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Bacterial reverse mutation assay (Ames test)

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using a dose range of 50 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy ofthe S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

In vitro mammalian cell gene mutation test (HPRT)

2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid 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 three independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post mitochondrial fraction (S-9), followed by a third confirmatory experiment conducted in the absence of S-9 only. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In Experiment 1 eleven concentrations, ranging from 0.4 to 4 µg/mL, were tested in the absence of S 9 and twelve concentrations, ranging from 5 to 80 μg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 2.4 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S 9, which gave 16% and 10% RS, respectively.

In Experiment 2 eleven concentrations, ranging from 0.5 to 4 µg/mL, were tested in the absence of S 9 and ten concentrations, ranging from 5 to 75 μg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed were 2.5 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, both of which gave 18%.

In Experiment 3, eleven concentrations, ranging from 0.5 to 3.5 µg/mL, were tested in the absence of S-9. Seven days after treatment, the highest concentration analysed was 3 µg/mL, which gave 9% RS. No concentration in Experiment 3 gave 10 20% RS: cultures analysed at 2.75 and 3 µg/mL gave 24% and 9% RS, respectively, therefore both concentrations were analysed.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4 nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiment 1 in the absence and presence of S-9 and in Experiment 2 in the presence of S-9 only, no statistically significant increases in mutant frequency were observed following treatment with test item at any concentration tested and there were no significant linear trends. A statistically significant increase in mutant frequency was observed at the highest concentration analysed (2.5 µg/mL) in the absence of S-9 in Experiment 2 with a significant linear trend.

In Experiment 3, a statistically significant increase in mutant frequency (MF) over the concurrent control was again observed at 2.5 µg/mL. However, two higher concentrations (2.75 and 3 µg/mL) were tested and the MF values at both concentrations were similar to (and not significantly different from) the vehicle control MF value and there was no statistically significant linear trend.

Small but significant increases in mutant frequency were therefore seen at 2.5 µg/mL in Experiments 2 and 3 in the absence of S-9. However, a statistically significant linear trend was observed only in Experiment 2 and in Experiment 3, no increases in mutant frequency were observed at two higher concentrations (2.75 and 3 µg/mL). The small statistical increases seen in Experiments 2 and 3 may therefore be considered of highly questionable biological relevance.

It is concluded that 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid induced small but significant increases in mutant frequency at a single concentration (2.5 µg/mL) in two out of three experiments performed in the absence of a rat liver metabolic activation system (S-9). However, a statistically significant linear trend was observed in only one experiment and in a confirmatory experiment, no increases in mutant frequency were observed at two higher concentrations (2.75 and 3 µg/mL). The small statistical increases may therefore be considered of highly questionable biological relevance. In the same test system, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid did not induce increases in mutant frequency in the presence of S-9 when tested up to toxic concentrations in two independent experiments.

In vivo mammalian cell micronucleus test

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 30 mg/kg and with 15 and 7.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil (two groups each of 7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 30 mg/kg in both the 24 and 48-hour groups, these were as follows: Hunched posture and ptosis.

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material was considered to be non-genotoxic under the conditions of the test.


Justification for selection of genetic toxicity endpoint
Several studies are necessary to evaluate the mutagenicity potential of 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid : the Ames test, HPRT test and the in vivo micronucleus assays.

Short description of key information:
The Ames test showed negative results in presence and in absence of metabolic activation (S9). The HPRT test is inconclusive without S9 and negative with S9. An in vivo study was available on 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid, it was a micronucleus study performed on rat by oral route; negative results were obtained in this test.
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 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The Ames test and the in vivo micronucleus assay in rats test showed negative results in presence and in absence of metabolic activation.

Inconclusive results were observed in the HPRT test but results suggests a negative result.

Based on the weight of evidence of all three tests, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid is considered to be not mutagenic, no classification for genotoxicity is required for 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid, according to the Regulation EC 1272/2008 and the Directive EEC/67/548.