Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Gene Mutation Study in Bacteria
Negative, OECD 471, EU Method B.13/14, Thompson 2012

In vitro Gene Mutation
Negative, dioctyltin oxide, L5178Y TK+/-, OECD 476, EU Method B.17, EPA OPPTS 870.5300, Flanders 2012


In vitro Gene Mutation in Bacteria (Krul, 2002)


Under the conditions of the test, the results obtained with the test material in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and in the Escherichia coli strain WP2uvrA, in both the absence and presence of metabolic activation (S9-mix), the test material was not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
The target substance is a mono-constituent organotin substance that consists of a tin as central metal element with two octyl-ligands. The source substance Dioctyltin oxide (DOTO) (EC Number 212-791-1 and CAS 870-08-6) is also an organotin compound that has the identical structure elements as the target substance in respect of the tin-alkyl moiety.
According to WHO IPCS CIRCAD (2006) organotin compounds are characterized by a tin–carbon bond and have the general formula RxSn(L)(4−x), where R is an organic alkyl or aryl group and L is an organic (or sometimes inorganic) ligand. The organotin moiety is significant toxicologically. The anionic ligand influences physicochemical properties but generally has little or no effect on the toxicology.
Since the target substance and the source substances share the identical organotin moiety, and the organotin moiety is generally recognized as the relevant toxophore of organotins and the toxicity estimates (AE) respectively toxicity limits for organotins are expressed as tin, the overall ecotoxicity/systemic toxicity of the target can be interpolated by assessing the (eco-)toxicity of the source (WHO IPCS CIRCAD, 2006, BAUA AGS TRGS 900, 2014, Summer KH, Klein D and Greim H, 2003).
The purity of the source and target substance are expected to be similar, based on the manufacturing method. The impurity profile is not expected to have strong effects on substance properties and any impurity of (eco-)toxicological relevance of the source substances is expected to be present in the target substance. Consequently, the hazard profiles of the source substances, including those of their impurities, are intrinsically covered. Differences in impurities are not expected and thus do not have an impact on the (eco-)toxic properties.

References
BAUA (Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (Federal Institute for Occupational Safety and Health)) AGS (Ausschuss für Gefahrstoffe (Committee on Hazardous Substances)) TRGS (Technical Rules for Hazardous Substances) 900 (2014). Begründung zu n-Octylzinnverbindungen, April 2014.
Summer KH, Klein D, Griem H (2003). Ecological and toxicological aspects of mono- and disubstituted methyl-, butyl-, octyl-, and dodecyltin compounds - Update 2002. GSF National Research Center for Environment and Health, Neuherberg, for the Organotin Environmental Programme (ORTEP) Association.
World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Concise International Chemical Assessment Document (CICAD) 73 Mono- and disubstituted methyltin, butyltin, and octyltin compounds (2006). Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals
Reason / purpose for cross-reference:
read-across source
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The target substance is a mono-constituent organotin substance that consists of a tin as central metal element with two octyl-ligands. The source substance Dioctyltin oxide (DOTO) (EC Number 212-791-1 and CAS 870-08-6) is also an organotin compound that has the identical structure elements as the target substance in respect of the tin-alkyl moiety.
According to WHO IPCS CIRCAD (2006) organotin compounds are characterized by a tin–carbon bond and have the general formula RxSn(L)(4−x), where R is an organic alkyl or aryl group and L is an organic (or sometimes inorganic) ligand. The organotin moiety is significant toxicologically. The anionic ligand influences physicochemical properties but generally has little or no effect on the toxicology.
Since the target substance and the source substances share the identical organotin moiety, and the organotin moiety is generally recognized as the relevant toxophore of organotins and the toxicity estimates (AE) respectively toxicity limits for organotins are expressed as tin, the overall ecotoxicity/systemic toxicity of the target can be interpolated by assessing the (eco-)toxicity of the source (WHO IPCS CIRCAD, 2006, BAUA AGS TRGS 900, 2014, Summer KH, Klein D and Greim H, 2003).
The purity of the source and target substance are expected to be similar, based on the manufacturing method. The impurity profile is not expected to have strong effects on substance properties and any impurity of (eco-)toxicological relevance of the source substances is expected to be present in the target substance. Consequently, the hazard profiles of the source substances, including those of their impurities, are intrinsically covered. Differences in impurities are not expected and thus do not have an impact on the (eco-)toxic properties.

References
BAUA (Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (Federal Institute for Occupational Safety and Health)) AGS (Ausschuss für Gefahrstoffe (Committee on Hazardous Substances)) TRGS (Technical Rules for Hazardous Substances) 900 (2014). Begründung zu n-Octylzinnverbindungen, April 2014.
Summer KH, Klein D, Griem H (2003). Ecological and toxicological aspects of mono- and disubstituted methyl-, butyl-, octyl-, and dodecyltin compounds - Update 2002. GSF National Research Center for Environment and Health, Neuherberg, for the Organotin Environmental Programme (ORTEP) Association.
World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Concise International Chemical Assessment Document (CICAD) 73 Mono- and disubstituted methyltin, butyltin, and octyltin compounds (2006). Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals
Reason / purpose for cross-reference:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2012 to 03 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Additional strain / cell type characteristics:
other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiments 1 and 2
0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test material was insoluble in dimethyl sulphoxide at 50 mg/mL; it was soluble in dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. Acetone was selected as the most appropriate vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
- EXPERIMENT 1
METHOD OF APPLICATION: in agar (plate incorporation)
2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli), 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation) were thoroughly mixed and the mix was immediately poured onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.05 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
Evaluation criteria:
The mutagenicity study was considered valid if the mean colony counts of the control plates were within acceptable ranges and if the results of the positive controls met the criteria for a positive response. Furthermore, all tester strains must have demonstrated the required characteristics as determined by their respective strain checks. There should be a minimum of four non-toxic test material dose levels and no evidence of excessive contamination.

A test material was considered to be positive if the increase in the mean number of revertant colonies on the test plates was concentration-related or if a reproducible two-fold or more increase was observed compared to that of negative control plates.
A test substance was considered to be negative in the bacterial gene mutation test if it produced neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRE-STUDY CHECKS
The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

PRELIMINARY TOXICITY TEST
The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA). The test material formulation and S9-mix used in the experiment were both shown to be sterile.

MUTATION TEST
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Results for the negative controls were considered to be acceptable. Furthermore, all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1: Experiment 1 - Mean Number of Revertants

Dose (µg/plate)

 

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

0

Mean

115

118

24

12

38

51

35

36

13

12

SD

13.3

14.6

4.5

0.0

4.6

6.4

5.3

6.1

4.4

3.5

50

Mean

103

103

20

9

42

47

32

31

9

12

SD

6.1

8.1

1.0

2.0

1.7

6.2

7.5

2.0

5.9

1.2

150

Mean

112

113

22

10

43

45

24

27

8

12

SD

0.6

2.1

1.7

4.2

4.6

5.5

1.2

6.7

1.0

0.6

500

Mean

99

99

22

12

31

36

35

28

13

8

SD

4.5

4.5

2.6

1.0

5.2

8.5

3.8

10.3

6.0

5.0

1500

Mean

108 P

107 P

23 P

11 P

42 P

41 P

30 P

30 P

12 P

8 P

SD

1.7

4.0

3.5

1.7

5.6

10.5

10.5

4.0

8.0

6.4

5000

Mean

96 P

75 P

22 P

11 P

35 P

41 P

30 P

34 P

10 P

11 P

SD

8.1

6.2

1.5

2.0

3.6

6.1

3.2

2.5

1.2

4.5

Positive Control

Substance (µg/plate)

ENNG

3

2AA

1

ENNG

5

2AA

2

ENNG

2

2AA

10

4NQO

0.2

BP

5

9AA

80

2AA

2

Mean

409

1513

141

288

550

427

137

195

561

278

SD

82.4

77.9

9.9

24.1

28.1

19.4

12.7

3.1

202.6

3.1

 

Table 2: Experiment 2 - Mean Number of Revertants

Dose (µg/plate)

 

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

0

Mean

110

99

22

11

40

39

19

31

12

10

SD

10.6

20.1

4.9

3.8

9.0

6.7

4.0

10.2

4.0

5.2

50

Mean

97

106

23

12

45

50*

19

32

8

12

SD

2.9

10.1

0.6

3.1

0.0

4.6

4.0

7.0

4.6

7.2

150

Mean

100

112

20

10

35

46

30

31

13

8

SD

12.9

11.6

4.6

1.2

4.0

4.2

7.6

6.8

5.5

1.0

500

Mean

108

103

21

15

43

48

16

28

10

11

SD

20.3

5.0

2.0

4.6

11.6

7.0

4.6

4.5

4.0

3.2

1500

Mean

104 P

105 P

14 P

12 P

33 P

46 P

20 P

20 P

12 P

14 P

SD

3.8

11.7

6.6

0.0

2.1

2.1

2.3

1.2

0.0

2.1

5000

Mean

103 P

91 P

20 P

9 P

32 P

52 P*

15 P

24 P

17 P

8 P

SD

14.2

0.6

3.6

2.6

3.0

4.0

8.5

7.8

1.7

3.1

Positive Control

Substance (µg/plate)

ENNG

3

2AA

1

ENNG

5

2AA

2

ENNG

2

2AA

10

4NQO

0.2

BP

5

9AA

80

2AA

2

Mean

466

1474

180

280

596

341

129

669

463

236

SD

24.9

187.6

48.6

29.3

47.4

31.9

17.1

41.4

87.2

19.7

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4 -nitroquinoline-1 -oxide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

BP = benzo(a)pyrene

P = precipitate

* = p≤0.05

SD = standard deviation

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under the conditions of this study, the test material was not mutagenic to S. typhimurium strains TA1535, TA1537, TA98 and TA100 and in the E. coli strain WP2uvrA in both the absence and presence of metabolic activation.
Executive summary:

The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
26 March 2002 to 10 April 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with GLP and in accordance with the standardised guidelines OECD 471 and US EPA OPPTS 870.5100. The study was performed to a high standard sufficient to assess the quality of the presented results. The test material DOTO (di-n-octyltin oxide) is in the same category of substances as the registration substance, as such it is considered acceptable to use as additional information to address this endpoint and to provide support in the read-across approach.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine requirement in the Salmonella typhimurium strains
Tryptophan requirement in the Escherichia coli strain
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: Stored as nutrient broth cultures with 7.4 % DMSO at <60 °C.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Stored as nutrient broth cultures with 7.4 % DMSO at <60 °C.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range finding test: 0, 0.3, 0.8, 2.3, 7, 21, 62, 185, 556, 1667 and 5000 µg/plate.
Definitive test: 0, 62, 185, 556, 1667 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Methanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: N-ethyl-N-nitrosourea and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
2 mL molten top agar (0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium stains, and supplemented with 0.05 mM tryptophane for the E.coli WP2 uvrA strain), maintained at 46 °C. 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test material solution, or the solvent or positive control substance and 0.5 mL S9-mix for the plates with metabolic activation or 0.5 mL sodium phosphate 100 mM (pH 7.4) for without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose).

DURATION
- Exposure duration: 48-72 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
Evaluation criteria:
The mutagenicity study was considered valid if the mean colony counts of the control values of the strains were within acceptable ranges, if the results of the positive controls met the criteria for a positive response, and if no more than 5 % of the plates were lost through contamination or other unforseen events.
A test substance was considered to be positive if the increase in the mean number of revertant colonies on the test plates was concentration-related or if a reproducible two-fold or more increase was observed compared to that of negative control plates.
A test substance was considered to be negative in the bacterial gene mutation test if it produced neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Statistics:
No statistical analysis was performed.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test material was found to precipitate at all concentrations

RANGE-FINDING/SCREENING STUDIES: A dose range finding test was performed with TA 98 in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.3-5000 µg/plate. Dioctyloxostannane (dioctyltin oxide) was not toxic at any concentration both in the absence and presence of S9-mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A slightly more dense background lawn of the bacterial growth was observed at the dose levels at which the slight increases were observed; however, these occured alongside precipitation of the test material.

Table 2: Results of range finding test

Dose µg/plate

TA 98

Without S9-mix

With S9-mix

0

38

52

36

46

29

48

Mean

34

49

SD

5

3

0.3

46

57

48

61

40

46

Mean

45

55

SD

4

8

0.8

46

69

44

57

26

60

Mean

39

62

SD

11

6

2.3

52

52

42

55

34

57

Mean

43

55

SD

9

3

7

27

59

42

59

46

60

Mean

38

59

SD

10

1

21

31

53

27

42

36

47

Mean

31

47

SD

5

6

62

42

56

33

47

40

60

Mean

38

54

SD

5

7

185

45

58

36

63

39

46

Mean

40

56

SD

5

9

556

34

38

39

45

35

40

Mean

36

41

SD

3

4

1667

45

59

44

57

39

68

Mean

43

61

SD

3

6

5000

37

57

41

52

38

50

Mean

39

53

SD

2

4

Positive control

1170

1090

1225

1051

1201

1074

Mean

1199

1072

SD

28

20

*The positive controls employed are detail in table 1.

Table 2: Results of the definitive test

Dose µg/plate

TA 1535

TA 1537

TA 98

TA 100

E. coli

Without S9-mix

With S9-mix

Without S9-mix

With S9-mix

Without S9-mix

With S9-mix

Without S9-mix

With S9-mix

Without S9-mix

With S9-mix

0

15

17

8

22

29

77

165

156

**

31

21

28

13

6

32

51

186

189

23

26

16

15

13

15

27

36

163

175

37

25

Mean

17

20

11

14

29

55

171

173

30

27

SD

3

7

3

8

3

21

13

17

10

3

62

15

20

5

15

16

60

151

170

40

46

18

24

15

14

27

40

148

166

46

39

20

22

12

8

24

51

173

198

40

42

Mean

18P

22

11

12

22

50

153

178

42*

42

SD

3

2

5

4

6

10

14

17

3

4

185

8

23

11

16

25

57

159

181

35

41

23

24

6

4

28

55

149

161

36

34

20

13

4

9

28

56

211

182

31

47

Mean

17P

20

7

10

27

56

173

175

34*

41

SD

8

6

4

6

2

1

33

12

3

7

556

17

20

9

9

31

38

183

144

49

37

30

23

9

8

26

53

168

155

36

33

14

25

8

11

29

35

170

159

30

49

Mean

20P

23

9

9

29

42

174

153

38*

40

SD

9

3

1

2

3

10

8

8

10

8

1667

20

11

5

6

48

31

146

162

33

50

28

20

18

8

35

37

146

152

41

38

22

31

18

12

36

85

150

170

67

27

Mean

23P

21

14

9

40

51

147

161

47*

38

SD

4

10

8

3

7

30

2

9

18

12

5000

31

20

8

11

31

40

153

166

46

49

34

19

7

14

51

42

137

161

46

39

30

28

11

15

26

48

131

161

42

41

Mean

32*

22

9

13

36

43

140

163

45*

43

SD

2

5

2

2

13

4

11

3

2

5

Positive control

442

365

1053

211

1105

634

744

1245

287

698

444

345

863

212

1185

738

704

940

221

677

427

374

913

216

1186

555

708

1012

242

715

Mean

438

361

943

213

1159

642

719

1066

250

697

SD

9

15

98

3

46

92

22

159

34

19

P: Precipitation of test material

* : Precipitation of test material and slightly more dense background lawn of bacterial growth than in concomitant control plates.

**: Not counted; no bacteria on the agar plate

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under the conditions of the test, the results obtained with the test material in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and in the Escherichia coli strain WP2uvrA, in both the absence and presence of metabolic activation (S9-mix), the test material was not mutagenic.
Executive summary:

The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with the OECD guideline 471 and EPA OPPTS 870.5100 and to GLP. The cultures were exposed to 0, 62, 185, 556, 1667 and 5000 µg/plate. Precipitation of the test material was observed at a number of the concentrations tested. In some of the plates a slight dose-dependant increase in the number of revertants was noticed, however this was accompanied by an increased background lawn and precipitation. This was therefore not attributed to the mutagenic potential of the test material. Under the conditions of the study, the results obtained with the test material in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and in the Escherichia coli strain WP2uvrA, in both the absence and presence of metabolic activation (S9-mix), the test substance was not mutagenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April 2012 to 31 July 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with GLP, in accordance with the standardised guidelines OECD 476, EU Method B.17 and US EPA OPPTS 870.5300 and was performed to a high standard. The test material DOTO (di-n-octyltin oxide) is in the same category of substances as the registration substance and it is therefore considered to be acceptable to use a read-across approach to address this endpoint.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK+/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), sodium pyruvate (1 mM), amphoterin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media)
- Properly maintained: Yes. A bank of L5178Y TK+/- cells were stored in a liquid nitrogen freezer. Following removal from liquid nitrogen, the cultures were kept at 37 °C under an atmosphere of 5 % CO2 in air
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test - with and without S9 mix
0, 3.53, 7.05, 14.10, 28.20, 56.41, 112.81, 225.63, 451.25, 902.50 µg/mL

Experiment 1 - with and without S9 mix
0, 3.5, 7, 14, 28, 42, 56, 84, 112 µg/mL test material and 400 µg/mL of ethylmethanesulphonate (EMS) as a positive control in the absence of S9 mix and 2 µg/mL of cyclophosphamide (CP) as a positive control in the presence of S9 mix
acetone as solvent control

Experiment 2 - without S9-mix
0, 0.63, 1.25, 2.5, 5, 7.5, 10, 15, 20 µg/mL test material and 150 µg/mL of ethylmethanesulphonate (EMS) as a positive control
acetone as solvent control

With S9-mix
0, 20, 30, 40, 50, 60, 70, 80, 90 µg/mL test material and 2 µg/mL of cyclophosphamide (CP) as a positive control
acetone as solvent control

Experiment 3 - with S9-mix
0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 µg/mL test material and 2 µg/mL of cyclophosphamide (CP) as a positive control
acetone as solvent control
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
The test material was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test material was formulated within 2 hours of it being applied to the test system. Vehicle and positive controls were used in parallel with the test material. Test material dilutions were dosed at 0.1 mL per culture and positive control solutions were dosed at 0.2 mL per culture.

Exponentially growing suspension cultures of L5178Y cells were treated in duplicate with the solvent control, positive controls or a range of concentrations of the test material for 4 or 24 hours in the presence and/or absence of S9-mix. The cells were then cultured to allow any induced mutations to be expressed. During this expression time the growth rate was monitored and, where appropriate, the cells subcultured daily. At the end of the 48-hour expression time, samples were grown both in selective and non selective medium, and the results obtained used to determine the mutant frequency per viable cell.


DURATION
- Exposure duration: 4 hours (Experiment 1; Experiment 2 with S9 mix and Experiment 3); 24 hours (Experiment 2 without S9 mix)
- Expression time (cells in growth medium): The post-treatment cultures were incubated at 37 °C with 5 % CO2 in air for a 48 hour expression period. To maintain exponential growth during the expression time, each culture was counted and, where appropriate, diluted daily to give approximately 2 x 10^5 cells per mL.
- Selection time (if incubation with a selection agent): On day 2, for the assessment of mutants, a sample of each of the post-expression cultures was diluted to 1 x 10^4 cells per mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine in 96-well microtitre plates.

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: Duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Survival was measured by relative total growth (RTG). RTG is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control.

DETERMINATION OF VIABILITY:
For the assessment of viability, cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

DATA EVALUATION:
Cell growth in individual microwell plates was assessed after 10-14 days using a magnifying mirror box. The survival plates and viability plates were scored for the number of wells containing positive wells (wells with colonies) together with the total number of scorable wells (normally 96 per plate). The numbers of small (colonies less than 25 % of the average area of the large colonies)and large colonies (colonies covering ¼ to ¾ of the surface of the well and generally no more than 2 cells thick)were also recorded. To assist the scoring of the colonies 5-trifluorothymidine (TFT) mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for approximately 2 hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a blue/black colour, aiding visualisation of mutant colonies.

CALCUALTIONS:
Viability calculations were based on P(0), the proportion of wells in which a colony had not grown:

P(0) = number of negative wells/total wells plated

%V = -ln P(0)/number of cells per well

Relative suspension growth (RSG) is defined as the relative total two day suspension growth of the test culture compared to the total two-day suspension growth of the vehicle control. Relative total growth (RTG) is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control. The RSG of each test culture was multiplied by the relative cloning efficiency of the test culture at the time of mutant selection and expressed relative to the cloning efficiency of the vehicle control. The highest concentration assayed was designed to reduce RTG to 10 %-20 % of the solvent control culture values unless limited by solubility, pH or osmolarity effects or a limit concentration of 5 µL/mL, 5000 µg/mL or 10 mM (whichever is the lowest).

MUTANT FREQUENCY (M.F.)
The mutant frequency for each culture was then calculated:

M.F. =-[ ( ln P(0) selective medium) / cells per well in selectibe medium) ] / surviving fraction in non-selective medium
Evaluation criteria:
CRITERIA FOR A POSITIVE RESPONSE
A statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value by the Global Evaluation Factor of 126 x 10^-6, for the microwell method, is required. The assay must also demonstrate a positive linear trend.

CRITERIA FOR A NEGATIVE RESPONSE
A negative response is obtained when there is no reproducible statistically significant dose-related increase in mutant frequency. When a test material induces a modest reproducible increase in mutant frequency that do not exceed to GEF value then scientific judgement is required. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

CRITERIA FOR SCORING MUTATION PLATES
Each well of mutation plates was scored as containing either a small colony or a large colony
Small colony: an average area less than 25 % of the area of the well, usually observed to be more than two cells thick
Large colony: Average coverage ¼ to ¾ of the surface of the well and are generally no more than 2 cells thick
An empty well was one which contained no cell growth.
Statistics:
Mutant frequency experimental data was analysed using the Mutant 240C (York Electronic Research) which follows the statistical guidelines recommended by UKEMS.

The distribution of colony-forming units over the wells is described by the Poisson distribution, viability on day 2 was therefore calculated using the zero term of the Poisson distribution [P(0)].
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Test Material solubility: The molecular weight of the test material was 361, therefore, the maximum proposed dose level in the solubility test was initially set at 3610 µg/mL, the 10 mM limit dose. However, due to formulation difficulties, the maximum achievable dose level suitable for dosing was set at 1805 µg/mL. Acetone is toxic to L5178Y at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test material was formulated at 180.5 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 902.5 µg/mL. The purity of the test material was accounted for when the dosing solutions were formulated.
- Precipitation: Precipitate of the test material was observed at and above 28.20 µg/mL in both the 4 hour exposure groups and at 56.41 µg/mL and above in the 24 hour exposure group of the preliminary toxicity test. In experiment 1, precipitate of the test material was observed at 28 µg/mL and above, at 70 µg/mL and above in experiment 2 and at 60 µg/mL and above in experiment 3.
- pH and osmolality: The effect of the test material on the pH and osmolarity of the treatment medium was investigated. There were no marked change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm in the solubility test.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period with and without metabolic activation (20 % S9-mix), and at 1.5 x 10^5 cells/mL using a 24 hour exposure period without metabolic activation. The dose range used in the preliminary toxicity test was 3.53 to 902.5 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10 medium, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 hours by counting and diluting to 10^5 cells/mL. After a further 24 hours, the cultures were counted and discarded. The cell counts were used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post tratment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control was performed to give a percentage Relative Suspension Growth (%RSG) value. Results from the preliminary toxicity test were used in conjuction with evaluations of the solubility of the test material to determine the concentrations used in the mutagenicity tests.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Preliminary toxicity test: In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. Precipitate of the test material was observed at and above 28.20 µg/mL in both of the 4 hour exposure groups, and at and above 56.41 µg/mL in the 24 hour exposure group. Based on the %RSG values observed, the maximum dose levels in the subsequent mutagenicity test were limited by test material-induced toxicity.

- Experiment 1: There was evidence of marked dose-related toxicity following exposure to the test material in both the absence and presence of S9 mix as indicated by the RTG, %RSG and %V values. The levels of toxicity observed were similar to those noted during the preliminary toxicity test. It should, however, be noted that the reductions were only observed at dose levels that had been excluded from the statistical analysis due to the toxicity exceeding the upper limit of 90 %. Excessive toxicity was noted at 112 µg/mL in both the absence and presence of metabolic activation resulting in this dose level not being plated for viability or 5-TFT resistance.

- Experiment 2: There was evidence of marked toxicity following exposure to the test material in the absence of metabolic activation, even more marked for the extended 24 hour exposure, as indicated by the RTG and %RSG values. The expected levels of toxicity were not achieved in the presence of metabolic activation and only modest levels of toxicity were achieved. It was considered that this may have been due to the lowering of the S9 concentration to 1 % in this experiment compared to the 2 % S9 concentration in the preliminary toxicity test and in experiment 1. The most marked reduction in the presence of metabolic activation was observed at the penultimate dose level and the RTG value appeared to increase slightly at the maximum dose level indicating that maximum exposure had been achieved due to the presence of precipitate effectively reducing exposure of the test material to cells. It was therefore considered appropriate to perform a confirmatory Experiment 3 using the same exposure conditions and a narrower dose interval in the presence of metabolic activation.There was no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation. The excessive toxicity observed at and above 15 µg/mL in the absence of metabolic activation, resulted in these dose levels not being plated for viability or 5 -TFT resistance.

-Experiment 3: As was seen in Experiment 2, the levels of toxicity observed, as indicated by the RTG and %RSG values were very modest and differed from those of the preliminary toxicity test and Experiment 1 where 2 % S9 had been used. The levels of toxicity observed were very similar to those observed in the presence of 1 % metabolic activation in Experiment 2. The most marked toxicity was once again observed at the penultimate dose level and a much greater increase in %RSG and RTG was observed at the maximum dose level. This was taken to confirm that maximum exposure to the test material to the cells had been achieved due to the presence of precipitate effectively reducing exposure of the test material to the cells. This also confirmed that the difference in toxicity observed in the preliminary toxicity and Experiment 1 was caused by the lowering of the S9 concentration from 2 % to 1 %. The test material was therefore considered to have been adequately tested.

Mutagenicity Test

- Experiment 1:The test material did not induce any statistically significant or dose related increases in the mutant frequency x 10^-6 per viable cell either in the absence or presence of metabolic activation. A very modest but statistically significant dose related increase in mutant frequency was observed in the presence of metabolic activation, at one dose level, however, the GEF was not exceeded and there was no evidence of any marked increase in the absolute number of mutant colonies. In both the presence and absence of metabolic activation, a marked increase in mutant frequency was observed in the dose level that was plated out that exceeded the upper limit of acceptable toxicity. However, there was again no evidence of an increase in absolute numbers of mutant colonies, there was no evidence of a shift towards small colony formation that would have indicated a clastogenic response, and the viability values were low resulting in a falsely high mutant frequency value. The increases in mutant frequency were therefore considered to be due to cytotoxicity and not a true genotoxic response and were, therefore considered artefactual and of no toxicological significance.

Table 1: Experiment 1 Summary of Results

Treatment (µg/mL)

4 hours -S9

Treatment (µg/mL)

4 hours +S9

% RSG

RTG

MF §

% RSG

RTG

MF §

0

100

1.00

129.11

0

100

1.00

134.75

3.5 Ø

96

3.5 Ø

88

7

102

1.02

145.06

7

99

1.05

145.87

14

88

0.80

048.54

14

83

0.90

156.85

28

73

0.78

134.95

28

68

0.86

129.35

42

78

0.77

143.34

42

61

0.66

175.58

56

34

0.35

146.99

56

34

0.30

215.14 *

84 X

9

0.01

540.45

84 X

15

0.04

318.87

112 Ø

2

112 Ø

5

Linear trend

NS

Linear trend

*

EMS (400)

77

0.49

1081.21

CP (2)

48

0.29

1833.97

-Experiment 2:The test material did no induce any statistically significant or dose related increases in the mutant frequency x 10^-6 per viable cell in ether the absence or presence of metabolic activation.

Table 2: Experiment 2 Summary of Results

Treatment (µg/mL)

4 hours -S9

Treatment (µg/mL)

4 hours +S9

% RSG

RTG

MF §

% RSG

RTG

MF §

0

100

1.00

175.05

0

100

1.00

162.07

0.63

90

0.92

151.31

20 Ø

89

1.25

77

0.75

189.17

30 Ø

94

2.5

69

0.88

152.31

40

99

0.91

149.64

5

38

0.59

150.95

50

87

0.73

172.61

7.5

20

0.36

142.04

60

81

0.71

171.65

10

17

0.29

158.60

70

80

0.63

159.50

15 Ø

3

80

57

0.46

174.73

20 Ø

0

90

59

0.55

139.04

Linear trend

NS

Linear trend

NS

EMS (150)

49

0.52

1260.54

CP (2)

64

0.34

2212.67

-Experiment 3:The test material did not induce any statistically significant or dose related increases in the mutant frequency x 10 ^-6 per viable cell in the presence of metabolic activation. precipitate of the test material was observed at and above 60 µg/mL.

Table 3: Experiment 3 Summary of Results

Treatment (µg/mL)

4 hours +S9

% RSG

RTG

MF §

0

100

1.00

126.04

20 Ø

104

30 Ø

94

40 Ø

104

50 Ø

85

60

82

0.91

112.86

70

71

0.71

139.88

80

78

0.77

137.90

90

62

0.63

139.56

100

52

0.54

150.21

110

69

0.88

101.91

Linear trend

NS

CP (2)

75

0.46

1429.48

In all of the Experiments, none of the vehicle control mutant frequency values were outside the acceptable range and all positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Key to tables

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

CP = cyclophosphamide

EMS = ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/ 10^6 viable cells 2 days after treatment

Ø = not plated for viability or 5-TFT resistance

X = treatment excluded from test statistics due to toxicity

NS = Not Significant

* = P < 0.05

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under the conditions of the assay, the test material was determined not to be mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L5178Y TK +/- mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxiliary metabolic system (S9 mix). Large and small mutant colonies were scored for all cultures in each experiment.

Initially, two independent experiments were performed. In Experiment 1, cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation. However, due to a marked difference in toxicity in the 4-hour exposure groups in the presence of metabolic activation between Experiment 1 and 2, and an apparent maximum exposure being achieved at the penultimate dose level in Experiment 2, a confirmatory Experiment 3 was performed using a 4-hour exposure group at ten dose levels in the presence of metabolic activation (1 % S9) only.

 

The dose range of the test material was selected following the results of a preliminary toxicity test and was 3.5 to 112 µg/mL in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 0.63 to 20 µg/mL in the absence of metabolic activation, and 20 to 90 µg/mL in the presence of metabolic activation. In Experiment 3 the dose range was 20 to 110 µg/mL in the presence of metabolic activation only.

 

Under the conditions of the test, the maximum dose levels used in the mutagenicity test were limited by test material-induced toxicity. Overall, precipitate of test material was observed at and above 28 µg/mL in the mutagenicity test. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK+/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first, second or third experiment. The test material is therefore considered to be non-mutagenic to L5178Y cells under the conditions of this assay.

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

Genetic toxicity in vivo

Description of key information

In vivo Clastogenicity Study
Negative, dioctyltin oxide, OECD 474, de Vogel 2004

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The target substance is a mono-constituent organotin substance that consists of a tin as central metal element with two octyl-ligands. The source substance Dioctyltin oxide (DOTO) (EC Number 212-791-1 and CAS 870-08-6) is also an organotin compound that has the identical structure elements as the target substance in respect of the tin-alkyl moiety.
According to WHO IPCS CIRCAD (2006) organotin compounds are characterized by a tin–carbon bond and have the general formula RxSn(L)(4−x), where R is an organic alkyl or aryl group and L is an organic (or sometimes inorganic) ligand. The organotin moiety is significant toxicologically. The anionic ligand influences physicochemical properties but generally has little or no effect on the toxicology.
Since the target substance and the source substances share the identical organotin moiety, and the organotin moiety is generally recognized as the relevant toxophore of organotins and the toxicity estimates (AE) respectively toxicity limits for organotins are expressed as tin, the overall ecotoxicity/systemic toxicity of the target can be interpolated by assessing the (eco-)toxicity of the source (WHO IPCS CIRCAD, 2006, BAUA AGS TRGS 900, 2014, Summer KH, Klein D and Greim H, 2003).
The purity of the source and target substance are expected to be similar, based on the manufacturing method. The impurity profile is not expected to have strong effects on substance properties and any impurity of (eco-)toxicological relevance of the source substances is expected to be present in the target substance. Consequently, the hazard profiles of the source substances, including those of their impurities, are intrinsically covered. Differences in impurities are not expected and thus do not have an impact on the (eco-)toxic properties.

References
BAUA (Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (Federal Institute for Occupational Safety and Health)) AGS (Ausschuss für Gefahrstoffe (Committee on Hazardous Substances)) TRGS (Technical Rules for Hazardous Substances) 900 (2014). Begründung zu n-Octylzinnverbindungen, April 2014.
Summer KH, Klein D, Griem H (2003). Ecological and toxicological aspects of mono- and disubstituted methyl-, butyl-, octyl-, and dodecyltin compounds - Update 2002. GSF National Research Center for Environment and Health, Neuherberg, for the Organotin Environmental Programme (ORTEP) Association.
World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Concise International Chemical Assessment Document (CICAD) 73 Mono- and disubstituted methyltin, butyltin, and octyltin compounds (2006). Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals
Reason / purpose for cross-reference:
read-across source
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 October 2003 to 11 December 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted in accordance with the standardised testing guideline OECD 474 and in line with GLP with no deviations thought to affect the quality of the presented data. The study was reported to a high standard. The test material DOTO (di-n-octyltin oxide) is in the same category of substances as the registration substance and it is therefore considered to be acceptable to use a read-across approach to address this endpoint.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: Approximately 34 g
- Fasting period before study: 2 hours and 45 minutes in the range finding test and 3 hours and 30 minutes in the main test.
- Diet: ad libitum
- Water: Tap water, ad libitum delivered in polypropylene bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): At least 30 % not exceeding 70 %
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 4 November 2003 To: 7 November 2003
MAIN TEST IN-LIFE DATES: From: 25 November 2003 To: 28 November 2003
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 100, 50 and 25 mg/mL
- Amount of vehicle: 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The day before dosing, the test material was suspended in corn oil at a stock concentration of 100 mg/mL and stirred overnight on a magnetic stirrer. The two lower concentrations of 50 and 25 mg/mL were prepared prior to dosing on day 0.
Duration of treatment / exposure:
One treatment
Frequency of treatment:
A single exposure was used
Post exposure period:
At 24 hours after treatment, 5 animals of each dose level of the test material, 5 negative control animals and 5 positive control animals were sacrificed by cervical dislocation. After 48 hours the remaining animals of the high dose and and the negative control group were sacrificed.
Remarks:
Doses / Concentrations:
0 (A), 500 (B), 1000 (C) and 2000 (D) mg/kg bw
Basis:
nominal conc.
MAIN TEST
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
Range Finding Test
No. of animals per sex per dose:
In the range finding test, 2 males and 2 females were used in each dose group to determine the toxicity of the test material.
In the main test, 10 male animals were used in each of the high dose group and the negative control group and 5 in all other dose groups and the positive control.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.75 mg/kg bw (10 mL/kg bw) in saline
Tissues and cell types examined:
From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears. Two smears per animal were prepared.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels and sex chosen for the main test were based on the clinical signs observed during the dose range finding test. The range-finding test demonstrated an absence of toxicity and no sex difference, therefore, the main test was carried out with male mice only and graded dose levels up to the limit dose of the guideline were used.

DETAILS OF SLIDE PREPARATION: The smears were air-dried and fixed in methanol. One smear per animal was stained with May-Grünwald Giemsa solution. The pther slide was stored as a reserve slide.

METHOD OF ANALYSIS: The slides were read by moving from the begining of the smear to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE were scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was therefore recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
Evaluation criteria:
The study was considered to be valid if the positive controls gave a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were in the historical range.
A response was considered positive if the mean number of MPE/2000 PE was statistically significantly higher in comparison to the vehicle controls.
The test material was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if a clear dose-related increase in the mean numbers of MPE/2000 PE was observed, when compared to the vehicle controls.
A test material was considered to be negative if it produced no positive response at any of the doses and time points analysed.
The test material or its metabolites was considered to have reached the general circulation and thereby the bone marrow, if the test material statistically reduced the mean number of PE/E or caused systemic toxicity.
Both statistical significance and biological relevance were considered when evaluating the responses.
Statistics:
At 24 hours, data on MPE and PE were evaluated using One Way Anova with a factor group (A, B, C and D). If the Anova demonstrated a significant effect (p<0.05), pooled error variance t-tests were performed, or if the variances were not homogenous, separate variance t-tests. These t-tests were applied to the negative control group (A) versus treatment groups B (500), C (1000) and D (2000). Furthermore, the positive control group E and the negative control group A were compared using the same process. A linear trend test (orthogonal contrasts) was also applied across groups A-D.
At 48 hours after administration, for treatment groups A and D, data on MPE and PE were analysed using pooled error variance t-tests, or if variances were not homogenous, separate variance t-tests.
All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 and 2000 mg/kg bw, dosed as 20 mL/kg bw
- Clinical signs of toxicity in test animals: Female 3 died 24 hours post dosing due to a failed oral administration. No severe clinical signs were observed as a results of test material administration.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: At both sacrifice times, the two way ANOVA did not demonstrate a statisitically significant effect for MPE and PE. There was no observable genotoxicity or clastogenicity up to a dose of 2000 mg/kg bw. At 24 hours, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the vehicle control. The test system was therefore considered to be valid.

Table 1: Results of range finding test

Test material concentration

1 h post administration

4 h post administration

24 h post administration

48 h post administration

Animal number and sex

500 mg/kg bw

sl; se; br

sl; se; p; br; †

Male 2

Male 4

Female 1

Female 3

1000 mg/kg bw

Male 6

Male 8

Female 5

Female 7

2000 mg/kg bw

Male 10

Male 12

Female 9

Female 11

Empty cells = no clinical signs observed

sl = Sluggishness

se = slit-eyes

p = piloerection

br = irregular breathing

† = animal died

 

Table 2: Results of the main test

Group

Bodyweight (g)

Mouse no.

Observation time (h)

PE

NE

MPE

MNE

A

36.1

2

24

87

113

2

0

32.2

4

24

74

126

2

0

35.7

6

24

93

107

3

0

37.6

8

24

113

87

1

0

32.7

10

24

79

121

2

0

34.5

12

48

98

102

1

1

36.0

14

48

96

104

2

0

35.6

16

48

93

107

2

0

32.8

18

48

79

121

4

0

35.1

20

48

74

126

2

0

34.83 ± 0.56

Mean ± S.D.

24

89.2 ± 15.2

 

2.0 ± 0.7

48

88.0 ± 10.8

 

2.2 ± 1.1

B

33.1

22

24

101

99

3

0

34.7

24

24

63

137

1

0

33.8

26

24

89

111

2

0

35.6

28

24

81

119

4

0

34.7

30

24

92

108

3

0

34.38 ± 0.43

Mean ± S.D.

 

85.2 ± 14.3

 

2.6 ± 1.1

C

34.6

42

24

77

123

2

0

32.8

44

24

90

110

2

0

35.7

46

24

109

91

2

0

33.9

48

24

77

123

2

0

33.6

50

24

102

98

1

0

34.12 ± 0.49

Mean ± S.D.

 

91.0 ± 14.4

 

1.8 ± 0.4

D

36.3

62

24

66

134

2

0

34.7

64

24

111

89

2

0

31.5

66

24

81

119

1

0

33.6

68

24

99

101

1

1

33.7

70

24

103

97

1

0

37.8

72

48

83

117

1

0

33.8

74

48

77

123

2

0

33.2

76

48

73

127

2

0

33.3

78

48

74

126

2

0

34.3

80

48

69

131

0

0

34.22 ± 0.55

Mean ± S.D.

24

92.0 ± 18.2

1.4 ± 0.5

48

75.2 ± 5.2

1.4 ± 0.9

E

33.8

82

24

81

119

50

0

34.1

84

24

78

122

64

0

32.3

86

24

66

134

57

1

34.4

88

24

74

126

40

0

33.9

90

24

86

114

41

0

33.70 ± 0.36

Mean ± S.D.

 

77.0 ± 7.6

50.4 ± 10.3*

 

A = Vehicle control (corn oil)

B = 500 mg/kg bw test material

C = 1000 mg/kg bw test material

D = 2000 mg/kg bw test material

E = Mitomycin C 0.75 mg/kg bw in saline i.p. injection

Body weight = Bodyweight prior to dosing

PE = number of PE scored per 200 E scored

NE = number NE scored per 200 E scored

MPE = number of MPE scored per 2000 PE scored

MNE = number MNE scored per number of NE scored

* P<0.001 (t-tests) n = 5

 

Conclusions:
Interpretation of results: negative
Under the conditions of the study, the test material was found not to produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow cells of mice.
Executive summary:

The potential genotoxicity and clastogenicity of the test material to the bone marrow cells of Swiss male mice in vivo was assessed in a study conducted in accordance with OECD 474 and to GLP. At oral doses up to 2000 mg/kg bw (the limit dose recommended by the guideline) no chromosomal damage or damage to the mitotic spindle apparatus was noted. Under the conditions of the study the test material was found to be non-genotoxic and non-clastogenic.

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

Additional information

In Vitro Gene Mutation Study in Bacteria


In the key study, the mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.


 


The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control. Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.


 


The study was both performed and reported to a high standard. As such it was deemed acceptable to assign the study a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997).


 


Supporting information is available on the structural analogue dioctyltin oxide (DOTO). In the supporting study, the mutagenicity of DOTO was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with the OECD guideline 471 and EPA OPPTS 870.5100 and to GLP. The cultures were exposed to 0, 62, 185, 556, 1667 and 5000 µg/plate. Precipitation of the test material was observed at a number of the concentrations tested. In some of the plates, a slight dose-dependant increase in the number of revertants was noticed; however this was accompanied by an increased background lawn and precipitation. This was therefore not attributed to the mutagenic potential of the test material. Under the conditions of the study, the results obtained with the test material in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and in the Escherichia coli strain WP2uvrA, in both the absence and presence of metabolic activation (S9-mix), the test material was not mutagenic. As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with acceptable restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997).


 


In vitro Gene Mutation Study


The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L5178Y TK+/- mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxiliary metabolic system (S9 mix). Large and small mutant colonies were scored for all cultures in each experiment. Initially, two independent experiments were performed. In Experiment 1, cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation. However, due to a marked difference in toxicity in the 4-hour exposure groups in the presence of metabolic activation between Experiment 1 and 2, and an apparent maximum exposure being achieved at the penultimate dose level in Experiment 2, a confirmatory Experiment 3 was performed using a 4-hour exposure group at ten dose levels in the presence of metabolic activation (1 % S9) only.


 


The dose range, selected following a preliminary toxicity test, was 3.5 to 112 µg/mL in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 0.63 to 20 µg/mL in the absence of metabolic activation and 20 to 90 µg/mL in the presence of metabolic activation. In Experiment 3 the dose range was 20 to 110 µg/mL in the presence of metabolic activation only.


 


Under the conditions of the test, the maximum dose levels used in the mutagenicity test were limited by test material-induced toxicity. Overall, precipitate of test material was observed at and above 28 µg/mL in the mutagenicity test. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK+/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first, second or third experiment. The test material is therefore considered to be non-mutagenic to L5178Y cells under the conditions of this assay.


 


As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with acceptable restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.  


 


In vivo Cytogenicity Study


The potential genotoxicity and clastogenicity of the test material to the bone marrow cells of Swiss male mice was assessed in vivo in a study conducted in accordance with OECD 474 and to GLP. At oral doses up to 2000 mg/kg bw (the limit dose recommended by the guideline) no chromosomal damage or damage to the mitotic spindle apparatus was noted. Under the conditions of the study the test material was found to be non-genotoxic and non-clastogenic.


 


As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with acceptable restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.



Justification for selection of genetic toxicity endpoint
As multiple studies are presented to address genetic toxicity, no one study was selected as the key study as they represent different types of genetic toxicity and are therefore not comparable.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification and labelling as defined in Regulation (EC) No. 1272/2008 (CLP) the test material does not require classification for genetic toxicity.