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Administrative data

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:
15 October 2015 to 05 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dichlorobenzoyl chloride
EC Number:
220-813-6
EC Name:
3,5-dichlorobenzoyl chloride
Cas Number:
2905-62-6
Molecular formula:
C7H3Cl3O
IUPAC Name:
3,5-dichlorobenzoyl chloride
Test material form:
other: solid (unspecified)
Details on test material:
- Appearance: White solid
- Storage conditions of test material: At room temperature

Method

Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Enriched nutrient broth, Oxoid LTD
- Properly maintained: Yes. The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- Storage conditions: Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Enriched nutrient broth, Oxoid LTD
- Properly maintained: Yes. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
- Storage conditions: Stock cultures were stored in liquid nitrogen (-196 °C).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST
- With and without S9-mix (direct plate assay): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (TA100 and WP2uvrA)
- With and without S9-mix (pre-incubation assay): 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate (TA100 and WP2uvrA)

FIRST EXPERIMENT (direct plate assay)
- With and without S9-mix: 17, 52, 164, 512 and 1600 μg/plate (TA1535, TA1537 and TA98)

SECOND EXPERIMENT (pre-incubation assay)
- Without S9-mix: 5.4, 17, 52, 164, 512 and 890 μg/plate (TA1535, TA1537 and TA98)
- With S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate (TA1535, TA1537 and TA98)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was soluble in acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation and pre-incubation

DOSE RANGE FINDING TEST
Selection of an adequate range of doses for the direct plate assay 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 μg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent direct plate assay was 1600 μg/plate.
Selection of an adequate range of doses for the pre-incubation assay was based on a dose range finding test with the strains TA100 and WP2uvrA. Seven concentrations, 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent pre-incubation assay was 890 μg/plate in the absence of S9-mix and 512 μg/plate in the presence of S9-mix.
At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain in the absence and presence of S9-mix.
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 above mentioned dose range finding study with two tester strains was reported as a part of the direct plate assay. In the second part of this experiment, the test material 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 (10⁹ cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test material in acetone 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 EXPERIMENT: PRE-INCUBATION ASSAY
The dose range finding study with two tester strains is reported as a part of the pre-incubation assay. In the second part of this experiment, the test material 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 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 (10⁹ cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test material in acetone. 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 were counted.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with test material precipitate sufficient to interfere with automated colony counting were counted manually. Evidence of test material 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:
ACCEPTABILITY OF THE ASSAY
The 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.
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 should be repeated.

DATA EVALUATION
In addition to the criteria stated below, 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.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is not greater than 2 times the concurrent control, and the total number of revertants in the tester strains TA1535, TA1537 or TA98 is not greater than 3 times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is greater than 2 times the concurrent control, or the total number of revertants in the tester strains TA1535, TA1537 or TA98 is greater than 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:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
FIRST EXPERIMENT: DIRECT PLATE ASSAY
- Precipitate
Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 512 μg/plate and above at the end of the incubation period. In the dose range finding, in the presence of S9-mix, no precipitation of the test material on the plates was observed at the start of the incubation period.

- Toxicity
To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the micro-colonies and the reduction of the revertant colonies were determined. Toxicity was observed in all tester strains.

- Mutagenicity
In the direct plate test, no increase in the number of revertants was observed upon treatment with the test material under all tested conditions.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- Precipitate
In the dose range finding test precipitation of the test material on the plates was observed at the start and at the end of the incubation period at the concentration of 1600 μg/plate.
In the second mutation experiment precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.

- Toxicity
To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the micro-colonies and the reduction of the revertant colonies were determined. Toxicity was observed in all tester strains except TA98 in the presence of S9 mix.

- Mutagenicity
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative control values were within the laboratory historical control data ranges.
The 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, except the response for the tester stain TA1537 in the second experiment in the absence of S9-mix. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Dose range finding test - Direct plate assay (mean number of revertant colonies/3 replicate plates)

Without S9-mix

Dose (μg/plate)

TA100

WP2uvrA

Positive control*

601

1530

Solvent control

89

37

1.7

81

23

5.4

83

29

17

87

34

52

96

28

164

74

26

512

70 s

22

1600

49 m

23

5000

28 e

13

With S9-mix

Positive control**

1269

174

Solvent control

86

39

1.7

93

37

5.4

100

34

17

87

34

52

103

28

164

87

34

512

67 s

27 s

1600

64 m

55 m

5000

25 e

26 e

 *TA100: Methylmethanesulfonate (MMS) 650 µg/plate; WP2uvrA: 4-Nitroquinoline N-oxide (4 -NQO) 10 µg/plate

**TA 100: 2 -Aminoanthracene (2AA) 1 µg/plate; WP2uvrA: 2AA 15 µg/plate

s: Bacterial background lawn slightly reduced

m: Bacterial background lawn moderately reduced

e: Bacterial background lawn extremely reduced

Table 2: Experiment 1 - Direct plate assay (mean number of revertant colonies/3 replicate plates)

Without S9-mix

  Dose (μg/plate)

TA1535

TA1537

TA98

Positive control*

948

636

862

Solvent control

15

8

15

17

23

6

15

52

14

6

19

164

12

7

17

512

9 s

7 s

13 s

1600

8 m

5 m

12 m

With S9-mix

Positive control**

259

439

1294

Solvent control

16

10

33

17

12

7

39

52

12

13

43

164

13

6

38

512

14 s

8 s

28 s

1600

4 m

8 m

20 m

 *TA1535: Sodium azide (SA) 5 µg/plate; TA1537: ICR-191 2.5 µg/plate; TA98: 2-Nitrofluorene (NF) 10 µg/plate

**TA 1535 and TA1537: 2AA 2.5 µg/plate; TA98: 2AA 1 µg/plate

s: Bacterial background lawn slightly reduced

m: Bacterial background lawn moderately reduced

 

Table 3: Dose range finding - Pre-incubation assay (mean number of revertant colonies/3 replicate plates)

Without S9-mix

Dose (µg/plate)

TA100

WP2uvrA

Positive control*

555

343

Solvent control

75

27

1.7

62

20

5.4

62

20

17

75

21

52

47

22

164

103

22

512

39 m

23

1600

0 a

19 m

With S9-mix

Positive control**

1536

547

Solvent control

95

16

1.7

78

18

5.4

102

26

17

90

18

52

92

21

164

35 m

15

512

e

29

1600

e

10 m

*TA100: MMS 650 µg/plate; WP2uvrA: 4 -NQO 10 µg/plate

**TA 100: 2AA 5 µg/plate; WP2uvrA: 2AA 15 µg/plate

m: Bacterial background lawn moderately reduced

e: Bacterial background lawn extremely reduced

a: Bacterial background lawn absent

 

Table 4: Experiment 2 - Pre-incubation assay (mean number of revertant colonies/3 replicate plates)

Without S9-mix

Dose (μg/plate)

TA1535

TA1537

TA98

Positive control*

796

45

1014

Solvent control

7

7

11

5.4

3

10

8

17

4

8 s

9

52

2 m

4 e (mc)

13 s

164

1 m

e (mc)

e (mc)

512

0 a

1 a

0 a

890

0 a

0 a

0 a (mc)

With S9-mix

Positive control**

140

194

702

Solvent control

5

8

21

1.7

4

6

24

5.4

7

8

18

17

5

16

17

52

9

7

17

164

8

11

16

512

10 s

9 m

15

*TA1535: Sodium azide (SA) 5 µg/plate; TA1537: NF 15 µg/plate; TA98: NF 10 µg/plate

**TA 1535 and TA1537: 2AA 2.5 µg/plate; TA98: 2AA 1 µg/plate

s: Bacterial background lawn slightly reduced

m: Bacterial background lawn moderately reduced

e: Bacterial background lawn extremely reduced

a: Bacterial background lawn absent

mc: Microcolonies

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: negative with and without metabolic activation

Under the conditions of the study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test material in two independent experiments; a direct plate assay was performed followed by a pre-incubation assay, both in the absence and presence of metabolic activation (S9-mix).

In a dose range finding study, the test material was initially tested up to concentrations of 5000 μg/plate in acetone in the strains TA100 and WP2uvrA in the direct plate assay. The test material precipitated on the plates at dose levels of 512 μg/plate and above. Toxicity was observed in both tester strains.

In the first mutation experiment, the test material was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98. The test material precipitated on the plates at dose levels of 512 μg/plate and above. Toxicity was observed in all tester strains.

In the second mutation experiment, the test material was initially tested up to concentrations of 1600 μg/plate in the strains TA100 and WP2uvrA in the pre-incubation assay. The test material precipitated on the plates at the dose level of 1600 μg/plate. Toxicity was observed in both tester strains.

In the main experiment, the test material was tested in the strains TA1535, TA1537 and TA98 up to concentrations of 890 and 512 μg/plate in the absence and presence of S9-mix, respectively. The test material did not precipitate on the plates at the top dose of 890 μg/plate. Toxicity was observed in all tester strains, except for the tester strain TA98 in the presence of S9-mix.

The test material did not induce a significant dose-related increase in the number of revertant colonies in any of the strains both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, acceptable responses were obtained for the vehicle and positive controls, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of the study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

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