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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test, OECD 471): S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A: negative with and without metabolic activation

Mammalian cytogenicity (Micronucleus test, OECD 487): Chinese hamster lung fibroblasts (V79): negative with and without metabolic activation

Mammalian mutagenicity (Mouse lymphoma gene mutation assay, OECD 476): Mouse lymphoma L5178Y cells: negative with and without metabolic activation

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:
25 Feb - 01 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his/trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 156, 313, 625, 1250, 2500 and 5000 µg/plate (TA 98 and TA 1535 with and without metabolic activation)
Experiment I+II:
- 313, 625, 1250, 2500 and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle/solvent used: distilled water
- Justification for choice of solvent: Distilled water was used as solvent, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: daunomycin (DM), sodium azide (NaN), ICR 191 acridine (ICR), 4-nitroquinoline-1-oxide (4-NQO); +S9-mix: 2-aminoanthracene (2-AA), benzo(a)pyrene (BaP)
Remarks:
DM (6 µg/plate: TA 98); NaN (1.5 µg/plate: TA 100, TA 1535); ICR (1 µg/plate: TA 1537); 4-NQO (2 µg/plate: E.coli WP2 uvrA); 2-AA (10 µg/plate: TA 98, TA 100, TA 1535, TA 1537; 50 µg/plate: E.coli WP2 uvrA); BaP (20 µg/plate: TA 98, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 plates for each test concentration and control

DETERMINATION OF CYTOTOXICITY
- A preliminary experiment was conducted with the test material in tester strains TA 98 and TA 1535 either in the presence or in the absence of metabolic activation to determine cytotoxicity and solubility of the test substance using concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate.
- Method: Cytotoxicity is detected by reduction in the number of revertant colonies
Evaluation criteria:
Means of individual plate counts (triplicates) were calculated for test solutions and controls. In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose-response is a further criterion for mutagenic materials.
Statistics:
Mean values and standard deviation were calculated for each test and control concentration.
Species / strain:
other: TA 1535, TA 1537, TA 98 and 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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitations were seen in all test material concentrations which did not influence the results of the assay.

RANGE-FINDING/SCREENING STUDIES
As no relevant cytotoxicity was observed in the preliminary test (data not shown), the highest test concentration used in the main test was 5000 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY
No signs of cytotoxicity are reported.

COMPARISON WITH HISTORICAL CONTROL DATA
The colony counts of negative and positive controls were in the dimensions of historical and literature data.

Table 1: Test results - Experiment I - Preincubation method

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

79 ± 2

8 ± 0

13 ± 2

13 ± 4

8 ± 2

313

87 ± 7

11 ± 2

10 ± 2

12 ± 1

6 ± 1

625

69 ± 8

8 ± 2

13 ± 6

11 ± 4

6 ± 3

1250

74 ± 6

7 ± 3

15 ± 2

11 ± 4

3 ± 1

2500

89 ± 13

7 ± 5

11 ± 3

12 ± 5

4 ± 2

5000

85 ± 8

8 ± 1

11 ± 4

15 ± 6

7 ± 3

Positive controls, –S9

Name

NaN

NaN

4-NQO

DM

ICR

Concentrations

(μg/plate)

1.5

1.5

2

6

1

Mean No. of colonies/plate

(average of

3 ± SD)

499 ± 31

431 ± 26

839 ± 67

435 ± 95

2040 ± 59

+

0

68 ± 11

7 ± 2

10 ± 5

13 ± 3

4 ± 1

+

313

63 ± 6

6 ± 0

12 ± 2

17 ± 3

6 ± 1

+

625

59 ± 16

7 ± 1

12 ± 4

18 ± 4

4 ± 2

+

1250

68 ± 20

8 ± 4

11 ± 2

20 ± 5

5 ± 3

+

2500

70 ± 15

6 ± 2

11 ± 6

16 ± 2

4 ± 2

+

5000

72 ± 10

6 ± 4

10 ± 3

18 ± 2

4 ± 2

Positive controls, +S9 (10%)

Name

2-AA / BaP

2-AA

2-AA

2-AA / BaP

2-AA

Concentrations

(μg/plate)

10 / 20

10

50

10 / 20

10

Mean No. of colonies/plate

(average of

3 ± SD)

1807 ± 11 / 584 ± 19

56 ± 7

316 ± 62

2068 ± 60 / 290 ± 79

182 ± 25

DM: daunomycin

NaN: sodium azide

ICR: ICR 191 acridine

4-NQO: 4-nitroquinoline-1-oxide

2-AA: 2-aminoanthracene

BaP: benzo(a)pyrene

Table 2: Test results - Experiment II - Plate incorporation method

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

70 ± 12

8 ± 3

10 ± 5

29 ± 4

4 ± 1

313

81 ± 6

6 ± 3

12 ± 6

18 ± 8

4 ± 3

625

88 ± 13

8 ± 3

11 ± 1

17 ± 3

5 ± 1

1250

81 ± 9

7 ± 2

13 ± 2

10 ± 3

6 ± 1

2500

97 ± 12

6 ± 3

12 ± 3

17 ± 5

5 ± 2

5000

79 ± 7

6 ± 3

14 ± 2

16 ± 4

4 ± 2

Positive controls, –S9

Name

NaN

NaN

4-NQO

DM

ICR

Concentrations

(μg/plate)

1.5

1.5

2

6

1

Mean No. of colonies/plate

(average of

3 ± SD)

468 ± 37

385 ± 12

1185 ± 291

576 ± 99

82 ± 13

+

0

86 ± 12

4 ± 0

12 ± 6

12 ± 4

5 ± 2

+

313

72 ± 3

6 ± 3

12 ± 5

11 ± 6

4 ± 1

+

625

77 ± 4

7 ± 2

8 ± 3

22 ± 8

5 ± 2

+

1250

73 ± 10

5 ± 2

11 ± 2

19 ± 3

5 ± 1

+

2500

73 ± 16

7 ± 3

13 ± 2

14 ± 3

4 ± 2

+

5000

70 ± 5

5 ± 2

14 ± 1

12 ± 7

4 ± 2

Positive controls, +S9 (10%)

Name

2-AA / BaP

2-AA

2-AA

2-AA / BaP

2-AA

Concentrations

(μg/plate)

10 / 20

10

50

10 / 20

10

Mean No. of colonies/plate

(average of

3 ± SD)

856 ± 154 / 383 ± 9

45 ± 2

378 ± 48

1782 ± 201 / 136 ± 23

175 ± 49

DM: daunomycin

NaN: sodium azide

ICR: ICR 191 acridine

4-NQO: 4-nitroquinoline-1-oxide

2-AA: 2-aminoanthracene

BaP: benzo(a)pyrene

Table 3: Historical control data (negative control)

Strain

MIN

MAX

MEAN

SD

N

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

TA 98

13

21

54

65

29

38

16

19

5

5

TA 100

62

63

246

232

112

108

78

70

5

5

TA 1535

7

8

28

20

14

11

8

5

5

5

TA 1537

3

3

4

6

4

5

n.a.

n.a.

2

2

WP2 uvrA

13

13

17

20

15

18

2

3

4

4

SD: standard deviation

n.a.: not applicable

N: number of studies

Table 4: Historical control data (positive control)

Strain

Chemical

MIN

MAX

N

TA 98

2-Aminoanthracene

135

>1000

5

Daunomycin

198

>500

5

TA 100

2-Aminoanthracene

326

>500

5

Sodium azide

274

>500

5

TA 1535

2-Aminoanthracene

67

>300

5

Sodium azide

83

>300

5

TA 1537

2-Aminoanthracene

107

210

2

ICR 191 Acridine

126

283

2

WP2 uvrA

2-Aminoanthracene

251

407

4

4-Nitroquinoline-1-oxide

756

1532

4

N: number of studies

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb - 02 Jun 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test (2014)
Deviations:
yes
Remarks:
No cytotoxicity was determined either in a preliminary cytotoxicity test or within the main experiments, thus dose selection for the main experiment is questionable
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 medium supplemented with
- 10% (v/v) fetal bovine serum (FBS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary range-finding solubility test:
- 0.32, 1.6, 8, 40, 200 and 1000 µg/mL (4 and 24 h)
Main experiment:
- 0.178, 0.53 and 1.6 µg/mL (4 h exposure with and without metabolic activation)
- 0.178, 0.53 and 1.6 µg/mL (24 h exposure without metabolic activation)
Vehicle / solvent:
- Vehicle/solvent used: RPMI 1640 medium supplemented with 10% (v/v) FBS
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: 0.2 µg/mL colchicine; +S9-mix: 5 µg/mL benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 24 h (1.5-2.0 normal cell cycles); 24 h treatment: 24 h (1.5-2.0 normal cell cycles)

STAIN: Hoechst 33258 solution and Pyronin Y

NUMBER OF REPLICATIONS: duplicate cultures for each test concentration and control

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (relative increase in cell counts, RICC); cytotoxicity = 100% - RICC [%]
Evaluation criteria:
A test substance was classified as genotoxic, if at least a 3-fold increased number of micronuclei in relation to the negative control was counted and/or a dose-response relationship was observed.
Statistics:
Mean values were calculated for each test and control culture.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at the highest test concentration of 1.6 µg/mL 19.8% cytotoxicity was determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Because of the solubility as a limiting factor, 1.6 µg/mL was chosen as the maximum concentration. This concentration was the highest concentration at which minimal precipitate was visible by light microscopy.

RANGE-FINDING/SCREENING STUDIES
Prior to testing of solved sample material, a preliminary range-finding test for the maximal concentration was done because the test material was a slightly soluble inorganic salt. Concentrations of 0.32, 1.6, 8, 40, 200 and 1000 µg/mL were prepared and incubated at 37 ± 1 °C under moderate agitation at the same time for approximately 4 and 24 h. The estimation of particles was done by gravimetric sedimentation followed by a microscopically examination. The highest concentration was the solution with no or a minimal precipitate content in the cultures, thus 1.6 µg/mL was selected as the highest test concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Slight cytotoxicity (19.8%) was only recorded at the highest test concentration of 1.6 µg/ml..

Table 1: Test result - Micronucleus assay (in vitro)

Test item

Concentration

(µg/mL)

Threshold value* (3-fold of negative control)

Number of cells with micronuclei (%)

Exposure period 4 h, fixation time 24 h, without S9-mix

Vehicle

0

 

 

1.35

0.45

Colchicine

0.2

1.85

 

Test item

0.178

1.15

0.53

0.55

1.6

0.65

Exposure period 4 h, fixation time 24 h, with 2% (v/v) S9-mix

Vehicle

0

 

 

1.95

0.65

Benzo(a)pyrene

5

2.7

 

Test item

0.178

1.15

0.53

0.8

1.6

1.2

Exposure period 24 h, fixation time 24 h, without S9-mix

Vehicle

0

 

 

1.35

0.45

Colchicine

0.2

4.3

 

Test item

0.178

0.6

0.53

0.85

1.6

0.9

*: A 3-fold increasing of micronuclei in comparison with the average of negative controls was set as threshold value.

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jan - 03 Mar 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
No cytotoxicity was determined within the main experiments. No vehicle controls were used in a preliminary cytotoxicity tests, thus dose selection for the main experiment is questionable.
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 medium supplemented with
- 10% (v/v) heat inactivated horse serum (cultivation and exposure medium)
- 20% (v/v) heat inactivated horse serum (cloning and mutant selection medium)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary range-finding test I+II:
- 0.32, 1.6, 8, 40, 200, 1000 and 5000 µg/mL (with and without metabolic activation)
- 125, 250, 500, 1000 and 2000 µg/mL (with and without metabolic activation)
Main and Confirmatory test:
- 31.25, 62.5, 125 and 250 µg/mL (with and without metabolic activation)
Vehicle / solvent:
- Vehicle/solvent used: RPMI 1640 medium supplemented with 10% (v/v) heat inactivated horse serum
- Justification for choice of solvent/vehicle: RPMI 1640 cell culture medium was used as vehicle/solvent, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Remarks:
Methylmethanesulfonate (10 µg/mL, -S9-mix); Benzo(a)pyrene (1.5 µg/mL, +S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates containing TFT selective medium. The microtitre plates were incubated for 7 days (cloning efficiency) or 11 - 14 days (mutation frequency).
- Selection time: 11 - 14 days

SELECTION AGENT: 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures for each test concentration and the negative control, single culture for the positive control

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The cloning efficiency (CE) of cells and the spontaneous mutation frequency (MF) of negative controls should be within their limit values (CE ≥50%; MF (negative control) 50 - 300 x 1E-06). The MF of positive controls should be increased 2 times or more than the MF in corresponding negative controls in each experiment. The results of negative and positive controls will confirm the sensitivity and accuracy of the test system according to the criteria of acceptance of literature and SOP.
Statistics:
No statistics were performed since only cloning efficiency and mutation frequency were calculated.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH during the exposure of the cells to the test substance did not change (colour of the pH indicator in the culture medium).
- Precipitation: Slight precipitations were seen in all test material concentrations which did not influence the results of the assay.

RANGE-FINDING/SCREENING STUDIES
Preliminary experiments were conducted (with and without metabolic activation) at concentrations of 0.32, 1.6, 8, 40, 200, 1000 and 5000 µg/mL (test I) and 125, 250, 500, 1000 and 2000 µg/mL (test II) to determine cytotoxicity and solubility of the test material. A concentration of 250 µg/mL was chosen as the highest concentration for the main and confirmatory experiment based on cytotoxicity (44/45% for CE 1 and 13/0% for CE 2 (with/without S9-mix)) and precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA
The observed cloning efficiency (CE 1 and CE 2) and the mutation frequency (MF) were in the range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In all test concentrations, negative and positive controls either both in the presence and absence of metabolic activation (1% (v/v) S9-mix) the cloning efficiency (CE 1 and CE 2) was not significantly different compared to the vehicle control group. No relevant cytotoxicity was determined within the experiments.

Table 1: Results of the preliminary cytotoxicity/solubility test

Test concentration (µg/mL)

CE 1

CE 2

without S9-mix

with S9-mix

without S9-mix

with S9-mix

Test I

0.32

0.65

0.71

1.01

0.92

1.6

0.57

0.87

0.68

0.56

8

0.73

0.56

0.85

0.69

40

0.70

0.89

0.69

0.85

200

0.87

1.12

0.66

0.84

1000

0.26

0.43

0.76

0.67

5000

0.12

0.28

0.47

0.53

Test II

125

1.09

0.94

1.03

0.97

250

0.57

0.56

1.04

0.87

500

0.29

0.28

0.64

0.85

1000

0.23

0.21

0.94

0.73

2000

0.20

0.18

0.86

0.77

Table 2: Results of the Main test and Confirmatory Test (CE 1 and CE 2)

Test concentration (µg/mL)

CE 1

CE 2

without S9-mix

with S9-mix

without S9-mix

with S9-mix

Main Test

Vehicle

0.93

0.86

0.84

1.00

31.25

1.00

0.88

0.85

0.98

62.5

0.95

0.65

0.80

0.88

125

0.83

0.57

0.79

1.03

250

0.73

0.67

0.94

0.92

MMS

0.87

-

0.58

-

BaP

-

0.68

-

0.92

Confirmatory Test

Vehicle

0.96

1.42

0.77

0.94

31.25

0.71

1.05

0.92

1.01

62.5

0.73

1.07

1.14

0.87

125

0.76

1.22

0.99

0.82

250

0.71

0.86

1.17

0.89

MMS

0.71

-

0.66

-

BaP

-

0.88

-

0.91

MMS: methylmethanesulfonate

BaP: benzo(a)pyrene

Table 3: Results of the Main test and Confirmatory Test (MF)

Test concentration (µg/mL)

MF

Big colonies

Small colonies

without S9-mix

with S9-mix

without S9-mix

with S9-mix

Main Test

Vehicle

67.18

98.16

60.25

72.66

31.25

51.50

95.96

78.48

102.05

62.5

59.00

91.35

65.22

75.93

125

69.20

63.61

62.44

62.33

250

69.14

65.80

55.79

62.62

MMS

297.67

-

1087.05

-

BaP

-

290.25

-

683.79

Confirmatory Test

Vehicle

106.21

106.05

70.15

56.92

31.25

59.95

113.08

39.60

61.40

62.5

80.52

97.70

54.92

48.39

125

97.70

112.44

48.39

51.77

250

71.24

115.04

51.13

60.37

MMS

363.50

-

1757.64

-

BaP

-

289.87

-

730.98

MMS: methylmethanesulfonate

BaP: benzo(a)pyrene

Table 4: Historical control data (CE 1, CE 2 and MF)

Controls

CE 1

CE 2

MIN

MAX

MEAN

SD

N

MIN

MAX

MEAN

SD

N

Medium (-S9-mix)

0.88

1.25

0.84

0.15

5

0.90

1.22

0.86

0.17

5

Medium (+S9-mix)

0.87

1.05

0.87

0.09

5

0.93

1.24

0.85

0.16

5

MMS (-S9-mix)

0.63

1.01

1.04

0.14

5

0.61

1.08

1.16

0.27

5

BaP

(+S9-mix)

0.80

1.00

0.99

0.13

5

0.73

1.12

1.06

0.11

5

MMS: methylmethanesulfonate

BaP: benzo(a)pyrene

SD: standard deviation

N: number of studies

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity (mutagenicity) in bacteria in vitro

A reliable bacterial gene mutation study (Ames Test) performed according to OECD TG 471 and in compliance with GLP is available with boron orthophosphate (CAS 13308-51-5) (Kolp, 2015a). The Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA were tested according to the pre-incubation (experiment I) and plate incorporation (experiment II) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix (10% (v/v))). Two independent experiments were conducted in the concentration range from 313 to 5000 µg/plate (experiment I and II). No relevant cytotoxicity was observed in a preliminary toxicity test performed with tester strains TA 98 and TA 1535 after treatment with the test item up to 5000 µg/plate in the absence and presence of metabolic activation. Slight precipitation was seen in all test material concentrations which did not influence the results of the assay. Appropriate solvent (distilled water) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of the test.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro Micronucleus Test equivalent or similar to OECD TG 487 and in compliance with GLP was performed with boron orthophosphate (CAS 13308-51-5) (Prietzsch, 2015). Chinese hamster lung fibroblasts (V79) were treated in duplicate cultures with the test material or vehicle (RPMI 1640 medium supplemented with 10% (v/v) FBS) in the absence or presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix 2% (v/v)). Short-term (4 hours with and without S9 mix) and long-term (24 hours without S9 mix) experiments were conducted at concentrations of 0.178, 0.53 and 1.6 µg/mL. Fixation of the cells was performed 24 hours (approx. 1.5 to 2.0 normal cell cycles) after start of exposure with the test material. Appropriate solvent and positive controls were included in the test and gave the expected results. The number of micronucleated cells found after treatment with the test item was within the normal range of the negative control and thus no genotoxic effects were recorded either in the presence or in the absence of metabolic activation. No cytotoxicity was determined either in a preliminary cytotoxicity test or within the main experiments, thus dose selection for the main experiment is questionable. Only slight cytotoxicity (19.8%) and precipitation were reported at the highest test concentration of 1.6 µg/mL without any details given. Nevertheless, based on the results of the study the test material is considered not to be clastogenic under the conditions of this in vitro study.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro Mammalian Cell Gene Mutation Test was performed with boron orthophosphate (CAS 13308-51-5) in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) equivalent or similar to OECD TG 476 and under GLP (Kolp, 2015b). The cells were treated with the test substance in duplicate cultures, together with vehicle (RPMI 1640 medium supplemented with 10% (v/v) heat inactivated horse serum) and positive controls (single cultures). The cells were exposed to the test substance for 3 hours in the absence and presence of metabolic activation (Aroclor-1254-induced rat liver S9-mix (1% (v/v)) in the main and the confirmatory test. The concentration range of the test material in both experiments was 31.25 to 250 µg/mL following the results of a preliminary toxicity test. Slight precipitations were seen in all test material concentrations which did not influence the results of the assay. The vehicle controls (RPMI 1640 medium supplemented with 10% (v/v) heat inactivated horse serum) were within the normal range for the L5178Y cell line at the TK locus. The positive controls induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. No cytotoxicity was determined within the main experiments. Moreover, no vehicle controls were used in a preliminary cytotoxicity tests, thus dose selection for the main experiment is considered to be questionable. Nevertheless, the test material did not induce any toxicologically significant increases in the mutant frequency at any concentration level up to and including 250 μg/mL. The distribution of small and big colonies showed neither chromosome nor gene mutations. Thus, the test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

Conclusion

Taken together, the available data on genetic toxicity with boron orthophosphate (CAS 13308-51-5) do not indicate any mutagenic and clastogenic potential. Thus, the test substance is not to be classified.

Justification for classification or non-classification

The available data on genetic toxicity with boron orthophosphate (CAS 13308-51-5) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.