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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Oct 2017 until the 22 Nov 2017.
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dilithium tetraborate
EC Number:
234-514-3
EC Name:
Dilithium tetraborate
Cas Number:
12007-60-2
Molecular formula:
B4Li2O7
IUPAC Name:
dilithium(1+) bicyclo[3.3.1]tetraboroxane-3,7-bis(olate)
Test material form:
solid: bulk

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S-9 MIX
Test concentrations with justification for top dose:
In the dose range finding test eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate using TA100 and the WP2uvrA, both with and without S9-mix.
The highest concentration of Dilithium tetraborate used in the subsequent mutation assay was 5000 µg/plate. 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.

Vehicle / solvent:
Milli-Q water
Controls
Untreated negative controls:
yes
Remarks:
Milli-Q water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:

METHOD OF APPLICATION: Agar plates

DURATION
- Preincubation period: 30 ± 2 minutes
- Exposure duration: 48 ± 4 h
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn was observed in tester strains TA1535 and TA1537 (absence of S9-mix). No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed in the other tester strains.

METABOLIC ACTIVATION SYSTEM
Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Evaluation criteria:
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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The bacterial lawn was slightly reduced
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The bacterial lawn was slightly reduced
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. Dilithium tetraborate 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. In all tester strains, no increase in the number of revertants was observed.Precipitation of Dilithium tetraborate on the plates was not observed at the start or at the end of the incubation period in any tester strain.

In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Dilithium tetraborate did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn was observed in tester strains TA1535 and TA1537 (absence of S9-mix).

In tester strain TA100, the test item induced up to 2.3-fold increases in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In all other tester strains, no increase in the number of revertants was observed. The results in TA100 are therefore considered equivocal.


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 WP2uvrA, absence of S9-mix in the second experiment. The results in TA100 are therefore considered equivocal.
Remarks on result:
other:
Remarks:
Note: The number of revertant colonies was within the historical control data for all concentrations

Any other information on results incl. tables

Table1          
Experiment 1: Mutagenic Response of Dilithium tetraborate in theSalmonella typhimuriumReverse Mutation Assay



Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains ofSalmonella typhimurium.

 


TA1535


TA1537

 


TA98

 

 Without S9-mix

 

Positive control

838

±

27

 

1028

±

50

 

1001

±

37

 

Solvent control

8

±

3

 

3

±

3

 

11

±

3

 

188

11

±

3

 

6

±

2

 

16

±

6

 

375

11

±

4

 

4

±

4

 

11

±

4

 

750

11

±

3

 

6

±

2

 

13

±

7

 

1500

7

±

3

 

4

±

1

 

14

±

5

 

3000

5

±

5

 

3

±

2

n

12

±

5

 

5000

6

±

5

NP n

0

±

0

NP a

17

±

4

NP n

 

 

 

 

 

 

 

 

 

 

 

 

 

 With S9-mix1

 

Positive control

277

±

21

 

421

±

22

 

1043

±

21

 

Solvent control

8

±

2

 

4

±

1

 

16

±

5

 

188

9

±

4

 

6

±

2

 

15

±

1

 

375

11

±

2

 

4

±

4

 

17

±

2

 

750

15

±

5

 

4

±

1

 

14

±

5

 

1500

14

±

2

 

5

±

3

 

18

±

6

 

3000

10

±

3

 

3

±

0

n

16

±

7

 

5000

8

±

2

NP n

0

±

0

NP a

18

±

2

NP n

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

Plate incorporation assay (5% S9)

NP

No precipitate

a

Bacterial background lawn absent

n

Normal bacterial background lawn

 

Table2         
Experiment 1A: Mutagenic Response of Dilithium tetraborate in theSalmonella typhimuriumReverse Mutation Assay

 


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (±S.D.) with
Salmonella typhimuriumTA1537.

 


TA1537



 



 

 Without S9-mix

 

Positive control

566

±

35

 

 

 

 

 

 

 

 

 

Solvent control

5

±

4

 

 

 

 

 

 

 

 

 

188

6

±

2

 

 

 

 

 

 

 

 

 

375

5

±

3

 

 

 

 

 

 

 

 

 

750

6

±

3

 

 

 

 

 

 

 

 

 

1500

0

±

0

a

 

 

 

 

 

 

 

 

3000

6

±

3

 

 

 

 

 

 

 

 

 

5000

4

±

4

n NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 With S9-mix1

 

Positive control

378

±

21

 

 

 

 

 

 

 

 

 

Milli-Q water

6

±

2

 

 

 

 

 

 

 

 

 

188

4

±

5

 

 

 

 

 

 

 

 

 

375

8

±

4

 

 

 

 

 

 

 

 

 

750

9

±

6

 

 

 

 

 

 

 

 

 

1500

0

±

0

a

 

 

 

 

 

 

 

 

3000

4

±

3

 

 

 

 

 

 

 

 

 

5000

3

±

3

n NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

Plate incorporation assay (5% S9)

NP

No precipitate

a

Bacterial background lawn absent

n

Normal bacterial background lawn

 

Table 3        
Experiment 2: Mutagenic Response of Dilithium tetraborate in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay




Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains ofSalmonella typhimuriumand oneEscherichia colistrain.

 


TA1535


TA1537

 


TA98


TA100


WP2uvrA

 Without S9-mix

 

Positive control

1019

±

61

 

190

±

55

 

1356

±

31

 

602

±

111

 

75

±

18

 

Solvent control

9

±

1

 

4

±

4

13

±

7

 

103

±

14

 

22

±

10

 

188

12

±

2

 

4

±

1

 

10

±

1

 

104

±

24

 

26

±

11

 

375

8

±

4

 

5

±

2

 

15

±

4

 

112

±

12

 

22

±

6

 

750

9

±

6

 

6

±

5

 

14

±

3

 

111

±

25

 

29

±

7

 

1500

12

±

4

 

4

±

1

 

14

±

5

 

117

±

20

 

38

±

6

 

3000

5

±

2

n

4

±

1

n

10

±

5

 

165

±

8

 

23

±

4

 

5000

7

±

2

s NP

2

±

1

s NP

12

±

4

n NP

178

±

22

n NP

22

±

8

n NP

 

 With S9-mix1

 

Positive control

171

±

16

 

108

±

22

 

651

±

67

 

1359

±

383

 

597

±

21

 

Solvent control

12

±

9

 

5

±

1

 

15

±

3

 

69

±

13

 

29

±

4

 

188

12

±

10

 

3

±

2

 

24

±

8

 

69

±

2

 

30

±

1

 

375

8

±

2

 

5

±

2

 

14

±

2

 

83

±

11

 

31

±

10

 

750

7

±

0

 

4

±

1

 

17

±

2

 

99

±

3

 

32

±

4

 

1500

10

±

4

 

5

±

2

 

9

±

1

 

108

±

10

 

32

±

4

 

3000

5

±

2

 

3

±

1

 

16

±

3

 

142

±

18

 

42

±

10

 

5000

7

±

4

n NP

3

±

2

n NP

12

±

2

n NP

161

±

21

n NP

36

±

7

n NP

 

 

1

Pre-incubation assay (5% S9)

NP

No precipitate

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

 

 


Applicant's summary and conclusion

Conclusions:
In an OECD TG 471 study dilithium tetraborate induced dose related increases in tester strain TA100 in the absence and presence of S9-mix in two independent experiments (2.0 and 2.3-fold), respectively. The number of revertant colonies was within the historical control data range for all concentrations, with the exception of the highest concentration in the presence of S9-mix which was just above the historical control data range.

The test result was equivocal in tester strain TA100 of the Salmonella typhimurium reverse mutation assay. However, the test item was not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA98) or the Escherichia coli reverse mutation assay using strain WP2uvrA.      
Executive summary:

This OECD TG 471 in vitro study was used determine the potential of Dilithium tetraborate and/or its metabolites upto concentrations of 5000 µg/plate 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 ofEscherichiacoli(E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test result was equivocal in tester strain TA100 of the Salmonella typhimurium reverse mutation assay. However, the test item was not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA98) or the Escherichia coli reverse mutation assay using strain WP2uvrA.