

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

30% test item, non-irradiated

No. with + reactions:

11

Total no. in group:

15

Clinical observations:

Scattered mild redness was observed. Bodyweight gains were within normal limits

Remarks on result:

not determinable

Reading:

1st reading

Hours after challenge:

24

Group:

other: vehicle control

Dose level:

Irradiated

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

Bodyweight gains were within normal limits.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

other: vehicle control

Dose level:

Non-irradiated

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

Bodyweight gains were within normal limits

Remarks on result:

no indication of skin sensitisation

Eleven of fifteen animals showed scattered mild redness (score 1) at both the irradiated and corresponding non-irradiated test material sites; no reactions were observed at the vehicle control site of these animals. The reactions are considered to be as a result of primary skin irritation caused by the test material at the challenge concentration of 30% (caused by a lowered threshold level for skin irritancy for these animals) or as a result of contact sensitisation caused by the test material.

Table 1. Photosensitisation results

Guinea pig number	30% test item, irradiated	Vehicle control, irradiated	30% test item, non-irradiated	Vehicle control, non-irradiated
1 -0	0	0	0	0
1 -1	0	0	0	0
1 -2	1	0	1	0
1 -3	1	0	1	0
2 -0	0	0	0	0
2 -1	1	0	1	0
2 -2	0	0	0	0
2 -3	1	0	1	0
3 -0	1	0	1	0
3 -1	1	0	1	0
3 -2	1	0	1	0
3 -3	1	0	1	0
4 -0	1	0	1	0
4 -1	1	0	1	0
4 -2	1	0	1	0

Interpretation of results:

study cannot be used for classification

Conclusions:

In a phototoxicity and photosensitisation test in guinea pigs, eleven of fifteen animals showed scattered mild redness following exposure to a 30% test item concentration challenge dose. The study director attributed the skin reactions to mild irritation.

Executive summary:

The photosensitisation potential of the test item was determined in female albino Dunkin-Hartley guinea pigs. On Day 1, the test item at 30% concentration was applied on the clipped and depilated nuchal region of 15 guinea pigs. Thirty minutes after application, the guinea pigs were exposed to ultraviolet irradiation for 30 minutes. The procedure was repeated on Days 4 and 7. On Day 28, a challenge dose of 30% test item concentration and a vehicle control were applied on the clipped and depilated dorsal regions of the guinea pigs. Thirty minutes after application, the guinea pigs were exposed to ultraviolet irradiation for 30 minutes. The degree of erythema was assessed 24 hours after application.

 

Eleven of fifteen animals showed scattered mild redness at both the irradiated and corresponding non-irradiated test material sites. The cause for minor skin reaction is undetermined, as the reactions may be as a result of primary skin irritation (caused by a lowered threshold level for skin irritancy for these animals) or as a result of contact sensitisation. The study is considered reliable with restriction (Klimisch 2) as it was GLP-compliant and the experimental design and reporting were adequate.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the test item was determined in a local lymph node assay (LLNA) in female CBA/JcrHSD mice (2016). Doses of 2.5, 5, 10, 25, 50 and 100% test item were 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) and is sufficient to meet the Annex VII information requirement.

The photosensitisation potential of the test item was determined in female albino Dunkin-Hartley guinea pigs (1984). On Day 1, the test item at 30% concentration was applied on the clipped and depilated nuchal region of 15 guinea pigs. Thirty minutes after application, the guinea pigs were ultraviolet irradiated for 30 minutes. The procedure was repeated on Days 4 and 7. On Day 28, the test item at 30% concentration and a vehicle control were applied on the clipped and depilated dorsal regions of the guinea pigs. Thirty minutes after application, the guinea pigs were ultraviolet irradiated for 30 minutes. The degree of erythema was assessed 24 hours after application. Eleven of fifteen animals showed scattered mild redness at both the irradiated and corresponding non-irradiated test material sites. The cause for minor skin reaction is undetermined, as the reactions may be as a result of primary skin irritation (caused by a lowered threshold level for skin irritancy for these animals) or as a result of contact sensitisation. However, in light of the 2016 study, the effects were likely to have been as a result of sensitisation. The study is considered reliable with restriction (Klimisch 2).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

One key study (Klimisch 1) is available for skin sensitisation classification (2016). The EC3 value was calculated to be 9.5% in a LLNA study, and in accordance with the CLP Regulation (EC) No. 1272/2008, the test item is a Category 1B skin sensitiser.