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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only one strain under study: E. coli
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
only one strain under study: E. coli
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
only one strain under study: E. coli
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Tryptophan-Locus
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient broth
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix ( Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
Range finding: seven concentrations up to 5000 µg/plate
Main test: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of the test material
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
Solved in DMSO; without S9 mix; 0,05 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (CAS NO.: 613-13-8)
Remarks:
Solved in DMSO; with S9 mix; 10 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Preincubation period: 30 min., in glass vessels, with test material in solution
- Incubation time: 72 hours (petri dishes containing minimal agar without tryptophan)

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY in the range finding test
- Method: cloning efficiency
Evaluation criteria:
The mutagenic activity of a test substance was assessed by applying the following criteria:
- increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-response relationship (...)
- If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.
Statistics:
The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett's test.
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Visible thinning of the background lawn of non-revertant cells was obtained following exposure to BC-300 at 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results : negative

It is concluded that, under the test conditions employed, the test material BC-300 showed no evidence of mutagenic activity in this bacterial system.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
First Addendum to OECD Guidelines No. 4 71, "Salmonella typhimurium, Reverse Mutation Assay", adopted May 26, 1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 84/449, L 251, B 14, p. 143-145
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus in selected strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient medium: 8 g Difco Nutrient Broth, 5 g NaCl in 20 ml (for 0,5 ml bacterial suspension)
- Storage: stock cultures in ampoules with nutrient broth and 5 % DMSO in liquid nitrogen
- Properly maintained: yes

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al. (1970, 1977) . In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
Pre-study with strain TA 98 and TA 100: 1 – 5000 µg/plate

Main-experiments:
Exp. I : TA 1535, TA 1537, TA 98, TA 100:
10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg /plate
Exp. II : TA 1535, TA 98, TA 100:
10.0; 33.3; 100.0; 333.3; 1000.0 and 5000.0 µg/plate
Exp. II: TA 1537:
10.0; 33.3; 66.6; 333.3; 100.0; 666.6 and 1000.0 µg/plate
Vehicle / solvent:
Solvent: Methanol. On the day of experiment, the test article was dissolved.
Justification: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
sodium azide for strains: TA 1535, TA 100 without metabolic activation, 10 µg/plate, dissolved in aqua dest..
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
4-nitro-o-phenylene-diamine for strains TA 1537, TA 98 without metabolic activation, dissolved in DMSO, 50 µg/plate.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-aminoanthracene for all strains with metabolic activation, dissolved in DMSO, 10 µg /plate.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72h

NUMBER OF REPLICATIONS: 3/strain/dose for each of the two experiments.
Evaluation criteria:
The test material is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A significant response is described as follows:
The test material is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test material regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method was available.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
partial or complete reduction in the number of revertants, at the higher dose levels with and without metabolic activation in experiment I and II in all strains used
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration- dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Table 1: Results WITHOUT S9 mix (/ = not performed)

Dose µg/plate

Strains

Experiment

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Neg. control

9

9

10

6

24

21

107

96

Solvent control

8

7

6

7

23

16

99

94

10.0

10

13

5

6

24

19

111

98

33.3

8

6

6

7

32

16

99

99

66.6

/

/

/

10

/

/

/

/

100.0

8

7

6

9

21

15

78

76

333.3

8

8

5

7

23

17

75

44

666.6

/

/

/

2

/

/

/

/

1000.0

5

5

3

0

11

9

29

8

5000.0

3

2

0

/

0

1

24

4

Pos. contr.: Sodium azid 10 µg/plate

122

964

 

 

 

 

29

8

Pos. control:

4-Nitro-o-phenylene-diamine 50 µg/plate

 

 

193

318

1981

1913

 

 

Table 2: Results WITH S9 mix (/ = not performed)

Dose µg/plate

Strains

Experiment

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Neg. control

11

13

5

8

43

20

95

93

Solvent control

9

13

7

11

43

32

99

115

10.0

11

13

12

11

44

27

128

120

33.3

10

10

11

14

46

27

147

102

66.6

/

/

/

9

/

/

/

/

100.0

11

12

8

10

30

28

112

122

333.3

12

15

7

10

38

21

132

126

666.6

/

/

/

6

/

/

/

/

1000.0

7

11

4

5

27

18

123

86

5000.0

4

1

0

/

1

0

27

13

Pos. contr.: 2-Amino-anthracene 10 µg/plate

126

117

111

132

772

753

783

926

Conclusions:
Interpretation of results : negative

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
First Addendum to the OECD Guideline 473, adopted May 26, 1983
Qualifier:
according to
Guideline:
other: EEC Directive 84/449, L 251, B 10, p. 131-133
Qualifier:
according to
Guideline:
other: EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, "In vitro mammalian cytogenetics"
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Stored in liquid nitrogen, thawed stock cultures are propagated at 37°C in 80 cm2 plastic flasks
- Cell culture medium: MEM (minimal essential medium; SEROMED; 12247 Berlin) supplemented with 10% fetal calf serum (FCS; SEROMED).
- Properly maintained: yes. The cells are subcultured twice weekly. The cell cultures are incubated at 37°C and 4.5 % carbon dioxide atmosphere.
- Checked for Mycoplasma contamination and for karyotype stability: yes, before freezing.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
Pre-experiment:
without S9 mix:
7 h: 1.0; 1.5; 2.0; 3.0 µg/ml
18 h: 0.1; 0.3; 1.0; 1.5; 2.0; 3.0 µg/ml
28 h: 1.0; 1.5; 2.0; 3.0 µg/ml

with S9 mix:
7 h: 4.0; 6.0; 8.0; 10.0 µg/ml
18 h: 0.4; 1.0; 4.0; 6.0; 8.0; 10.0 µg/ml
28 h: 4.0; 6.0; 8.0; 10.0 µg/ml

Main-experiment:

without S9 mix:
7 h: 1.5 µg/ml
18 h: 0.1; 1.0; 2.0 µg/ml
28 h: 2.0 µg/ml

with S9 mix:
7 h: 4.0 µg/ml
18 h: 0.4; 4.0; 8.0 µg/ml
28 h: 8.0 µg/ml
Vehicle / solvent:
Solvent used: Ethanol
Justification for the choice of solvent: The solvent was chosen according to its solubulity properties and its non-toxcicity for the cells. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent: ethanol
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix; dissolved in nutrient medium, final conc.: 0.72 mg/ml (5.76 mM)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix; dissolved in nutrient medium, final conc.: 1.40 ~g/ml (5.00 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in serum free medium containing the test article, either without S9 mix or with 20 µl S9 mix
DURATION
- Preincubation period: 48h and 55h
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 7h, 28 h (48h preincubation), 18h (55h preincubation)

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/ml culture medium) added after 5, 15.5 and 25.5 h (2-2.5h before fixation)
STAIN (for cytogenetic assays): Giemsa (Merck)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The test material is classified as mutagenic
- if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or
- a significant positive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (P < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
colony forming ability reduced > 0,1 µg/ml without S9 mix, > 3,0 µg/ml with S9 mix (results taken from the pre-experiment).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING: in a pre-experiment using the plating efficiency assay as indicator for toxicity response.
Treatment with concentrations higher than 0.1 µg/ml (without S9 mix) and 3.0 µg/ml (with S9 mix) reduced clearly the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix.
With higher concentrations >2.0 µg/ml (without S9 mix) and >8.0 µg/ml (with S9 mix)) no or not enough scorable cells could be found.

The aberration rates of the cells after treatment with the test article (0.0 % - 3.5 %) were in or near to the range of the control values: 0.5 % - 2.0 %. There was no relevant increase in cells with structural aberrations after treatment with the test material at any fixation interval either without or with metabolic activation by S9 mix.

Table 1: Mutagenicity data

Assay

Fixation interval: 7h

Number of cells analysed

Conc. µg/ml

S9 mix

Per cent aberrant cells

incl. gaps

excl. gaps

exchanges

Solvent control

200

0,0

-

2,00

1,50

0,00

Test material

200

1,5

-

6,50

3,50

0,00

Solvent control

200

0,0

+

5,50

2,00

0,,50

Test material

200

4,0

+

5,50

1,00

0,00

Assay

Fixation interval:18h

Number of cells analysed

Conc. µg/ml

S9 mix

Per cent aberrant cells

Incl. gaps

excl. gaps

exchanges

Negative control

200

0,0

-

2,00

1,50

0,50

Solvent control

200

0,0

-

4,00

1,50

0,00

Positive control EMS

200

0,72

-

14,00

9,00

5,50

Test material

200

0,1

-

3,50

2,00

0,50

Test material

200

1,0

-

4,00

2,50

0,00

Test material

200

2,0

-

5,00

2,50

1,50

Negative Control

200

0,0

+

2,50

0,50

0,00

Solvent control

200

0,0

+

1,00

1,00

0,50

Positive Control CPA

200

1,4

+

22,50

20,50

9,00

Test material

200

0,4

+

3,00

1,00

0,00

Test material

200

4,0

+

1,50

0,00

0,00

Test material

200

8,0

+

1,00

0,50

0,,50

Assay

Fixation interval:28h

Number of cells analysed

Conc. µg/ml

S9 mix

Per cent aberrant cells

 

incl. gaps

excl. gaps

exchanges

Solvent control

200

0,0

-

4,00

2,00

1,50

Test material

200

2,0

-

3,50

1,50

0,50

Solvent control

200

0,0

+

4,00

2,00

1,50

Test material

200

8,0

+

5,00

3,00

0,00

Conclusions:
Interpretation of results : negative

It can be stated that in the study described and under the experimental conditions reported, the test material did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.
Therefore, NA-11 is considered to be non-mutagenic in this chromosomal aberration test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Second Addendum, Section 4, No. 476, adopted April 4, 1984
Qualifier:
according to
Guideline:
other: EEC Directive 87/302, L 133, p. 61 - 63
Qualifier:
according to
Guideline:
other: EPA; 40 CFR; Ch.I; Part 798; Detection of gene mutation in somatic cells in culture; pp. 717-720 (7-1-86 Edition)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Gene for HGPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Stored in liquid nitrogen, thawed stock cultures are propagated at 37°C in 80 cm2 plastic flasks
- Cell culture medium: MEM (minimal essential medium; SEROMED; D-12247 Berlin) supplemented with 10% fetal calf serum (FCS; SEROMED).
- Properly maintained: yes. The cells are subcultured twice weekly. The cell cultures are incubated at 37°C and 4.5 % carbon dioxide atmosphere.
- Checked for Mycoplasma contamination and for karyotype stability: yes, before freezing.
- For the selection of mutants, the medium is supplemented with 11 µg/ml thioguanine.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (The S9 mix preparation was performed according to Ames et al.(1977)).
Test concentrations with justification for top dose:
Pre-experiment for toxicity (colony forming ability):
Without and with metabolic activation 0.01, 0.10, 1.00, 3.00, 10.00, 30.00, 60.00, 100.00 µg/ml

Main-Experiment:
with S9 mix: 0.10, 0.30, 1.00, 2.00, 6.00, 10.00 µg/ml
without S9 mix: 0.03, 0.10, 0.30, 1.00, 2.00, 3.00 µg/ml
Vehicle / solvent:
Solvent used: ethanol (test material was dissolved on the day of the experiment)
Justification for the solvent: The solvent was chosen according to its solubility properties and its relative nontoxicity for the cells.
The final concentration of the solvent in the culture medium did not exceed 1 % v/v.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
EMS without S9 mix, dissolved in nutrient medium 1 mg/ml (8mM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
with DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
DMBA with S9 mix, dissolved in DMSO (Dimethylsulfoxid), conc. in nutrient medium: 15.4 µg/ml (60,1 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: test material in serum free medium is given to a cell suspension

DURATION
- Preincubation period: 24h, seeded in MEM with 10% FCS (complete medium).
- Exposure duration: 4h in serum-free medium containing the test material, either without S9 mix or with 20 µg/ml S9 mix.
- Expression time (cells in growth medium): day 1-9
- Selection time (if incubation with a selection agent): mutant selection medium day 9 - 17
- Cloning efficiency: day 9 - 16
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days for plating efficiency and dose relationsship, 15 days for cloning efficiency determinations, 16 days for mutant selection.

SELECTION AGENT (mutation assays): thioguanine (6TG)
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution.

NUMBER OF REPLICATIONS:
determination of toxicity: in duplicate per experimental point.
determination of mutation rates: 1 flask per experimental point.

DETERMINATION OF CYTOTOXICITY
- Method: concentration-related cloning efficiency: day 8: fixation and staining of colonies.
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10e6 cells found in the negative and/or solvent controls fall within the laboratory historical control range data : 0-45 mutants/10e6 cells.
b) the positive control substances must produce a significant increase in mutant colony frequencies.
c) the colonic efficiency (absolute value) of the negative and/or solvent controls must exceed 50% .

A significant response is described as follows:
The test article is classified as mutagenic
- if it induces reproducibly with at least one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment or
- if there is a reproducible concentration - related increase in the mutation frequency.

Such evaluation may be considered independently of an enhancement factor for induced mutants.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found (5 - 45 mutants per 106 cells) a seemingly concentration-related increase of the mutations within this range will be regarded as to be irrelevant.
Statistics:
No appropriate statistical method is available.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
; reduced plating efficiency >0,10 µg/ml without S9 mix, >3,00µg/ml with S9 (values taken from the pre-experiment)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The study was performed in two independent experiments using identical procedures, both with and without liver microsomal activation.

Table 1: Mutagenity data - Experiment I

Concentration µg/ml

S9

Mean number of mutant colonies per flask

Mutant colonies per 10e6 cells

 

 

 

 

Negative control 0,00

-

 7,8

 33,6

Positive contr.- EMS

-

 151,6

 631,6

Test material 0,03

-

 12,6

 40,9

 0,10

 -

 10,8

 33,8

 0,30

 8,6

 26,9

 1,00

 0,0

 0,0

 2,00

 -

 severe toxic effects

 

 3,00

 -

 severe toxic effects

 

 

 

 

 

Negative control

+

8,8 

 29,5

Negative control-DMSO

+

11,4

 40,2

Positive control - DMBA

+

 67,2

 183,0

Test material 0,10

+

 5,4

 18,0

 0,30

+

 6,6

 22,9

 1,00  +  14,4  37,7
 3,00  +

6,6

 12,5
 6,00  + severe toxic effects  
 10,00  +

severe toxic effects

 

Table 2: Mutagenity data - Experiment II


Concentrationµg/ml

 

S9

Mean number of mutant colonies per flask

Mutant colonies per 10e6 cells

Negative control

-

2,4

8,6

Positive control - EMS

-

220,0

962,4

Test material         0,03

-

13,4

49,5

0,10

-

15,6

48,2

0,30

-

15,0

65,8

1,00

-

6,2

24,1

2,00

-

7,0

22,5

3,00

-

7,4

29,8

 

 

 

 

Negative control

+

8,6

41,3

Negative control- DMSO

+

14,2

65,9

Positive control- DMBA

+

46,8

239,0

Test material        0,10

+

6,0

26,8

0,30

+

10,6

51,2

1,00

+

8,0

27,0

3,00

+

8,8

46,8

6,00

+

6,6

28,9

10,00

+

1,2

4,8

The solvent control for the positive control of the second experiment was excluded because the mutation rate (65.9 mutants/106 cells) exceeded the range of the historic controls (5 - 45 mutants/ 106 cells.). This has no influence on the'result of the test since all evaluations are based on the solvent control.

Conclusions:
Interpretation of results: negative

Under the conditions of this study, NA-11 did not induce gene mutations at the HPRT locus in Chinese hamster V79 cells either with or without metabolic activation.
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 negative results attained in all in vitro genotoxicity studies NA-11 is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [ REGULATION (EC) 1272/2008].