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Bacterial reverse mutation assay (Ames test) (read-across with Ethoxylated bisphenol A diacrylate)

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium (OECD 471).

After a preliminary assay, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. The test item Ethoxylated bisphenol A diacrylate was dissolved in dimethylsulfoxide (DMSO).

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

Since the test item was found freely soluble and non-cytotoxic in the preliminary test, the highest selected dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains used in both mutagenicity experiments, with and without S9 mix. A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in the experiments performed using the direct plate incorporation method and at dose-levels ≥ 2500 µg/plate in the experiment performed using the pre-incubation method. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any tested dose-level towards the five strains used, in any experiments, either with or without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix. The test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium, either in the presence or in the absence of liver metabolizing system.

In vitro mammalian cell gene mutation test (HPRT) (read-across with Ethoxylated bisphenol A diacrylate)

Ethoxylated bisphenol A diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for all experiments. For Experiment 1 eleven concentrations, ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentration analysed in the absence of S-9 was 40 µg/mL, which gave 21% RS and was considered acceptably close to the target toxicity range of 10-20% RTG. In the presence of S-9, steep concentration related toxicity was observed between 200 and 250 µg/mL, which gave 44% and 2% RS, respectively. Marked heterogeneity was observed at 250 µg/mL but the concentration was included in the analysis for comparative purposes. In Experiment 2 twelve concentrations, ranging from 5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 275 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 45 µg/mL in the absence of S-9 and 210 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively. In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Ethoxylated bisphenol A diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. It is concluded that Ethoxylated bisphenol A diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

In vitro mammalian cell micronucleus test (read-across with Ethoxylated bisphenol A diacrylate)

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells, in L5178Y TK+/- mouse lymphoma cells (OECD 487).

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254: first experiment of 3 h treatment/24 h recovery, with and without S9 mix; second experiment of 24 h treatment/20 h recovery without S9 mix, and 3 h treatment/24 h recovery with S9 mix.

 In order to check the reliability of the significant increase in the frequency of micronucleated cells noted in the second experiment without S9 mix (24 h treatment + 20 h recovery) and in order to exhibit about 55% toxicity, a third experiment was performed using eight dose-levels of the test item (two cultures/dose-level) without metabolic activation and using a 24 h treatment + 20 h recovery period.

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account. The test item was dissolved in dimethylsulfoxide (DMSO).

Since the test item was found severely cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be tested in the main experiments was based on the level of precipitation for the first experiment with S9 mix and on the level of cytoxicity for all the other experiments, according to the criteria specified in the international guidelines.

Following the 3-hour treatment without S9, no noteworthy increase in the frequency of micronucleated cells was noted in comparison to the vehicle control cultures. Following the 24-hour treatment (without S9) in the second experiment, an increase in the frequency of micronucleated cells (reaching or exceeding the threshold of 2.5-fold the vehicle control value) was observed at dose-levels >= 12.5 µg/mL. This increase was dose-related and reached statistical significance at the dose-level of 25 µg/mL (p < 0.05). Also, the corresponding frequencies of micronucleated cells exceeded the historical data range of the vehicle control (3 and 5 micronucleated cells in 1000 cells at 12.5 and 25 µg/mL, respectively,versus 0.5 to 2 for the historical data range). Moreover, this increase was found to be reproducible since significant increases in the frequency of micronucleated cells exceeding the threshold of 2.5-fold the vehicle control value were observed at 25 and 30 µg/mL in a third experiment performed under the same experimental conditions with a narrower range of dose-level. Statistical significance was reached at 25 µg/mL. Therefore, these increases being reproducible in independent experiments, they were considered as biologically significant.

With S9 mix, In the first experiment, a marked toxicity was observed at 200 µg/mL as shown by a 77% decrease in the PD. In the second experiment with S9 mix, no noteworthy toxicity was observed at any tested dose-levels as shown by the absence of notably decrease in the PD.

In the first experiment, non dose-related increases in the frequency of micronucleated cells, exceeding the threshold of 2.5-fold the vehicle control value were observed at all analyzed dose‑levels. At 6.25 µg/mL, the increase was statistically significant (p < 0.05). At all analyzed dose-levels, the frequencies of micronucleated cells remained within the historical data range of the vehicle control (up to 4 micronucleated cells in 1000 cellsversus0.5 to 5 for the historical data range). Moreover, these increases were neither dose-related nor reproducible since no increase in the frequency of micronucleated cells was noted in the second experiment performed under the same experimental conditions with a narrower range of dose-level. Consequently, these increases, being neither reproducible nor dose-related, were considered as non-biologically relevant.

To conclude, the test item induced chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells in the absence of rat metabolizing system. In the presence of metabolic activation, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in L5178Y TK+/- cultured mouse lymphoma cells.

Justification for selection of genetic toxicity endpoint
Three studies are available for the genotoxicity endpoint on Ethoxylated bisphenol A diacrylate (read-across). There are three in vitro studies.

Short description of key information:
The three in vitro recommended tests were performed on Ethoxylated Bisphenol A diacrylate (read-across). The Ames test and the HPRT test showed negative results in presence and in absence of metabolic activation. However, the in vitro micronucleus test showed a positive response without metabolic activation. An in vivo micronucleus test was proposed to conclude on the genotoxicity potential of Ethoxylated Bisphenol A diacrylate.

Endpoint Conclusion: No study available (further information necessary)

Justification for classification or non-classification

All key studies were performed on Ethoxylated bisphenol A diacrylate. The Ames test and the HPRT test showed negative results in presence and in absence of metabolic activation. However, the in vitro micronucleus test showed a positive response without metabolic activation. No in vivo study are available.

The supporting studies were performed on 2 moles Ethoxylated bisphenol A dimethacrylate, and showed negative results with and without metabolic activation in all three tests (Ames, HPRT and in vitro micronucleus tests).

Based on the available data, it was not possible to conclude on the genotoxicity potential of Ethoxylated Bisphenol A diacrylate, and therefore of Ethoxylated bisphenol A dimethacrylate. It is necessary to perform a vivo study. As the in vitro micronucleus assay was positive, an in vivo micronucleus test on rat (OECD 474) is proposed to ECHA on Ethoxylated bisphenol A diacrylate.