Reaction productReaction product of 1,1´-Methylenebis(4-substituted cyclohexane), [2-(substitutedmethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate and [3-prop-2-substituted-2,2-bis(prop-2-enoyloxymethyl)propyl] prop-2-enoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2011 - 19 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Reaction products of 1,1’-methylenebis (4-isocyanatocyclohexane) and pentaerythritol triacrylate and pentaerythritol tetraacrylate

- Substance type: Clear colourless or slightly yellow, sticky resin
- Physical state: solid
- Purity: 98.5% (heating residue)
- Lot/batch No.: 90501
- Expiration date of the lot/batch: 24 April 2012
- Storage condition of test material: At room temperature protected from light

Study specific test substance information
- General information Avoid heat, direct sunlight, acid and base
- Hygroscopic No
- Volatile No
- Test substance handling Use amber-coloured glassware or wrap container in tin-foil.
- Specific Gravity / Density 1.10-1.20
- Stability at higher temperatures Yes, maximum temperature: 50°C, maximum duration: 1 month
- Stability in vehicle:
• Methyl ethyl ketone At least 1 month
- Solubility in vehicle:
• Methyl ethyl ketone 0-100%

The test substance used for the formulations of 25% and 50% in the initial pre-screen test was stored in the dark instead of protected from light.
Evaluation: Although the test substance for the initial pre-screen study was not fully protected from light at all times, it was stored in the dark and the test substance was never exposed to direct sunlight. In addition, the dose levels of the main study were selected on the additional pre-screen study and the test substance for the additional pre-screen study was protected from light. Therefore, the deviation in the initial pre-screen test did not affect the main study.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeld makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9 - 23.1
- Humidity (%): 41-70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

A temporary deviation from the minimum level of temperature occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 23-nov-2012 to 19-dec-2012

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0, 1, 2.5, 5%

No. of animals per dose:

5

Details on study design:

The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest
concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2 (see section 6.7) and/or an increase
in ear thickness < 25%) and is the highest possible concentration that can technically be applied.

Initially, two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids).

The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

Based on the results of the initially treated animals, two additional concentrations (5% and 10%) were tested in a similar manner at a later stage.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

Ref. 1: Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.Methyl ethyl ketone (Merck, Darmstadt, Germany).

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance was weighed and stored protected from light using containers wrapped in tin foil (test substance used for the initial pre-screen test was not stored in containers wrapped in tin foil, but was stored in the dark*). The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 45ºC for a maximum of 20 minutes. The test substance formulations were allowed to cool down to a temperature of maximally 39.5ºC prior to dosing.

* Evaluation: Although the test substance for the initial pre-screen study was not fully protected from light at all times, it was stored in the dark and the test substance was never exposed to direct sunlight. In addition, the dose levels of the main study were selected on the additional pre-screen study and the test substance for the additional pre-screen study was protected from light. Therefore, the deviation in the initial pre-screen test did not affect the main study.


Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a
description of all other (local) effects was recorded according to guidelines.
Ear thickness: On Day 1 (prior to dosing) and on Day 6, ear thickness measurements were conducted for control animals (animals 1-5) and animals treated at 5% (animals 16-20) using a digital thickness gauge.

Ear thickness measurements were not performed for animals treated at 1% and 2.5% (animals 6-15) in the main study.
Evaluation: Sufficient data was available to evaluate the irritation of the ears in the main study.


Necropsy: All animals surviving to the end of the study were sacrificed by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal). (no necropsy was conducted on the animals of the pre-screen test).

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 1, 2.5 and 5% were 11.4, 18.5 and 24.7 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 1, 2.5 and 5% were 1058, 1707 and 2279 DPM respectively. The mean DPM/animal value for the vehicle control group was 92 DPM.

Any other information on results incl. tables

Results Pre-screen test:

Initially, test substance concentrations of 25% and 50% were tested. The ear thickness of animals treated at 25% and 50% were increased on Day 3 and, more pronounced, on Day 6 when compared to theirDay 1 pre-dose values. The average increase of ear thickness on Day 6 exceeded 25% for both test substance concentrations of 25% and 50%. Very slight erythema of the ears was observed for animals at both concentrations on Days 3-6 and for animals at 50% also on Day 2.

Based on the results of the initial pre-screen test, test concentrations of 5% and 10% were also tested in additional animals.The ear thickness of animals treated at 5% and 10% were increased on Day 3 and, more pronounced, on Day 6 when compared to theirDay 1 pre-dose values. The average increase of ear thickness on Day 6 exceeded 25% for the 10% test substance concentration, but not for the 5% test substance concentration. Very slight erythema of the ears was observed for animals at both concentrations on Days 3-6.

No signs of systemic toxicity were observed in any of the animals examined.

Based on these results, the highest test substance concentration selected for the main study was a 5% concentration.

Other results - main study:

Very slight erythema of the ears was observed for animals treated at 5% on Days 3-6. No erythema of the ears was observed for any of the other animals. The ear thickness of animals treated at 5% were increased on Day 6 when compared to theirDay 1 pre-dose values, but the increase was less than 25%. The ear thickness of control animals was similar on Day 1 (pre-dose) and on Day 6.The irritation of the ears as shown by the animals at 5% was considered not to have a toxicologically significant effect on the activity of the nodes.

 

All auricular lymph nodes of animals treated with the test substance appeared larger in size as compared to those of control animals. The largest auricular lymph nodes were found in the higher dose groups.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for one control animal was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The SI values calculated for the substance concentrations 1, 2.5 and 5% were 11.4, 18.5 and 24.7 respectively.
These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 1%.
Based on these results:
- according to the recommendations made in the test guidelines, Reaction products of 1,1’-methylenebis(4-isocyanatocyclohexane) and pentaerythritol triacrylate and pentaerythritol tetraacrylate would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), Reaction products of 1,1’-methylenebis(4-isocyanatocyclohexane) and pentaerythritol triacrylate and pentaerythritol tetraacrylate should be classified as strong skin sensitizer (Category 1, Sub-category 1A).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Reaction products of 1,1’-methylenebis(4-isocyanatocyclohexane) and pentaerythritol triacrylate and pentaerythritol tetraacrylate should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.