Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The DPRA test (OECD 442C) cannot be performed on UVCB substance. So an in vitro skin sensitization study (KeratinoSens, OECD 442D) was performed and showed positive evidence of skin sensitization. A second in vitro skin sensitization study (h-CLAT, OECD 442E) was not performed because the results of this test will not change the needing to perform an in vivo study (OECD 429, LLNA). The mouse study showed no evidence of skin sensitization. As the in vivo studies are considered to be more relevant to conclude on the skin sensitization classification, the positive results obtained in vitro are considered to be false positive results and 2 moles Ethoxylated Bisphenol A methacrylate is considered to be not skin sensitizer.


 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
July 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
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)
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
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
- Morality/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
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
2% concentration
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5% concentration
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.

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.

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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

LLNA study (CRL 2021)


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. 


In vitro skin sensitisation study (CRL 2021)


In an in vitro (KeratinoSensTM) study, performed according to OECD guideline 442D and in accordance with GLP principles, the ability of the test substance to activate the antioxidant/electrophil responsive element (ARE)-dependent pathway was determined. The test item was dissolved in dimethyl sulfoxide (DMSO) at 40 mg/mL. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.20 – 400 μg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. Precipitation was observed in the 96-well plates in experiment 1 (200 and 400 μg/mL at the start and end of the incubation period) and experiment 2 (50 to 400 μg/mL at the start of the incubation period and 100 to 400 μg/mL at the end of the incubation period).


Overall it is concluded that the test conditions were adequate and that the test system functioned properly.


The test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, doserelated induction of the luciferase activity (EC1.5 values of 1.6 μg/mL and 3.8 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 8.50-fold and 6.64-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μg/mL with a cell viability of >70% compared to the vehicle control.


In conclusion, Ethoxylated Bisphenol A dimethacrylate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.


Guinea pigs study (Huntingdon 1978)


The method employed for the detection of delayed contact hypersensitivity (Kynoch 1978) was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman (1970). Ten male albino guinea pigs were used for assessment of the sensitizing potential of 2 moles Ethoxylated bisphenol A dimethacrylate.


No dermal irritation was observed following intradermal injections (induction) of test substance 5% in liquid paraffin. No dermal irritation was seen following the topical application of undiluted test substance (topical induction). No dermal irritation was seen in any of guinea-pigs following the challenge application (25% v/v of test substance).


According to this preliminary study, 2 moles Ethoxylated bisphenol A dimethacrylate is not sensitizing for skin in guinea pigs.


 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

 


 Based on the in vivo skin sensitization study (LLNA), no classification for skin sensitisation is required for 2 modes ethoxylated bisphenol A dimethacrylate according to the Regulation EC N°1272/2008.