Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jun 2017 - 10 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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:
Rat liver microsomal enzymes (S9 homogenate), prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the 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 item used in the subsequent mutation assays 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.
Vehicle / solvent:
The vehicle of the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA).
Untreated negative controls:
yes
Remarks:
Milli-Q water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 (Sigma), 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD, EC).
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid

Dose-Range Finding Test: Mutagenic Response of MEA Polyborate 1:1 in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Direct plate assay


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (
±S.D.) with one Salmonella typhimurium and one Escherichia colistrain.

 


TA100


WP2uvrA



 

Without S9-mix

Positive control

722

±

32

 

1272

±

76

Solvent control

101

±

7

 

19

±

6

1.7

99

±

7

 

19

±

1

5.4

108

±

5

 

18

±

3

17

101

±

25

 

25

±

6

52

117

±

9

 

23

±

0

164

111

±

29

 

21

±

5

512

100

±

16

 

22

±

8

1600

110

±

6

 

20

±

5

5000

128

±

11

n NP

19

±

6

n NP

With S9-mix

Positive control

475

±

106

 

420

±

13

Solvent control

92

±

6

 

29

±

10

1.7

107

±

15

 

27

±

4

5.4

95

±

12

 

25

±

4

17

110

±

12

 

28

±

8

52

111

±

16

 

29

±

11

164

112

±

14

 

24

±

5

512

107

±

9

 

27

±

6

1600

101

±

11

 

27

±

2

5000

126

±

17

n NP

38

±

4

n NP

NP

No precipitate

n

Normal bacterial background lawn

Experiment 1: Mutagenic Response of MEA Polyborate 1:1 in the Salmonella typhimurium Reverse Mutation Assay

Direct plate assay

Dose

(µg/plate)


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

 


TA1535


TA1537

 


TA98

 

 Without S9-mix

Positive control

790

±

47

 

985

±

99

 

1193

±

86

 

Solvent control

11

±

2

 

6

±

2

 

16

±

3

 

52

16

±

3

 

6

±

2

 

11

±

6

 

164

11

±

4

 

4

±

1

 

15

±

5

 

512

11

±

7

 

6

±

2

 

25

±

5

 

1600

11

±

4

 

6

±

2

 

16

±

9

 

5000

6

±

4

n NP

4

±

1

n NP

16

±

2

n NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With S9-mix

Positive control

328

±

21

 

546

±

104

 

1022

±

61

 

Milli-Q water

10

±

3

 

8

±

4

 

24

±

11

 

52

13

±

2

 

7

±

2

 

29

±

5

 

164

10

±

2

 

6

±

3

 

25

±

9

 

512

12

±

1

 

7

±

3

 

30

±

6

 

1600

13

±

3

 

5

±

0

 

24

±

3

 

5000

7

±

2

n NP

8

±

4

n NP

21

±

2

n NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NP

No precipitate

n

Normal bacterial background lawn

Experiment 2: Mutagenic Response of MEA Polyborate 1:1 in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay


Pre-incubation 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

886

±

21

 

1183

±

75

 

1211

±

169

 

708

±

171

 

202

±

35

 

 

Solvent control

7

±

2

 

9

±

1

 

20

±

5

 

118

±

4

 

36

±

9

 

 

52

13

±

1

 

8

±

2

 

34

±

21

 

114

±

13

 

25

±

10

 

 

164

12

±

2

 

8

±

4

 

19

±

7

 

104

±

51

 

29

±

4

 

 

512

15

±

5

 

8

±

3

 

18

±

4

 

112

±

20

 

31

±

4

i

 

1600

9

±

6

 

11

±

6

 

12

±

4

 

149

±

8

 

31

±

13

 

 

5000

9

±

3

n SP

5

±

2

n SP

15

±

5

n SP

179

±

17

n SP

38

±

17

n SP

 

 

With S9-mix

Positive control

217

±

3

 

177

±

16

 

495

±

86

 

1008

±

343

 

541

±

13

 

Milli-Q water

19

±

5

 

4

±

0

 

26

±

2

 

118

±

21

 

42

±

9

 

52

8

±

1

 

8

±

2

 

28

±

13

 

116

±

10

 

41

±

6

 

164

11

±

4

 

6

±

5

 

28

±

2

 

135

±

27

 

61

±

7

 

512

10

±

2

 

8

±

4

 

25

±

7

 

145

±

27

 

54

±

9

 

1600

13

±

1

 

10

±

8

 

23

±

9

 

148

±

11

 

48

±

9

 

5000

7

±

2

n SP

5

±

3

n SP

21

±

4

n SP

189

±

18

n SP

52

±

5

n SP

 

SP

Slight Precipitate

i

Plate infected

n

Normal bacterial background lawn

 

Conclusions:
Based on the results of a GLP OECD 471 study it is concluded that MEA Polyborate 1:1 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-12 to 2010-07-28
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Only 64 metaphase spreads examined
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes were routinely prepared from adult male Wister rats (6), which wre obtained from Charles Reiver (Sulzfeld, Germany)
Test concentrations with justification for top dose:
First cytogenic assay
Without S9-mix: 1000, 3330, 4000 and 5000 µg/ml culture medium ( 3h exposure time, 24 h fixation time)
With S9-mix : 1000, 3330 and 5000 µg/ml culture medium ( 3h exposure time, 24 h fixation time)
Second cytogenic assay
Without S9-mix: 50, 100, 200, 300, 500, 750 and 1000 µg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).
With S9-mix: 1000, 3330 and 5000 µg/ml culture medium (3 h exposure time, 48 h fixation time).
Third cytogenic assay
Without and with S9-mix: 5000 µg/ml culture medium (3 h exposure time, 24 h fixation time).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [RPMI 1640 medium]
- Justification for choice of solvent/vehicle:none given
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
- Exposure duration:3 hour
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hour

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):colchcine (0.5
STAIN (for cytogenetic assays):5% (v/v) Glemsa (Merck) solution in tap water

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:carried out
- Determination of endoreplication:carried out
- Other:

OTHER:
Evaluation criteria:
A substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one sided , p<0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
Statistics:
The parameter used was the increase in the number of cells with chromosome aberrations. The statistical significance was given by Chi-square test, one sided , p<0.05.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Clastogenicity was observed in the first cytogentic assay. This was assumed to be due to the high pH in that assay.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Based on the results of this study, it is concluded that MEA Polyborate 1:1 has shown a clastogenic response in the chromosome aberration study in experiments where the pH of the treated cultures was above the pH of the solvent control. The clastogenicity was only observed in the first cytogenetic assay. Due to fact that the adjustment of the pH of the cultures treated with MEA Polyborate 1:1 did not show a clastogenic response in the third cytogenetic assay, it has to be assumed that the high pH is responsible for the detected clastogenic activity.
Executive summary:

Evaluation of the ability of MEA Polyborate 1:1 to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiments). This report describes the effect of MEA Polyborate 1:1 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of MEA Polyborate 1:1 was tested in three independent experiments. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch EU-SMG 01695 of MEA Polyborate 1:1 was a clear colourless liquid. MEA Polyborate 1:1 was dissolved in RPMI 1640 medium. In the first cytogenetic assay, MEA Polyborate 1:1 was tested up to 5000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest concentration that should be tested according to the guidelines. In the second cytogenetic assay, MEA Polyborate 1:1 was tested up to 1000 µg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. In the presence of S9-mix MEA Polyborate 1:1 was tested up to 5000 µg/ml for a 3 h exposure time with a 48 h fixation time. In the third cytogenetic assay, MEA Polyborate 1:1 was tested at the concentration of 5000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. In this cytogenetic assay the pH of the cultures which were treated with MEA Polyborate 1:1 was adjusted to the pH of the solvent control. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. No biologically relevant effects of MEA Polyborate 1:1 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 MEA Polyborate 1:1 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. In the first and second cytogenetic assay MEA Polyborate 1:1 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix. However, exchange figures were observed at all concentrations tested in the first cytogenetic assay. It should be noted that chromosomal exchanges are comparatively rare spontaneous events and therefore greater significance should be attached to the observation of exchange figures. In the first cytogenetic assay, in the presence of S9-mix, MEA Polyborate 1:1 induced a statistically significant, increase in the number of cells with chromosome aberrations at the highest tested concentration of 5000 µg/ml, both when gaps were included and excluded. In the second cytogenetic assay, in the presence of S9-mix, MEA Polyborate 1:1 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. The statistically significant increase in the number of cells with chromosome aberrations and the exchange figures were observed in cultures treated with MEA Polyborate 1:1 which had a pH (8.42 - 9.14) above the negative control (pH = 8.01). It is known that changes in pH induce significant increases in chromosome aberrations. Therefore, an additional third cytogenetic assay was performed in which the pH of the cultures treated with MEA Polyborate 1:1 was adjusted to the pH of the solvent control. In the third cytogenetic assay, MEA Polyborate 1:1 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. Moreover, no exchange figures were observed. Based on the results of this study, it is concluded that MEA Polyborate 1:1 has shown a clastogenic response in the chromosome aberration study in experiments where the pH of the treated cultures was above the pH of the solvent control. The clastogenicity was only observed in the first cytogenetic assay. Due to fact that the adjustment of the pH of the cultures treated with MEA Polyborate 1:1 did not show a clastogenic response in the third cytogenetic assay, it has to be assumed that the high pH is responsible for the detected clastogenic activity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the results of a GLP OECD 471 study it is concluded that MEA Polyborate 1:1 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Based on the results of this study, it is concluded that MEA Polyborate 1:1 has shown a clastogenic response in the chromosome aberration study in experiments where the pH of the treated cultures was above the pH of the solvent control. The clastogenicity was only observed in the first cytogenetic assay. Due to fact that the adjustment of the pH of the cultures treated with MEA Polyborate 1:1 did not show a clastogenic response in the third cytogenetic assay, it has to be assumed that the high pH is responsible for the detected clastogenic activity.

The substance is therefore not classified as mutagenic under GHS.