[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:

17 February 2021 - 09 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was required in the final ECHA decision CCH.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

July 2003

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

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 mass of (1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl) bismethacrylate and 2-{4-[2-(4-{2-[2-(methacryloyloxy)ethoxy]ethoxy}phenyl)propan-2-yl]phenoxy}ethyl methacrylate

EC Number:

939-702-5

Molecular formula:

C23H24O4 (C2H4O)n

IUPAC Name:

Reaction mass of (1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl) bismethacrylate and 2-{4-[2-(4-{2-[2-(methacryloyloxy)ethoxy]ethoxy}phenyl)propan-2-yl]phenoxy}ethyl methacrylate

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: yes
- Microbiological status of animals: SPF
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 20.7 to 26.3 g
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized wooden fibers as bedding material
- Diet: ad libitum; Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum; Municipal tap-water
- Contaminants: It is considered that there were no known contaminants in the water or feed that would interfere with the objectives of the study.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23°C (actual range)
- Humidity (%): 38 - 42% (actual range)
- Air changes (per hr): 10 or greater air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 17 February 2021 To: 8 March 2021

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

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.

Concentration:

Pre-screen test: 100, 50, 25 and 10%.
Main study: 10, 5 and 2%.

No. of animals per dose:

5 in main study; pre-screen test 2

Details on study design:

PRE-SCREEN TESTS:
- Initially, two test item concentrations were tested; 50% and 100%. The highest concentration was the maximum concentration as required in the test guidelines. Based on the results, two additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

MAIN STUDY
- Induction: The dorsal surface of both ears was topically treated (25 µL/ear) with the test item, at
approximately the same time on days 1, 2 and 3. 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
- Tissue processing for radioactivity: Day 6
- Radioactivity measurements: Day 7

OBSERVATIONS
- Mortality/moribundity checks: Twice daily, in the morning and at the end of the working day
- Post-dose observations: Once daily on days 1-6 (on days 1-3 at least 3 hours after dosing).
- Body weights: Individually weighed on day 1 and 6
- Irritation: Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) (table 1).

TERMINAL PROCEDURES
- No necropsy was performed, since all animals survived until the end of the observation period.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Amber glassware was used for the preparation of formulations.
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.

Positive control substance(s):

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

Statistics:

no

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

2% concentration

Results 2

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

5% concentration

Results 3

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

10% concentration

Cellular proliferation data / Observations:

PRE-SCREEN TEST
Test item concentrations selected for the main study were based on the results of a pre-screen test. At 100%, 50% and 25%, a clinical sign of systemic toxicity (hunched posture) was noted in all animals. Therefore, these concentrations did not meet the selection criteria. At 10%, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

MAIN STUDY
SKIN REACTIONS/IRRITATION
The very slight irritation of the ears as shown by all animals treated at 10% on Days 3 and 4 was considered not to have a toxicologically significant effect on the activity of the nodes. No irritation was observed in the other animals.

SYSTEM 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
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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 2, 5 and 10% were 312, 563 and 437 DPM, respectively. The mean DPM/animal value for the vehicle control group was 286 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.1, 2.0 and 1.5, respectively.

Any other information on results incl. tables

Table 1. Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

group	TS ^1(%)	animal	Size nodes ^2		DPM ^3 / animal	mean DPM ± SEM ^4			mean SI ± SEM
			left	right
1	0	1	n	n	228	286	±	92	1.0	±	0.3
		2	n	n	130
		3	n	n	262
		4	n	n	644
		5	n	n	168
2	2	6	n	n	326	312	±	20	1.1	±	0.1
		7	n	n	298
		8	n	n	365
		9	n	n	246
		10	n	n	326
3	5	11	n	n	753	563	±	120	2.0	±	0.4
		12	n	n	918
		13	n	n	463
		14	n	n	428
		15	n	n	251
4	10	16	n	n	519	437	±	80	1.5	±	0.3
		17	n	n	369
		18	n	n	382
		19	n	n	695
		20	n	n	221

¹   TS = test item (% w/w).

²   Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

³     DPM = Disintegrations per minute.

⁴     SEM = Standard Error of the Mean.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not skin sensitizer

Conclusions:

A Local Lymph Node Assay (LLNA) was performed according to OECD guideline 429 and in accordance with GLP principles. Since there was no indication that the test item elicits a SI = 3 when tested up to 10%, Ethoxylated Bisphenol A dimethacrylate was considered not to be a skin sensitizer.

Executive summary:

A Local Lymph Node Assay (LLNA) was performed according to OECD guideline 429 and in accordance with GLP principles. 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. At 100%, 50% and 25%, a clinical sign of systemic toxicity (hunched posture) was noted. Therefore, these concentrations did not meet the selection criteria. At 10%, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 or 10% 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.
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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 2, 5 and 10% were 312, 563 and 437 DPM, respectively. The mean DPM/animal value for the vehicle control group was 286 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.1, 2.0 and 1.5, respectively.
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, Ethoxylated Bisphenol A dimethacrylate was considered not to be a skin sensitizer.
Based on these results, Ethoxylated Bisphenol A dimethacrylate 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).