Registration Dossier

Administrative data

Description of key information

To evaluate the skin sensitisation potential of Ethoxylated (3) bisphenol A dimethacrylate, an in vitro skin sensitisation test was first performed. As the substance is an UVCB, the Direct peptide reactivity assay (DPRA, OECD 442C) cannot be done, an in vitro skin sensitisation test (KeratinoSens, OECD 442D) was performed and positive results were obtained.

The registrants decided to not perform the second in vitro skin sensitisation test (h-CLaT, OECD 442E), because of in case of positive, negative or inconclusive results in this test, an in vivo skin sensitisation study (OECD 429, LLNA) should be performed after. Indeed, in case of negative/inconclusive h-CLaT, no conclusion can be made with one positive KeratinoSens and one negative/inconclusive h-CLaT. In case of positive h-CLaT, the substance will be considered as skin sensitizer (with two positive in vitro tests) but a LLNA should be performed to determine the sub-category of classification (1B or 1A). That's why, LLNA is the second test performed and showed negative results. The results obtained in vitro are considered to be false positive results.

In conclusion, Ethoxylated (3) bisphenol A dimethacrylate is considered to be not a 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:
28 Aug 2019 - 18 Sep 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes
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
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 21.2 to 25.2 g.
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), provided ad libitum
- Water: municipal tap-water, provided ad libitum
- Acclimation period: the animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions: no, animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 44-76%
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 28 Aug 2019 To: 16 Sept 2019
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 and 100% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. There was no information available about the stability and the solubility of the test item in vehicle. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.

A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study.
- Tested concentrations: 50% and 100% (w/w)
- Irritation: at 100% test item concentration only slight irritation of the ears was observed.
- Systemic toxicity: not observed
- Ear thickness measurements: at 50%: between 0.210 and 0.235 on days 1-6, an increase of max. 10% was observed on day 6; at 100%: between 0.200 and 0.240 on days 1-6, an increase of max. 15% was observed on day 6.
- Erythema scores: at 50%: score 1 (barely perceptible erythema) on day 2, 3, 4, 5 and 6, at 100%: score 1 (barely perceptible erythema) on each day of treatment (day 1, 2 and 3).

MAIN STUDY
- INDUCTION (days 1, 2 and 3): The dorsal surface of both ears was topically treated (25 µL/ear) each day with the test item at concentrations of 0, 25, 50 and 100% (w/w).
- EXCISION OF NODES (day 6): Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine. After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. 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: 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) and then stored in the refrigerator until the next day.
- RADIOACTIVITY MEASUREMENTS: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail 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).

OBSERVATIONS:
- Mortality: animals were checked twice daily for general health/mortality and moribundity
- Clinical observations: once daily on day 1-6 (on days 1-3 between 3 and 4 hours after dosing)
- Bodyweight: animals were weighed individually on day 1 (predose) and 6 (prior to necropsy)
- Irritation: erythema and eschar formation observations were performed once daily on days 1-6 (on days 1-3 within 1 hour after dosing)

- Criteria used to consider a positive response:
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical analysis was performed.
Positive control results:
- Test date: July 2019
- SI values: 0.7, 2.2 and 6.4 for 5, 10 and 15% concentrations, respectively.
- EC3 value: 4.8-19.5% (within the acceptable range compared to reliability checks in recent years).
Based on this result, it was concluded that the Local Lymph Node Assay as performed at the test facility is an appropriate model for testing contact hypersensitivity.
Parameter:
SI
Value:
0.9
Test group / Remarks:
25%
Parameter:
SI
Value:
0.9
Test group / Remarks:
50%
Parameter:
SI
Value:
0.8
Test group / Remarks:
100%
Cellular proliferation data / Observations:
EC3 CALCULATION was not possible as the test item did not elicited a SI>3 when tested up to 100%.

IRRITATION: The slight irritation of the ears as shown by animals treated at 100% on days 1-3 was considered not to have a toxicologically significant effect on the activity of the nodes.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

MACROSCOPIC EXAMINATION: The auricular lymph nodes of the lowest dose group (25%) were considered normal in size. The lymph nodes of the 50% and 100% dose groups were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Interpretation of results:
GHS criteria not met
Remarks:
Not skin sensitizing
Conclusions:
Ethoxylated (3) bisphenol A dimethacrylate did not elicit a SI=3 when tested up to 100% and is therefore considered not to be a skin sensitizer.
Executive summary:

The objective of this study was to evaluate whether Ethoxylated (3) bisphenol A dimethacrylate 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 No.429 (2010) test guidelines.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 50% and 100% test item concentration, no signs of systemic toxicity were noted and only slight irritation of the ears was observed and therefore a 100% 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 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 lowest dose group (25%) were considered normal in size. The lymph nodes of the 50% and 100% dose groups 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 471, 470 and 392 DPM, respectively. The mean DPM/animal value for the vehicle control group was 498 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.9, 0.9 and 0.8, respectively.

Since there was no indication that the test item elicited a SI = 3 when tested up to 100%, Ethoxylated (3) bisphenol A dimethacrylate was considered not to be a skin sensitizer. Based on these results, Ethoxylated (3) bisphenol A dimethacrylate would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines

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

In vitro KeratinoSens test (2019)


The objective of this study was to evaluatethe potential of the test item to activate the Nrf2 transcription factor.This test is a part of a tiered strategy for the evaluation of skin sensitisation potential (OECD Guideline No. 442D).


This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.


The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence.In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent validated runs were performed as part of this study.


All acceptance criteria were met for the positive and negative controls in both runs, this study was therefore considered as validated.


This study was performed testing concentrations from 0.98 and up to 2000 µM in culture medium containing 1% DMSO.


 At these tested concentrations, statistically significant gene-fold inductions above the threshold of 1.5 were noted at concentrations= 15.63 µM with an apparent dose-response relationship, in both runs.


The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered toactivate the Nrf2 transcription factor.


 


In vivo LLNA (2019)


The objective of this study was to evaluate whether Ethoxylated (3) bisphenol A dimethacrylate 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 No.429 (2010) test guidelines.


Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 50% and 100% test item concentration, no signs of systemic toxicity were noted and only slight irritation of the ears was observed and therefore a 100% 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 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 lowest dose group (25%) were considered normal in size. The lymph nodes of the 50% and 100% dose groups 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 471, 470 and 392 DPM, respectively. The mean DPM/animal value for the vehicle control group was 498 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.9, 0.9 and 0.8, respectively.


Since there was no indication that the test item elicited a SI = 3 when tested up to 100%, Ethoxylated (3) bisphenol A dimethacrylate was considered not to be a skin sensitizer. Based on these results, Ethoxylated (3) bisphenol A dimethacrylate would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the reliable in vivo study (LLNA), Ethoxylated (3) bisphenol A dimethacrylate should not be classified as skin sensitizer according to the Regulation EC N°1272/2008.