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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
03 - 09 Nov 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study, tested with the source substance CAS 4193-55-9. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(no detrimental impact on the outcome of the study).
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
(no detrimental impact on the outcome of the study).
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
TA 1537, genotype his C 3076; rfa-; uvrB; frame shift mutations.
TA 98, genotype his D 3052; rfa-; uvrB; R-factor; frame shift mutations.
TA 1535, genotype his G 46; rfa-; uvrB; base-pair mutations.
TA 100, genotype his G 46; rfa-; uvrB; R-factor; base-pair mutations.
WP2, genotype trp; trp; uvrA; base-pair substitutions and others
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle:
On the day of the experiment, the test article was dissolved in deionised water.
The solvent was chosen because of its solubility properties and its relative non- toxicity to the bacteria.
No precipitation of the test article occurred up to the highest investigated dose.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene and methyl methane sulfonate
Remarks:
-S9: NaN3 for TA 1535, TA 100; 4-NOPD for TA 1537, TA 98; MMS for WP2 uvrA. +S9: 2-M for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA.
Details on test system and conditions:
PRECULTURE
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100.
This nutrient medium contains per litre:
8 9 Merck Nutrient Broth (MERCK, 0-64293 Darmstadt)
5 9 NaCI (MERCK, 0-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

Selective Agar: the plates with the selective agar were obtained from E. Merck, 0-64293 Darmstadt.

Overlay Agar: the overlay agar contains per litre:
for Salmonella strains:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
For Escherichia coli:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
2.5 mg Tryptophan*
* (MERCK, 0-64293 Darmstadt)
Sterilisations were performed at 121°C in an autoclave.

S9 PREPARATION
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to over- come this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm which received daily applications of 80 mg/kg b.w. Phenobarbital ip. dissolved in aqua deionised and β-Naphthotlavone orally dissolved in corn oil on three subsequent days. The livers were prepared 24 hours after the last treatment.
After decapitation of the anaesthetised animals the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at - 20 °C for up to one week before use. The protein content was determined using an analysis kit of BioRad Laboratories, D-80939 Munchen.
The protein concentration in the S9 preparation was 23.7 mg/ml.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT TOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

DOSE SELECTION
Based upon the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. Two independent experiments were performed.
According to the dose selection criteria the test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate .

TEST PERFORMANCE
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
Evaluation criteria:
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Summary of results

without S9 mix

Conc. µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

I

II

I

II

I

II

I

II

I

II

Negative control

16

12

20

9

22

15

81

110

40

34

Solvent control

14

13

16

10

19

16

99

131

47

38

Positive control*

1023

951

108

138

283

169

286

453

683

356

33

9

11

18

11

25

20

91

117

56

46

100

17

12

15

10

21

14

55

105

38

28

333

12

13

17

12

20

16

87

112

42

37

1000

14

18

14

11

19

14

46

127

40

35

2500

12

16

17

6

19

20

59

124

53

32

5000

18

18

20

10

19

15

94

127

48

31

with S9 mix

Conc. µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

I

II

I

II

I

II

I

II

I

II

Negative control

17

17

17

6

34

27

78

125

70

40

Solvent control

21

15

27

8

28

26

84

127

54

42

Positive control**

120

89

127

93

204

60

416

401

167

112

33

21

20

23

14

25

18

68

119

52

33

100

24

18

16

8

27

24

69

115

44

36

333

17

20

25

12

28

25

77

129

52

44

1000

19

15

22

13

20

20

78

115

50

38

2500

20

17

19

11

38

28

77

115

59

55

5000

19

19

17

9

40

26

81

125

56

49

*Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100

4 -nito-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate)

Methyl methane snlfonate (5 µl/plate) strain WP2 uvrA

**2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98 and TA 100

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

Method

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate

Results

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifcs in the genome of the strains used. Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.