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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A GLP OECD 471 Ames test was performed. 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.

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:
Experimental start date: 02 February 2018. Experimental completion date: 01 March 2018.
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:
Adopted July 21, 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:
1,5-hexadiene. 9.4% purity.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch:01 June 2018
- Purity test date: 26 June 2017

Analyses conducted to support the information cited in the certificate of analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality system. Concentration, stability and homogeneity of test item formulations were not determined in this study. However, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 4 hours after preparation of the formulation.
Target gene:
Histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Additional mutations: rfa (deep rough, defective lipolysaccharide cellcoat), gal (galactose metabolism), chl (nitrate reductase), bio (biotin synthesis), uvrB (loss of excision repair system)
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate (rat liver microsomal enzymes) prepared from male Sprague Dawley rats injected intraperitoneally with Aroclor 1254
Test concentrations with justification for top dose:
Direct plate assay (first experiment): 1,5-hexadiene was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ¿g/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 ¿g/plate.
Pre-incubation assay (second experiment): Based on the results of the first mutation assay, 1,5-hexadiene was tested up to the dose level of 5000 ¿g/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Pre-incubation assay (third experiment): Due to the cytotoxicity in the second experiment, only three analyzable dose levels were left for the determination of the mutagenicity of the test item in the tester strain TA1535 in the presence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix. Therefore a third experiment was performed with concentrations of 1.7, 5.4 and 17 ¿g/plate to provide additional information.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, without metabolic activation. 1-aminoanthracene, with metabolic activation.
Remarks:
Solvents for positive controls: DMSO; saline for sodium azide.
Details on test system and experimental conditions:
Dose-range finding test. Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ¿g/plate were tested in triplicate. The highest concentration of 1,5-hexadiene used in the subsequent mutation assays was 5000 ¿g/plate or the level at which the test item inhibited bacterial growth. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
First experiment: direct plate assay. The dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 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 (10exp9 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. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Second and third experiment: pre-incubation assay. The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°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 (10exp9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO. 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. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Colony counting. 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.
Rationale for test conditions:
Test conditions were as specified by the OECD guideline.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following 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 Charles River Den Bosch.
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.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.
Statistics:
Not required (mean values compared numerically).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed in the absence and presence of S9-mix at the highest dose level tested.
Vehicle controls validity:
valid
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:
Cytotoxicity observed in the absence and presence of S9-mix at the highest dose level tested.
Vehicle controls validity:
valid
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:
Cytotoxicity observed in the absence and presence of S9-mix at the highest dose level tested.
Vehicle controls validity:
valid
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:
Cytotoxicity observed at dose levels of 1600 and 5000 µg/plate in the absence and presence of S9-mix
Vehicle controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
Direct plate assay. 1,5-hexadiene was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ¿g/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and
5000 ¿g/plate. Precipitation of 1,5-hexadiene on the plates was observed at the start of the incubation period at concentrations of 512 and 1600 ¿g/plate and upwards in the dose range finding and first experiment, respectively and at 5000 ¿g/plate at the end of the incubation period. To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains except for tester strain WP2uvrA where no toxicity was observed at any of the dose levels tested.
Pre-incubation assay. To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, 1,5-hexadiene was tested up to the dose level of 5000 ¿g/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation of 1,5-hexadiene on the plates was not observed at the start of the incubation period. At the end of the incubation period, the test item precipitated on the plates at the top dose of 5000 ¿g/plate in tester strain TA100 in the absence and presence of S9-mix and in tester strains TA1535 and WP2uvrA in the presence of S9-mix. In tester strain TA1535 in the absence of S9-mix precipitation was observed at the dose levels of 1600 ¿g/plate and upwards. In all other tester strains no precipitation was observed. 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 in the absence and presence of S9-mix. Due to the cytotoxicity in the second experiment, only three analyzable dose levels were left for the determination of the mutagenicity of the test item in the tester strain TA1535 in the presence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix. Therefore a third experiment was performed with concentrations of 1.7, 5.4 and 17 ¿g/plate to provide additional information. Precipitation of 1,5-hexadiene on the plates was not observed at the start and the end of the incubation period. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Remarks on result:
other:

Dose-range finding test; direct plate assay. Mean = mean number of revertant colonies:

 

Without S9-mix

TA100

WP2uvrA

Dose (µg/plate)

Mean

SD

Mean

SD

Positive control

829

97

1325

78

Solvent control

88

4

29

8

1.7

89

2

26

4

5.4

97

9

17

6

17

78

14

27

8

52

83

6

27

8

164

78

8

20

1

512

84

6

18

4

1600

68

18

14

6

5000

Reduced bacterial background

16

3

 

WithS9-mix

TA100

WP2uvrA

Dose (µg/plate)

Mean

SD

Mean

SD

Positive control

1269

153

584

25

Solvent control

87

13

33

13

1.7

95

3

27

1

5.4

92

5

34

12

17

101

9

28

2

52

99

13

30

1

164

87

13

23

7

512

88

14

25

5

1600

58

6

21

8

5000

Reduced bacterial background

26

10

 

Experiment 1: mutagenic response in the Salmonella typhimurium reverse mutation assay. Direct plate assay. Mean = Mean number of revertant colonies.

 

Without S9-mix

TA1535

TA1537

TA98

Dose (µg/plate)

Mean

SD

Mean

SD

Mean

SD

Pos. ctrol.

1056

38

1183

126

1103

95

Solvent

11

4

6

4

12

4

17

14

2

5

1

15

8

52

9

3

8

5

11

6

164

12

1

7

2

15

5

512

12

5

6

5

11

4

1600

11

3

4

1

12

2

5000*

11

5

4

2

9

1

 

WithS9-mix

TA1535

TA1537

TA98

 

Dose (µg/plate)

Mean

SD

Mean

SD

Mean

SD

Pos. ctrol.

311

18

370

46

978

115

Solvent

11

1

4

2

22

2

17

11

1

7

2

19

6

52

13

4

5

1

15

5

164

15

4

4

1

19

4

512

11

4

8

5

15

4

1600

13

7

6

4

12

2

5000*

7

2

6

3

18

4

Slight precipitate and bacterial background lawn slightly reduced at 5000 µg/plate.

 

Experiment 2: mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Pre-incubation assay. Mean = Mean number of revertant colonies.

 

Without S9-mix

TA1535

TA1537

TA98

TA100

WP2uvrA

Dose (µg/plate)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Pos. ctrol.

1205

59

196

19

1605

69

818

24

157

39

Solvent

11

4

8

6

11

1

101

21

17

4

17

6

2

8

0

13

5

135

16

20

4

52

14

11

5

2

11

1

111

7

20

8

164

5

3

3

2

12

6

110

14

17

8

512

10*

4

*

-

10

3

108

14

16

6

1600

*

-

*

-

*

-

83

-

17

2

5000

*

-

*

-

*

-

*

-

20

4

 

WithS9-mix

TA1535

TA1537

TA98

TA100

WP2uvrA

Dose (µg/plate)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Pos. ctrol.

283

28

276

20

833

83

1332

438

680

34

Solvent

25

22

7

4

27

6

99

7

32

7

17

18

22

4

1

21

7

105

3

27

6

52

12

1

7

3

18

0

89

2

22

8

164

17

7

6

2

18

2

92

12

25

3

512

*

-

*

-

14

5

82

7

19

4

1600

*

-

*

-

*

-

*

-

19

6

5000

*

-

*

-

*

-

*

-

17

2

*Bacterial background extremely reduced.

Experiment 3: mutagenic response in the Salmonella typhimurium reverse mutation assay. Pre-incubation assay. Mean: mean number of revertant colonies.

 

Without S9-mix

TA1535

TA1537

Dose (µg/plate)

Mean

SD

Mean

SD

Positive control

-

-

151

23

Solvent control

-

-

10

4

1.7

-

-

10

2

5.4

-

-

9

1

17

-

-

7

2

 

WithS9-mix

TA100

WP2uvrA

Dose (µg/plate)

Mean

SD

Mean

SD

Positive control

247

3

221

6

Solvent control

13

3

4

1

1.7

8

4

8

4

5.4

9

3

6

3

17

12

4

7

1
Conclusions:
Based on the results of this study it is concluded that 1,5-hexadiene is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of 1,5-hexadiene 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 and third a pre-incubation assay. An additional pre-incubation assay was performed in the absence and presence of S9-mix.

The study procedures described in this report were based on OECD 471 guideline.

Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colourless liquid with a purity of 99.4%. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 ¿g/plate in the strains TA100 and WP2uvrA in the direct plate assay. 1,5-hexadiene precipitated on the plates at the top dose level of 5000 ¿g/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 1600 and 5000 ¿g/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 5000 ¿g/plate in the strains TA1535, TA1537 and TA98. 1,5-hexadiene precipitated on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix at the highest dose level tested.

In the second mutation experiment, the test item was tested up to concentrations of 5000 ¿g/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at the top dose of 5000 ¿g/plate in tester strain TA100 in the absence and presence of S9-mix and in tester strains TA1535 and WP2uvrA in the presence of S9-mix. In tester strain TA1535 in the absence of S9-mix precipitation was observed at the dose levels of 1600 ¿g/plate and upwards. In all other tester strains no precipitation was observed. 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 in the absence and presence of S9-mix.

Due to severe cytotoxicity in tester strain TA1535 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix in the pre-incubation assay, an additional experiment was performed. In the additional experiment the test item was tested up to concentrations of 17 ¿g/plate in tester strain TA1535 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix in the pre-incubation 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.

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 1,5-hexadiene 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)

Additional information

The objective of this study was to determine the potential of 1,5-hexadiene 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 and third a pre-incubation assay. An additional pre-incubation assay was performed in the absence and presence of S9-mix.

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

Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colourless liquid with a purity of 99.4%. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 ¿g/plate in the strains TA100 and WP2uvrA in the direct plate assay. 1,5-hexadiene precipitated on the plates at the top dose level of 5000 ¿g/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 1600 and 5000 ¿g/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 5000 ¿g/plate in the strains TA1535, TA1537 and TA98. 1,5-hexadiene precipitated on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix at the highest dose level tested.

In the second mutation experiment, the test item was tested up to concentrations of 5000 ¿g/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at the top dose of 5000 ¿g/plate in tester strain TA100 in the absence and presence of S9-mix and in tester strains TA1535 and WP2uvrA in the presence of S9-mix. In tester strain TA1535 in the absence of S9-mix precipitation was observed at the dose levels of 1600 ¿g/plate and upwards. In all other tester strains no precipitation was observed. 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 in the absence and presence of S9-mix.

Due to severe cytotoxicity in tester strain TA1535 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix in the pre-incubation assay, an additional experiment was performed. In the additional experiment the test item was tested up to concentrations of 17 ¿g/plate in tester strain TA1535 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix in the pre-incubation 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.

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 1,5-hexadiene is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Classification for germ cell mutation (according to CLP) is not required for hexa-1,5-diene based on the negative results reported in the Ames test.