Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 June 2017 - 17 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Butane-1,3-diyl diacrylate
EC Number:
243-105-9
EC Name:
Butane-1,3-diyl diacrylate
Cas Number:
19485-03-1
Molecular formula:
C10H14O4
IUPAC Name:
butane-1,3-diyl bisacrylate
Test material form:
liquid
Specific details on test material used for the study:
- No correction for purity required
- Stability at higher temperatures: yes, maximum temperature: 38°C
- Solubility and stability in vehicle: not indicated

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain
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: 52, 164, 512, 1600 and 5000 µg/plate
Second experiment: 17, 52, 164, 512, 1600 and 5000 µg/plate

The top dose of 5000 µg/plate is according to guideline OECD 471.
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the vehicle has been accepted and approved by authorities and international guidelines
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
For details see 'Any other information on materials and methods', table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation).
Glucose agar medium (25 mL) contained 18 g/L purified agar and 20g/L glucose. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine.
and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
- First experiment: direct plate assay
- Second experiment: pre-incubation assay

DURATION
- Preincubation period (experiment 2): 30 minutes by 70 rpm at 37°C
- Exposure duration: 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: 10^98 cells/mL

METHODS OF SLIDE PREPARATION:
- Experiment 1: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of
non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.
- Experiment 2: The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO (see study plan deviation). After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: all concentrations (without S9); Exp. 2: at 1600 (without S9) and 5000 (with and without S9) 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:
Exp. 1: all concentrations (without S9); Exp.2: at 1600 (without S9) and 5000 (with and without S9) 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:
Exp. 1: all concentrations (with and without S9); Exp. 2: at 1600 (without S9) and 5000 (with and without S9) 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:
Exp. 1: all concentrations (without and with S9); Exp. 2: at 1600 (without S9) and 5000 (with and without S9) 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:
cytotoxicity
Remarks:
Only in exp. 2: at 1600 (without S9) and 5000 (with and without S9) µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Precipitation: in the first experiment at the start of the incubation period at 5000 µg/plate in all tester strains and at the end of the incubation period at 5000 µg/plate in tester strain TA100 only.
No precepitation was seen in the second experiment.

- Other:
In the first experiment, in tester strain TA1537, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 1600 µg/plate in the presence of S9-mix. Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item, rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- 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.

- Cytotoxicity was observed as a reduction of revertant colonies in the direct plate assay in all Salmonella typhimurium tester strains in the absence of S9 and for tester strain TA100 and TA98 in the presence of S9. In the pre-incubation assay cytotoxicity was observed as absence or extreme reduction of bacterial background for all tester strains at a dose level of 1600 and 5000 µg/plate in the absence of S9 and at a dose level of 5000 µg/plate in the presence of S9.
- Mutagenicity: no mutagenicity was observed in any of the tester strains at any dose level, with and without S9.

Any other information on results incl. tables

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. 

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD guideline 471 and GLP principles, 1,3-butylene glycol diacrylate 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 1,3-BUTYLENE GLYCOL DIACRYLATE 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 KAA0109 of the test item was a light yellow liquid with a purity of 84.6%. 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 only precipitated on the plates at the dose level of 5000 µg/plate in tester strain TA100 in the absence of S9-mix. Cytotoxicity, as evidenced by a reduction in the bacterial background lawn and/or a decrease in number of revertants, was observed in tester strain TA100 in presence and absence of S9-mix at the highest tested concentrations. No toxicity was observed in tester strain WP2uvrA at any of the concentrations tested.

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. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all three tester strains in the absence of S9-mix and in tester strain TA98 in the presence of S9-mix.

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 tester strains at the dose levels of 1600 and 5000 µg/plate in the absence of S9-mix and at 5000 µg/plate 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.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that 1,3-BUTYLENE GLYCOL DIACRYLATE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.