3,6,9-trioxaundecamethylene dimethacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study in bacteria is available on tetraethyleneglycol dimethacrylate and showed negative results.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2017 - 16 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- No correction factor for purity was required
- Stability at higher temperatures: maximum of 38°C

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9) prepared from male Sprague Dawley rats injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose-range finding test (reported as part of the first experiment): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
First experiment (with and without S9): 52, 164, 512, 1600 and 5000 µg/plate
Second experiment (with and without S9): 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the vehicle has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

For details on positive control substances see 'Any other information on materials and methods', table 1

Details on test system and experimental conditions:

- First experiment: direct plate assay
- Second experiment: pre-incubation assay

DURATION
- Preincubation period (only for experiment 2): 30 minutes by 70 rpm at 37°C
- Exposure duration (both experiments): 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain, with and without metabolic activation.

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

DECISION CRITERIA:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

ACCEPTABILITY CRITERIA:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in experiment 2 without S9 at a concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in experiment 2 without S9 at a concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in experiment 2 without S9 at a concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in experiment 2 without S9 at a concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- No precipitation was observed in both experiments.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

HISTORICAL CONTROL DATA (see table 2 and table 3)
- The results of the positive and solvent controls were within the historical data range of the test facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Since all bacterial strains showed negative responses of the entire dose-range, no significant dose-related increase in the number of revertants was observed in the two independently repeated experiment.
- In tester strain TA1537, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed at the dose levels of 164 and 5000 µg/plate in the absence of S9-mix. Since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item, rather it is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.

Table 2 Historical data of the solvent control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 - 36	3 - 32	3 – 20	3 – 23	8 - 41	9 - 55	66 - 161	63 - 160	10 – 59	9 - 69
Mean	11	11	6	7	16	23	105	105	25	31
SD	4	4	3	3	5	7	19	20	7	8
n	2057	2039	1950	1931	2023	2083	2027	2033	1739	1745

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Table 3 Historical data of the positive controls

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	125 - 1381	78 - 1058	55 – 1324	55 – 1051	410 – 1995	250 - 1977	537 – 1848	408 - 2651	93 – 1951	93 - 1359
Mean	839	220	736	382	1369	929	908	1330	1128	422
SD	153	112	331	150	310	345	178	324	484	151
n	2065	1967	1740	1933	1920	2014	2007	2020	1679	1728

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, Tetraethylene glycol dimethacrylate was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with or without metabolic activation.

Executive summary:

The objective of this study was to determine the potential of Tetraethylene glycol dimethacrylate and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch KZD0599 of the test item was a clear colourless liquid. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence S9-mix and in tester strain TA100 in presence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Tetraethylene glycol dimethacrylate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

in vitro gene mutation study in bacteria/ OECD TG 471 (2017) :

The objective of this study was to determine the potential of Tetraethylene glycol dimethacrylate and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence S9-mix and in tester strain TA100 in presence of S9-mix.

In both experiments, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was

observed.

In conclusion, based on the results of this study it is concluded that Tetraethylene glycol dimethacrylate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the available data, no classification for genetic toxicity is required for tetraethyleneglycol dimethacrylate according to the Regulation EC N°1272/2008.