Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2020 - 07 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Section 4, Health Effects, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

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:

Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

EC Number:

951-458-1

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Test material form:

liquid

Details on test material:

Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil
EC Number: 951-458-1
Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil
Substance Name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
EC number: 951-458-1
Molecular formula: EtC(CH2OCOCH2CH2NMePh)3
Molecular weight: 617.79 g/mol


Solubility in vehicle:
Acetone/Olive oil (4:1 v/v) (AcOO) Not indicated

Stability in vehicle:
Acetone/Olive oil (4:1 v/v) (AcOO) Not indicated

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Source: Janvier, Le Genest-Saint-Isle, France
Females: Nulliparous and non-pregnant
Age at study initiation: Approximately 10 weeks old
Weight at study initiation: 19.9 to 26.9 g

Housing:
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Diet:
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment:
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Acclimation period:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Veterinary Care:
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Justification for Test System and Number of Animals
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.
The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in Appendix 4 of this report. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using a tail mark with indelible ink.


ENVIRONMENTAL CONDITIONS
Temperature: The actual daily mean temperature during the study period was 22 to 23°C.
Humidity: The actual daily mean relative humidity during the study period was 40 to 52%.
Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Photoperiod: A 12 hour light/12 hour dark cycle was maintained


IN-LIFE DATES
Study Initiation Date: 10 Apr 2020
Initiation of Dosing: 29 Apr 2020
Completion of In-life: 25 May 2020
Experimental Start Date: 29 Apr 2020
Experimental Completion Date: 26 May 2020



Rationale for Vehicle
The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw Data of these trials will be retained by the Test Facility. There was no information available about the stability and solubility of the test item in vehicle.

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
Any residual volumes were discarded.

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

Merck, Darmstadt, Germany and Acros Organics, Geel, Belgium

Concentration:

0%, 25%, 50%, 100%

No. of animals per dose:

5 animals per dose

Details on study design:

PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.

Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration that could technically be applied.

The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. 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 (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.

Animals were sacrificed after the final observation.

At a 50 and 100% test item concentration, no signs of systemic toxicity were noted and only very slight irritation were observed. Therefore, a 100% concentration was selected as highest concentration for the main study.


MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Allocation - see table in 'any other information' below

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
 
Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Statistics:

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by the test facility. In this study, performed in June 2020, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159 (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).

The SI values calculated for the test item concentrations 5, 10 and 25% were 2.4, 2.9 and 4.5, respectively. An EC3 value of 10.9% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
The raw data, study plan and report from this study are kept in the test facility archives. The test described above was performed in accordance with the test facility Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.8

Test group / Remarks:

25% concentration

Results 2

Parameter:

SI

Value:

1.4

Test group / Remarks:

50% concentration

Results 3

Parameter:

SI

Value:

1.1

Test group / Remarks:

100% concentration

Cellular proliferation data / Observations:

Skin Reactions / Irritation
The very slight irritation of the ears observed at 2, 50 and 100% on Days 2 and/or 3 was considered not to have a toxicologically significant effect on the activity of the nodes.
Transparent test item remnants were present on the dorsal surface of the ears of all animals at 100% on Days 1-3, which did not hamper scoring of the skin reactions.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
The auricular lymph nodes of the 100 and 25% group were considered normal in size. The nodes in the animals of 50% were slightly enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 410, 756 and 590 DPM, respectively. The mean DPM/animal value for the vehicle control group was 538 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.8, 1.4 and 1.1, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, the test item was considered not to be a skin sensitizer.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 4).
Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.

The study was carried out based on the guidelines described in:

- OECD, Section 4, Health Effects, No.429 (2010).

- EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

- EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Test item concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.  

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The auricular lymph nodes of the 100 and 25% group were considered normal in size. The nodes in the animals of 50% were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 410, 756 and 590 DPM, respectively. The mean DPM/animal value for the vehicle control group was 538 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.8, 1.4 and 1.1, respectively.

Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, the test item was considered not to be a skin sensitizer.

Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).