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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
According to the results of the presently available data, the test substance is not mutagenic in the Ames test under the experimental conditions chosen.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-07-02 to 1997-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study conducted under GLP.
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain characteristics:
other: Salmonella strains: defective excision repair System (uvrB) which prevents the repair of lesions which are induced in the DNA, and reduced hydrophilic polysaccharicle layer (rfa) which leads to an increase in permeability to lipophilic substances.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500, 5000 µg/plate (standard plate test)
0, 4, 20, 100, 500, 2500 µg/plate (preincubation test)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine (AAC) chloride monohydrate and N-ethyl-N'-nitro-N-nitrosoguanidine
Positive control substance:
other: see details
Details on test system and conditions:
I) Standard plate test with Salmonelly typhimurium

METHOD OF APPLICATION:
Test tubes containing 2 mL portions of soft agar kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activation) or
0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates.

DURATION:
- Exposure duration: After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent): 48 - 72 hours

- SELECTION AGENT: his- medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


II) Standard plate test with Escherichia coli

METHOD OF APPLICATION:
Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45°C and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activation) or
0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.

DURATION:
- Exposure duration: After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent): 48 - 72 hours

- SELECTION AGENT: trp- medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


III) Preincubation test

METHOD OF APPLICATION:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
- Preincubation period: 20 min
- Exposure duration: After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies are counted.
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent): 48 - 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Standard plate and preincubation tests:

In each experiment 3 test plates per control used. After incubation at 37 °C fo 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Positive controls:
with S-9 mix: 2.5 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535
60 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strain E. coli WP2 uvrA
without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537
10 µg N-ethyl-N'-nitro-N-nitroso-guanidin (ENNG) (dissolved in DMSO) for the strain E. coli WP2 uvrA

The Titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.



Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not data, buffered test medium was used
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no limitation within the boundaries of this test
- Precipitation: no precipitation
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:
1st Experiment: Doses 0, 20, 100, 500, 2500 and 5000 µg/plate
2nd Experiment: Doses 0, 4, 20, 100, 500 and 2500 µg/plate

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect (reduced his or trp background growth, decrease in the number of his or trp revertants, decreased titer) was observed depending on the strain and test conditions at doses >=2,500 µg/plate (standard plate test) or >= 500 µg/plate (preincubation test).

Standard Plate Test:

Concentration µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

I

II

I

II

I

II

I

II

I

II

Solvent Control

19

19

9

9

26

38

109

111

38

39

Positive Control

1535

268

318

139

866

496

1362

927

579

247

20

17

15

9

11

28

37

117

107

39

40

100

17

16

8

8

24

36

104

108

36

42

500

15

15

6

6

21

31

107

93

37

42

2500

13

18

5

6

17

30

102

100

28

30

5000

6

12

3

2

11

24

59

68

24

13

I without S9 mix

II with S9 mix

 

Preincubation Method:

 

Concentration µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

I

II

I

II

I

II

I

II

I

II

Solvent Control

19

19

9

10

26

32

106

108

42

33

Positive Control

1296

129

746

124

1305

1002

942

1360

825

177

4

15

18

11

10

28

28

107

123

32

33

20

16

13

8

11

24

34

120

102

34

32

100

16

17

7

13

28

29

124

99

30

24

500

14

11

6

7

25

22 B

107

86 B

36

22

2500

0 B

0 B

0 B

0 B

0 B

0 B

0 B

0 B

15

0 B

 

I  without S9 mix

II with S9 mix

B: reduced his- background growth

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames Test:

The substance Butandioldivinylether was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli - reverse mutation assay. The tests are carried out in accordance with the OECD guidelines for testing of chemicals No. 471 and No. 472. An increase in the number of his or trp revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance Butandioldivinylether is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen.


Justification for selection of genetic toxicity endpoint
Applicable to endpoint study, guideline-conform and conducted under GLP.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)
The available study is considered reliable and suitable for classification purposes under Directive 67/548/EEC. As a result based on this Ames test the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result based on this Ames test the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.