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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An OECD TG 473 in vitro test has been conducted to determine the possible clastogenicity of dilithium tetraborate using cultured human lymphocytes.  Both in the absence and presence of S9-mix Dilithium tetraborate did not induce any statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

 

In an OECD TG 471 in vitro study dilithium tetraborate induced dose related increases in tester strain TA100 (Salmonella typhimurium) in the absence and presence of S9-mix in two independent experiments (2.0 and 2.3-fold), respectively. However, 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 therefore equivocal in the Salmonella typhimurium tester strain TA100 under the conditions of the study.

Dilithium tetraborate was not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA98) or the Escherichia coli reverse mutation assay using strain WP2uvrA.      

Link to relevant study records

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

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

 

 


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.       

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 September 2017 to 31 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:

Dose-range finding study: age 29, AGT = 14.2 h
First cytogenetic assay: age 25, AGT = 13.8 h
Second cytogenetic assay: age 35, AGT = 15.8 h
Cytogenetic assay 2A: age 22, AGT = 13.2 h

Whole blood samples were used to prepare the lymphocyte cultures, treated with heparin and added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was also added.

All cultures were then incubated at 37°C± 1.0°C (actual range 34.7 - 37.1°C) in the dark in a humid atmosphere of 80 - 100% (actual range 33 - 89%), containing 5.0 ± 0.5% CO2 in air.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was prepared from male Sprague-Dawley derived rats, dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
In order to select the appropriate dose levels for the chromosome aberration test, cytotoxicity data were obtained in a dose-range finding test. Dilithium tetraborate was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. Blood cultures were treated with 62.5, 125, 250, 500, 1000 and 1688 µg Dilithium tetraborate/mL culture medium with and without S9-mix. At a concentration of 1688 µg/mL Dilithium tetraborate precipitated in the culture medium.

Based on the results of the dose-range finding test, an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 0.01 M (3 h exposure time) or had an inhibition of the mitotic index of 50% or greater. The dose levels selected for the first cytogenetic assay, without and with S9-mix, were 500, 1000 and 1691 µg/mL culture medium (3 h exposure time, 24 h fixation time).

To obtain more information about the possible clastogenicity of dilithium tetraborate, a second cytogenetic assay was also performed in which human lymphocytes were continuously exposed to dilithium tetraborate in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay; 100, 250, 500, 600, 700, 800 and 900 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time) without S9-mix.
Vehicle / solvent:
The vehicle for the test item was Milli-Q water.
Untreated negative controls:
yes
Remarks:
Milli-Q water in culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: The test item was applied in Milli-Q water to the lymphocyte cultures.

DURATION
- Preincubation period: Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h.

- Exposure duration: In the dose range finding test, the cultures were exposed to selected doses of Dilithium tetraborate for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.

In the first cytogenetic assay, lymphocytes were exposed to selected doses of Dilithium tetraborate for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.

In the second cytogenetic assay, lymphocytes were exposed in duplicate to selected doses of Dilithium tetraborate for 24 h and 48 h in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.


SPINDLE INHIBITOR (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic0.56% (w/v) potassium chloride (Merck) solution at 37°C.
After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. Slides were allowed to dry and thereafter stained for
10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8.

NUMBER OF REPLICATIONS: At least two slides were prepared per culture.

DETERMINATION OF CYTOTOXICITY
- Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay
The mitotic index of each culture was determined by counting the number of metaphases from at least 987 cells (1000 cells with a maximum deviation of 5%). Chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%.

- Analysis of Slides for Chromosome Aberrations
One hundred and thirteen metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test,
one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING STUDY: At a concentration of 1688 µg/mL Dilithium tetraborate precipitated in the culture medium. In the dose-range finding study, blood cultures were treated with 62.5, 125, 250, 500, 1000 and 1688 µg Dilithium tetraborate/mL culture medium with and without S9-mix. The mitotic index of cultures treated with various dilithium tetraborate concentrations or with the negative control item were determined.


FIRST CYTOGENETIC ASSAY
The mitotic index of cultures treated with various dilithium tetraborate concentrations or with the positive or negative control items was determined. All dose levels were selected for scoring of chromosome aberrations.

Both in the absence and presence of S9-mix, dilithium tetraborate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix, Dilithium tetraborate did not increase the number of polyploid cells and cells with endoreduplicated chromosomes..

SECOND CYTOGENETIC ASSAY
To obtain more information about the possible clastogenicity of dilithium tetraborate, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to dilithium tetraborate in the absence of S9-mix for 24 or 48 hours. The following dose levles were selected; 100, 250, 500, 600, 700, 800 and 900 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).

However, after the 24 h exposure period, no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 250 µg/ml not enough cytotoxicity was observed (34%), whereas the next higher concentration of 500 µg/ml was too toxic for scoring (64%).

The assay was therefore repeated using the following dose levels; 10, 100, 200, 300, 400, 500, 600 and 700 µg/ml culture medium (24 h exposure time, 24 h fixation time) without S9-mix.

The following doses were then selected for scoring of chromosome aberrations:10, 200 and 300 µg/mL culture medium (24 h exposure time, 24 h fixation time) and 100, 250 and 500 µg/mL culture medium (48 h exposure time, 48 h fixation time) without S9-mix.

Results showed that dilithium tetraborate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations after 24 hours treatment.

After 48 hours treatment, a significant increase was observed at the highest dose level tested. However, no statistically significant trend was observed (p=0.095) and in addition the number of cells with aberrations was within the historical control data of the solvent control. Therefore the increase was considered not biologically relevant.

The number of polyploid cells and cells with endoreduplicated chromosomes were also not increased following treatment with dilithium tetraborate.

POSITIVE CONTROL
Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

SOLVENT CONTROL
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.

Overall the results showed that both in the absence and presence of S9-mix, dilithium tetraborate did not induce any statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

Conclusions:
In conclusion, a well conducted in vitro test has been conducted to determine the possible clastogenicity of dilithium tetraborate. Both in the absence and presence of S9-mix Dilithium tetraborate did not induce any statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
Executive summary:

A study was conducted to evaluate the ability of dilithium tetraborate to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of dilithium tetraborate was tested in two independent experiments.

In the first cytogenetic assay, dilithium tetraborate was tested for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction at various concentrations. 

In the second cytogenetic assay, dilithium tetraborate was tested up to 300 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 500 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

The test conditions were deemed adequate and the metabolic activation system (S9-mix) functioned properly as results from the solvent control were within the 95% control limits of the distribution of the historical negative control database.  In addition, the positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. The number of cells with chromosome aberrations found in the positive control cultures were within the 95% control limits of the distribution of the historical positive control database. 

In both of the two independently performed experiments, dilithium tetraborate did not induce any statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

No effects of dilithium tetraborate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that dilithium tetraborate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

In conclusion, results indicate that dilithium tetraborate is not clastogenic in human lymphocytes under the experimental conditions described. 

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

An OECD TG 473 in vitro test has been conducted to determine the possible clastogenicity of dilithium tetraborate using cultured human lymphocytes.  Both in the absence and presence of S9-mix Dilithium tetraborate did not induce any statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

 

In an OECD TG 471 in vitro study dilithium tetraborate induced dose related increases in tester strain TA100 (Salmonella typhimurium) in the absence and presence of S9-mix in two independent experiments (2.0 and 2.3-fold), respectively. The results in TA100 are therefore considered equivocal.

On the basis of the equivocal results obtained in the bacterial reverse mutation assay (OECD TG 471) further testing is proposed.