Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A mammalian cell gene mutation assay was performed in accordance with OECD Guidelines for the testing of chemicals No. 476 and EU method B17 in TK +/- locus, L5178Y cells.
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in both the presence and absence of metabolic activation and is therefore considered to be non-mutagenic under the conditions of the test.

A bacterial reverse mutation assay (Ames test) was performed in S. typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 in accordance with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Annex V of EEC Commission Directive 84/449/EEC.
The test material was found to be non-mutagenic under the conditions of this test, in both the presence and absence of metabolic activation.

An in vitro chromosome aberration test in human lymphocytes was performed, the method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.
The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system. It was therefore considered to be non-clastogenic to human lymphocytes 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:
key study
Study period:
14 February 1989 - 3 March 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. 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.
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
other: Target substance
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine locus
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538 and TA98
Additional strain / cell type characteristics:
other: TA1535 and TA100 are sensitive to agents inducing base-pair substitutions. TA1537, TA1538 and TA98 are sensitive to agents inducing frame-shift mutations.
Metabolic activation:
with and without
Metabolic activation system:
S9. Prepared from the livers of male Sprague-Dawley rats that had each recieved a single i.p. injection of Aroclor 1254 at 500 mg/kg 5 days before S9 preparation.
Test concentrations with justification for top dose:
Experiment 1 ± S9: 0, 8, 40, 200, 1000, 5000 µg/plate
Experiment 2 ± S9: 0, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
The test material was accurately weighed and dissolved in sterile distilled water and appropriate dilutions made.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
A solvent treatment group was used as the negative control
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG); 9-Aminoacridine (9AA); 4-Nitro-O-phenylenediamine (4NOPD) and 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Test Procedure
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 mL of bacterial suspension (TA100) and 0.L ml of test solution were added to 2 mL of molten, trace histidine supplemented media (histidine/biotin & top agar) and overlayed onto sterile plates of Vogel-Bonner agar (minimal agar - 25 mL/ plate). Five doses of the test compound and a solvent control (distilled water) were tested in duplicate. After 48 - 72 hours incubation the plates were scored for revertant colonies and examined for a thinning of the background lawn.
b) Mutation Study
EXPERIMENT 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using microorganisms.
Test Material and Negative Controls
0.1 mL of the appropriately diluted test material or negative control solution was placed in sets of sterile test tubes containing 2.0 mL of molten, trace histidine supplemented, top agar at 45°C. These sets comprised of two test tubes for each bacterial tester strain. A 0.1 mL aliquot of one of the bacterial suspensions was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 liver microsome mix; in the other tube 0.5 mL of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
Positive Controls
Without Activation
0.1 mL of one of the positive control solutions (MNNG, 9AA, or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar. 0.1 mL of the appropriate bacterial suspension and 0.5 mL of pH 7.4 buffer was also added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.
With Activation
0.1 mL of 2AA solution was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar. 0.1 mL of one of the test bacterial suspensions and 0.5 mL of S9 mix were also added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.

The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37°C for at least 48 hours and the number of revertant colonies counted.
EXPERIMENT 2
The complete experiment was repeated using fresh bacterial cultures, test material and control solutions.
Statistics:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
a) Preliminary Toxicity Study
The dose range of Substance 1658/5 used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was nontoxic in the strain of Salmonella used (TA100).
b) Mutation Study
Checks were done on viability and spontaneous reversion rate for each tester strain. The overnight culture of each strain was found to be in the required range of 10^7 – 10^9 bacteria per mL and the spontaneous reversion rate for each was found to be within the expected range.
No toxicity was exhibited to any of the strains of Salmonella used.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella, at any dose level used, either with or without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1

 

Mutation Study Mean and Individual Plate Counts for Test Material and Negative Controls

Strain of Salmonella typhimurium

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

 

 

 

 

 

 

TA1535

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

14

18

13

15

20

15

 

11

12

13

15

10

13

 

 

 

 

 

 

TA1537

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

6

4

3

3

6

5

 

9

8

9

10

7

9

 

 

 

 

 

 

TA1538

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

8

13

11

9

8

11

 

16

18

14

19

20

18

 

 

 

 

 

 

TA98

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

17

23

15

17

19

20

 

25

29

20

26

21

28

 

 

 

 

 

 

TA100

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

85

84

95

89

93

98

 

94

93

102

104

102

85

 

Experiment 2

Mutation Study Mean and Individual Plate Counts for Test Material and Negative Controls

Strain of Salmonella typhimurium

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

 

 

 

 

 

 

TA1535

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

11

11

13

10

10

10

 

15

10

12

13

17

13

 

 

 

 

 

 

TA1537

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

7

8

8

7

5

8

 

9

11

8

8

9

11

 

 

 

 

 

 

TA1538

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

10

8

9

13

14

13

 

14

10

13

11

11

15

 

 

 

 

 

 

TA98

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

17

15

16

16

15

20

 

23

25

24

24

21

26

 

 

 

 

 

 

TA100

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

87

74

72

82

88

81

 

93

85

93

90

86

94

 

Positive Controls

Experiment 1

Mean and Individual Plate Counts for Concurrent Positive Controls

Strain of Salmonella typhimurium

Test Material

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

TA1535

TA1537

TA1538

TA98

TA100

TA1535

TA1537

TA1538

TA98

TA100

MNNG

9AA

4NOPD

4NOPD

MNNG

2AA

2AA

2AA

2AA

2AA

2

50

10

10

2

3.3

3.3

3.3

3.3

3.3

                -

-

-

-

-

+

+

+

+

+

45

316

655

751

519

65

41

241

551

970

 

Experiment 2

Mean and Individual Plate Counts for Concurrent Positive Controls

Strain of Salmonella typhimurium

Test Material

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

TA1535

TA1537

TA1538

TA98

TA100

TA1535

TA1537

TA1538

TA98

TA100

MNNG

9AA

4NOPD

4NOPD

MNNG

2AA

2AA

2AA

2AA

2AA

2

50

10

10

2

3.3

3.3

3.3

3.3

3.3

                -

-

-

-

-

+

+

+

+

+

103

401

509

678

394

124

219

335

375

558

 

REFERENCES

Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.D. Proc. Nat. Acad. Sci. USA (1970), 70, 2285

Ames, B.N., McCann, J. and Yamasaki, E., Mutation Research (1975), 31, 347.

McCann, J., Coi, E., Yamasaki, E., and Ames, B.N. Proc. Nat. Acad. Sci. USA (1975) 75, 5135.

Garner, R.C., Miller, E.C., and Miller, J.A. Cancer Res. (1972), 33, 2058.

Conclusions:
Interpretation of results (migrated information):
negative

The test material was found to be non-mutagenic under the conditions of this test.
Executive summary:

The test material, SUBSTANCE 1658/5, was found to be non-mutagenic under the conditions of this test, in both the presence and absence of metabolic activation. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Annex V of EEC Commission Directive 84/449/EEC.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 1995 - 17 December 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. 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.
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
other: Target substance
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
Sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation, hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell cycle time for the lymphocytes from the donor used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (ACT). Using the current batch of foetal calf serum the donor used in this study has an AGT of approximately 12.5 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared in-house, from the livers of male Sprague-Dawley rats that had received a single i.p injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
Test concentrations with justification for top dose:
The concentrations of test material were 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL both with and without S9 .
Vehicle / solvent:
The test material was accurately weighed, suspended in Minimal Essential Media (MEM) and serial dilutions prepared. The maximum dose level was 5000 µg/mL which was the maximum recommended dose level.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulphonate (EMS) 500 µg/mL for cultures in the absence of metabolising enzymes. It was dissolved in dimethyl sulphoxide. Cyclophosphamide (CP) 25 µg/mL for cultures where S9 was included. It was dissolved in culture medium without serum.
Details on test system and experimental conditions:
Cell Culture
Cells were grown in Eagle's minimal essential medium, (supplemented with sodium bicarbonate, L-glutamine, penicillin streptomycin, amphotericin B and 15% foetal calf serum) at 37°C with 5% CO2 in air. The lymphocytes in fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/ml final concentration.

Preparation of Test and Control Materials
The test material was accurately weighed, suspended in Minimal Essential Media (MEM) and serial dilutions prepared. The maximum dose level was 5000 µg/ml which was the maximum recommended dose level.
Vehicle and positive controls were used in parallel with the test material. The positive control materials were as follows:
Ethyl methanesulphonate (EMS) 500 µg/mL for cultures in the absence of metabolising enzymes. It was dissolved in dimethyl sulphoxide.
Cyclophosphamide (CP) 25 µg/mL for cultures where S9 was included. It was dissolved in culture medium without serum.

Microsomal Enzyme Fraction
Lot No. Aro. S9/31/05/95 SPL for Experiment 1 and Aro. S9/11/10/95A for Experiment 2 were prepared in-house, from the livers of male Sprague-Dawley rats weighing ~200g. These had received a single i.p injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.

Culture Conditions - Experiment 1
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving when dispensed in sterile plastic flasks for each dose level.
8.05 - 9.05 ml MEM, 15% (FCS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin-M
0.75 mL heparinised whole blood

After 48 hours incubation at 37°C, 5% CO2 in humidified air, the cells with-S9 cultures were centrifuged and all but approximately 1 mL of the culture medium removed, reserved and replaced with MEM. 1.0 mL of the appropriate solution of vehicle or test material was added to each culture or 0.1 mL of the positive control. The final concentrations of test material were 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL in both with and without S9 cultures. 1 mL of 10% S9 in standard co-factors was added to the with-S9 cultures. All the cultures were then returned to the incubator.

After 4 hours of treatment at 37°C the with-S9 cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 16 hours.

Culture Conditions - Experiment 2
These were as above except that further cultures of lymphocytes were harvested 44 hours after the initiation of treatment as well as a 20-hour harvest in Experiment 1. Positive controls were evaluated only in the 20-hour harvest cultures.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 8 mL 0.075M hypotonic KCI. After fifteen minutes (including five minutes centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCI suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4°C for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and cover-slipped using mounting medium.

Coding
After checking that the slide preparations were of good quality, the slides were coded using a computerised random number generator.
Evaluation criteria:
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 46 or more chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides.
Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using fisher's Exact test or Chi-squared test.
Species / strain:
other: human lymphocytes
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:
Chromosome Aberration Test - Experiment 1

Treatment Final concentration of Flamtard H (µg/mL)
20h -S9 0*, 39, 78.1, 156.25*, 312.5*, 625*, 1250, 2500, 5000
20h +S9 0*, 39, 78.1, 156.25*, 312.5*, 625*, 1250, 2500, 5000
* dose levels selected for metaphase analysis

The test material formed a precipitate which increased with dose level. The 1250, 2500 and 5000 µg/ml dose levels were unscorable due to the precipitate obscuring the metaphase cells. The maximum scorable dose level was 625 µg/mL in both treatment cases. The mitotic index data demonstrated some evidence of toxicity in the 20-hour group with S9-mix at 625 µg/mL, however this may have been due to the precipitate obscuring the metaphases because it was not replicated in Experiment 2.

The vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. Except for the with S9-mix control which was slightly outside the range for cells with gaps only, however, this was considered not to affect the integrity of the study.

The positive control treatments gave statistically significant increases in the frequency of cells with aberrations, indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.

The test material was seen to induce no statistically significant or dose-related increases in the frequency of cells with aberrations either in the presence or absence of S9.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment case.

Chromosome Aberration Test - Experiment 2

Treatment Final concentration of Flamtard H (µg/mL)
20h -S9 0*, 78.1, 156.25*, 312.5*, 625*
20h +S9 0*, 78.1, 156.25*, 312.5*, 625*
44h -S9 0*, 156.25, 312.5, 625*
44h +S9 0*, 156.25, 312.5, 625*
* dose levels selected for metaphase analysis

The dose selection was as in Experiment 1. The mitotic index data demonstrated a dose-related increase in toxicity in the 20-hour treatment without S9 which was not observed in Experiment 1 which suggests that it may have been related to the precipitate of test material obscuring the metaphase cells.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.

The positive control treatments gave significant increases in the frequency of cells with aberrations, indicating that the metabolic activation system was satisfactory and that the test material itself was operating as expected. The response to cyclophosphamide was relatively poor but was within the expected range and therefore considered to be acceptable.

The test material was seen to induce no significant increase in the frequency of cells with chromosome aberrations either in the presence or absence of S9 mix in either the 20 or 44-hour treatment groups. There were insufficient good quality metaphases to score the full compliment of 200 cells at 625 µg/mL in the 44-hour group without S9 mix, however, sufficient cells were scored for an adequate assessment to be made.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the treatment cases.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

REFERENCES

Savage, J.R.K. (1976) Annotation: Classification and relationships of induced chromosomal structural changes. J. Med. Genet., 13, 103 - 122.

 

Scott, D., Dean, B.J., Danford, N.D., and Kirkland, D.J.: Metaphase chromosome aberration assays in vitro. !n: Kirkland, D.J. Basic mutagenicity tests: UKEMS recommended procedures. Report. Part 1 revised. Cambridge University Press, 1990:62-84.

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system. Flamtard H was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations or polyploid cells in either the presence or absence of a liver enzyme metabolising system. It was therefore considered to be non-clastogenic to human lymphocytes in vitro. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

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:
22 April 2008 to 02 June 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with GLP to appropriate OECD and EU study guideline. Although the study is conducted upon the Zinc Hydroxystannate it is anticipated that the substances would behave identically in the aqueous environment, none the less the reliability rating of the study has been adjusted accordingly.
Reason / purpose:
other: Target substance
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.


Cell Culture
The stocks of cells are stored in liquid nitrogen at -196°C. 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), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.


Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β naphthoflavone activated rat liver S9
Test concentrations with justification for top dose:
Concentrations used in the preliminary toxicity test (with and without metabolic activation)
0, 11.17, 22.34, 44.69, 89.38, 178.75, 357.5, 715, 1430, 2860 µg/ml

Concentrations used in the main test (with and without metabolic activation)
0, 178.75, 357.5, 715, 1430, 2145, 2860 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test material was dissolved in DMSO prior to dilution to the test concentrations.
- Justification for choice of solvent/vehicle: Not reported.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "Any other information on materials and methods inc. tables" section
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period:
- Exposure duration: 4 hours (in both the presence and absence of S9) and 24hours (in the absence of S9 only)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 52 to 72 hours

SPINDLE INHIBITOR (cytogenetic assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): MTT

NUMBER OF REPLICATIONS: 2 per experiment

NUMBER OF CELLS EVALUATED: ca. 192000 (2000 cells/well, 96 wells)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002. It was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 at 126 x 10.6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive.

However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. 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.
Statistics:
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS

Calculation of % Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the % Relative Suspension Growth.

Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Plating Efficiency (PE)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the plating efficiency (PE), from which is derived the viability (%V), was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells/total wells plated
PE% = -ln P(0) x 100/number of cells per well

Calculation of Relative Total Growth (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = PE/Mean Solvent Control PE x 100%

Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The maximum dose level used was the 10 mM limit dose. A precipitate of test material was observed at and above 178.75 µg/ml. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials 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 increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that included the 10 mM limit dose.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1

Treatment (µg/ml)

4-Hours-S9

Treatment (µg/ml)

4-Hours +S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1

148.68

0

100

1

195.82

178.75

111

1.06

159.2

178.75

123

1.24

160.01

357.5

106

1.16

157.24

357.5

108

1.16

132.92

715

94

1

209.65

715

109

1.18

173.12

1430

80

0.95

132.72

1430

103

1.19

154.93

2145

72

0.81

111.64

2145

97

0.98

168.36

2860

71

0.75

108.05

2860

102

1.26

164.29

Linear trend

NS

Linear trend

NS

EMS

CP

400

83

0.6

646.27

0

61

0.27

992.73

Experiment 2

Treatment (µg/ml)

24-Hours-S9

Treatment (µg/ml)

4-Hours +S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1

86.16

0

100

1

102.03

178.75

144

1.18

76.1

178.75

122

1.17

69.1

357.5

157

1.34

65.37

357.5

116

1.05

93.45

715

150

1.19

100.71

715

105

1.21

73.23

1430

154

0.99

116.97

1430

97

1

79.94

2145

154

1.33

102.07

2145

102

0.94

82.48

2860

159

1.24

107.49

2860

91

0.99

69.77

Linear trend

*

Linear trend

NS

EMS

CP

150

83

0.48

918.42

0

57

0.33

599.73

 

%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

* = p<0.05

NS = Not significant

Conclusions:
Interpretation of results (migrated information):
negative Negative, both in the presence and absence of metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

In a mammalian cell gene mutation assay (0770/0004) performed in accordance with OECD Guidelines for the testing of chemicals No. 476 and EU method B17, TK +/- locus, L5178Y cells cultured in vitro were exposed to Zinc Hydroxy Stannate at concentrations of up to 2860 µg/mL (10 mM limit dose) in both the presence and absence of mammalian metabolic activation

The positive controls induced the appropriate response. The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

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

Genetic toxicity in vivo

Description of key information

The potential of the test material to cause genetic toxicity was investigated in an in vivo mouse micronucleus test. The test followed the OECD Guidelines for Testing of Chemicals No. 474 "Genetic Toxicology: Micronucleus Test" and Annex V method B12 of EEC Commission Directive 84/449/EEC.
The test material was considered to be non-mutagenic under the conditions of the study.

Link to relevant study records
Reference
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:
17 February 1989 - 21 March 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. 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.
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
other: Target substance
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian cell micronucleus test
Species:
mouse
Strain:
other: Albino BKW
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 – 8 weeks.
- Weight at study initiation: Males 22 – 28g; Females 20 – 26g
- Housing: The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding.
- Diet (e.g. ad libitum): ad libitum with the exception of a 3-4 hour fast immediately before dosing and for approximately two hours after dosing; free access to food (Rat and Mouse Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
- Water (e.g. ad libitum): ad libitum with the exception of a 3-4 hour fast immediately before dosing and for approximately two hours after dosing; free access to mains drinking water was allowed throughout the study.
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 21 °C
- Humidity (%): 52 – 60% relative
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.
Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
A confirmatory range-finding study was performed to determine a suitable dose level for the micronucleus study. The dose level selected was a maximum practical dose of 5000 mg/kg.
The study was performed using one dose level (the maximum practical dose of 5000 mg/kg) at three kill times of 24, 48 and 72 hours after dosing.
Duration of treatment / exposure:
One dose administered by gavage.
Frequency of treatment:
All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing.
Post exposure period:
Animals were observed 1 and 4 hours after dosing and subsequently once daily for 3 days.
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Three groups, each of ten mice (five males and five females) were dosed once only with test material at the "maximum practical dose level".
Control animals:
yes
Positive control(s):
Four further groups of ten mice (five males and five females) were included in the study; three groups were treated with the vehicle alone (distilled water) and the fourth group was treated with cyclophosphamide, a positive control material.
Details of tissue and slide preparation:
PROCEDURE
i) Range-finding Toxicity Study
A confirmatory range-finding study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum practical dose of 5000 mg/kg.
Groups of mice were dosed as follows:
DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF MICE
mg/kg mg/mL mL/kg MALE FEMALE

5000 500 10 7 7

All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing.

Animals were observed 1 and 4 hours after dosing and subsequently once daily for 3 days. Deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed. At 72 hours after dosing, up to 2 mice of each sex were sampled for bone-marrow cells to evaluate any specific bone-marrow toxicity in the absence of deaths.
ii) Micronucleus Study
The study was performed using one dose level (the maximum practical dose) at three kill times of 24, 48 and 72 hours after dosing.
Three groups, each of ten mice (five males and five females) were dosed once only with test material at the "maximum practical dose level". One group of mice was killed by cervical dislocation 24 hours following treatment, a second at 48 hours and a third at 72 hours. In addition, four further groups of ten mice (five males and five females) were included in the study; three groups were treated with the vehicle alone (distilled water) and the fourth group was treated with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle control groups were killed 24, 48 and 72 hours following treatment and positive control group animals were killed 24 hours following treatment.
All animals were observed for signs of overt toxicity and death 1 and 4 hours after dosing and then once daily as applicable.
iii) Slide Preparation
Immediately following sacrifice (i.e. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, and stained in May-Grünwald/Giemsa.
iv) Evaluation of Slides
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes PCE (blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes NCE (pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.
Evaluation criteria:
Interpretation of Results
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72 hour kill times.
If the above criteria are not demonstrated, then the test material is considered to be non-mutagenic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the treatment group mean normochromatic to polychromatic ratio is twice the vehicle control value or when a treatment related increase is shown to be statistically significant.
Statistics:
If necessary, and where possible, all data were statistically analysed using the Kruskal-Wallis one-way analysis of variance by ranks (Kruskal W.H. and Wallis W.A. 1952 J. Am. Statist. Soc. 47 583).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TOXICITY STUDY
The mortality data are summarised as follows:

Dose Level Sex Number of Deaths on day TOTAL DEATHS
mg/kg animals treated 0 1 2 3

5000 Male 7 0 0 0 0 0/14
Female 7 0 0 0 0

No adverse clinical symptoms were observed in any of the animals treated with Substance 1658/5 and there were no premature mortalities.
MICRONUCLEUS STUDY
i) Mortality Data and Clinical Observations
No adverse clinical symptoms were observed in any of the animals treated with Substance 1658/5 in the micronucleus study and there were no premature mortalities.
ii) Evaluation of Bone Marrow Slides
A summary of the results of the micronucleus study is given in Table 1.
There were no significant increases in the frequency of micronucleated PCE's in any of the groups of animals treated with Substance 1658/5 when compared to the corresponding vehicle control group. Furthermore, there was no increase in the frequency of micronucleated NCE's or in the NCE/PCE ratio after treatment with Substance 1658/5.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material, Substance 1658/5, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Summary of Group Mean Data (Males and Females Combined)

Table 1

Treatment group

Number of PCE with Micronuclei per 1000 PCE

Number of NCE with Micronuclei per 1000 PCE

NCE PCE Ratio

Group Mean

SD

Group Mean

SD

Group Mean

SD

Vehicle Control

72 hour sampling time

0.8

0.9

0.4

0.6

1.00

1.07

Vehicle Control

48 hour sampling time

0.6

0.7

0.3

1.0

0.59

0.30

Vehicle Control

24 hour sampling time

0.9

1.3

0.4

0.7

0.79

0.22

PositiveControl

24 hour sampling time

41.5

9.1

0.7

0.5

1.22

0.36

Substance 1658/5 5000mg/kg

72 hour sampling time

0.9

1.1

0.3

1.1

0.92

0.28

Substance 1658/5 5000mg/kg

48 hour sampling time

1.1

1.3

0.2

0.5

0.81

0.37

Substance 1658/5 5000mg/kg

24 hour sampling time

0.7

0.8

0.3

0.7

0.75

0.17

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-mutagenic under the conditions of the study.
Executive summary:

The test material was considered to be non-mutagenic under the conditions of the study. The test followed the OECD Guidelines for Testing of Chemicals No. 474 "Genetic Toxicology: Micronucleus Test" and Annex V method B12 of EEC Commission Directive 84/449/EEC.

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

Additional information

Justification for selection of genetic toxicity endpoint
No study selected since all key studies were negative.
The studies presented were all conducted in accordance with GLP and the appropriate standardised guidelines for the relevant endpoint.
All 4 key studies were awarded a reliability score of 2 in accordance with the principles of Klimisch (1997) due to the fact that the study is being used in a read-across capacity. The test was conducted on the analogous substance Zinc Hydroxystannate. The results are considered to be representative of the registered material.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity.