Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-07 to 2017-12-08
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
S. typhimurium: his operon
E. choli: trp operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:water
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 1) 4-nitro-o-phenylene-diamine 2) 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) for experiment 1; preincubation for experiment 2
- Cell density at seeding (if applicable): approx. 1E09 cells/mL

DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: at least 48h at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Rationale for test conditions:
According to OECD test guideline
Evaluation criteria:
Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Evaluation of Mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
experiment 1 & 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
experiment 1& 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
experiment 1& 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
experiment 1 & 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
experiment 1& 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a preexperiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The test item was tested in the pre-experiment at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Toxicity was detected at 316 µg/plate (TA 98 without met. act.) and 1000 µg/plate (TA 100 without met. act.) and at 5000 µg/plate for both strains with met. act..

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See table 1 under "any other information on results"
- Negative (solvent/vehicle) historical control data: See table 2 under "any other information on results"

Any other information on results incl. tables

Table 1:

Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) without S9 (-S9)

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Substance Conc./plate

4-NOPD 10 µg

NaN310 µg

NaN310 µg

4-NOPD 40 µg

MMS

1 µL

Mean

430.7

612.1

792.0

94.5

524.2

SD

155.5

220.0

299.5

22.7

113.5

Min

141

132

38

35

208

Max

1830

1423

1854

273

918

RSD [%]

36.1

35.9

37.8

24.0

21.7

n

971

1188

931

929

229

Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) with S9 (+S9)

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Substance Conc./plate

2-AA 2.5 µg

2-AA 2.5 µg

2-AA 2.5 µg

2-AA 2.5 µg

2-AA

10 µg

Mean

1880.5

1727.7

133.9

234.1

227.0

SD

708.5

522.0

134.9

101.4

83.7

Min

70

169

22

26

84

Max

3606

3132

1954

682

451

RSD [%]

37.7

30.2

100.8

43.3

36.9

n

966

1184

927

925

230

Table 2:

Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9)

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Mean

24.2

90.7

13.8

8.2

44.4

SD

6.7

15.6

6.7

2.9

6.9

Min

11

49

4

3

32

Max

58

155

41

35

66

RSD [%]

27.7

17.2

48.6

35.3

15.5

n

972

1191

929

931

231

Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9)

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Mean

29.0

96.4

10.5

8.3

48.3

SD

6.8

14.1

4.5

3.1

8.8

Min

15

62

3.0

3

30

Max

59

160

38

36

78

RSD [%]

23.4

14.6

42.7

37.4

18.3

n

967

1189

925

926

231

S9:        metabolic activation 

Mean: mean of revertants/plate

Min.:     minimum of revertants/plate

Max.:    maximum of revertants/plate

SD:       Standard Deviation RSD: Relative Standard Deviation

n:       Number of control values

Table 3: Number of revertants per plate (mean of 3 plates) Experiment 1

 

[TA98]

[TA100]

[TA1535]

[TA1537]

[WP2uvrA]

Conc.
[unit]

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

 23

 40

 no

 73

 111

 no

 20

 22

 no

32

28

 no

48

62

 no

3.16

 33

 34

 no

 91

 112

 no

 23

 21

 no

33

27

 no

50

54

 no

10.0

 31

 37

 no

 90

 106

 no

 20

 21

 no

27

25

 no

52

49

 no

31.6

 28

 41

 no

 78

109

 no

 22

 17

 no

30

31

 no

54

51

 no

100.0

 33

 30

 no

 76

 94

 no

 21

 23

 no

25

26

 no

59

46

 no

316

 37

 30

 yes

 46

 114

 no

 21

 23

 no

30

28

 no

64

59

 no

1000.0

4

24

yes

2

 104

yes

3

16

yes

23

27

no

48

35

no

2500

2

50

yes

0

151

yes

0

18

yes

10

8

yes

52

29

yes

5000

0

28

yes

0

133

yes

0

1

yes

0

0

yes

15

9

yes

Positive control

 1132

 2958

 

 604

 2223

 

 930

 317

 

167

231

 

596

171

 

*solvent control with .water

Table 4: Number of revertants per plate (mean of ... plates) Experiment 2

 

[TA98]

[TA100]

[TA1535]

[TA1537]

[WP2uvrA]

Conc.
[unit]

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

 24

26 

 no

 99

82 

 no

 13

13 

 no

21

29

 no

60

60

 no

3.16

 22

 29

 no

 113

 89

 no

 14

 12

 no

29

31

 no

53

55

 no

10.0

 28

 30

 no

 112

 81

 no

 10

 16

 no

29

28

 no

64

63

 no

31.6

 22

 25

 no

 102

 100

 no

 11

 13

 no

29

30

 no

58

65

 no

100.0

 28

 23

 no

 109

 75

 no

 11

 15

 no

26

26

 no

58

67

 no

316

 20

 31

 no

 104

 99

 no

 10

 13

 no

29

23

 no

60

64

 no

1000.0

11

27

yes

63

81

no

5

6

yes

28

26

no

56

55

no

2500

3

32

yes

17

133

yes

1

8

yes

26

28

no

63

51

no

5000

0

6

yes

0

42

yes

0

4

yes

0

0

yes

5

22

yes

Positive control

 347

 978

 

 474

 757

 

 934

 188

 

207

110

 

653

150

 

*solvent control with .water

 

Applicant's summary and conclusion

Conclusions:
The present study was conducted according to OECD guideline 471 (1997). Four Salmonella thyphimurium strains and one Escherichia coli strain were incubated with Tetrahydrofolic acid at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate for at least 48h at 37°C. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Tetrahydrofolic acid did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Tetrahydrofolic acid is considered to be non-mutagenic in this bacterial reverse mutation assay.