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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA with and without metabolic activation

HPRT (OECD 476): negative in V79 cells with and without metabolic activation

Chromosome Aberration test (OECD 473): negative in cultured human lymphocytes with metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
(200 metaphases were scored)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted Jul 2016
Deviations:
yes
Remarks:
(200 metaphases were scored)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted May 2008
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Food and Consumer Product Safety Authority (VWA), Utrecht, The Netherlands
Type of assay:
other: Chromosome aberration
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors: Two male donors, aged 32 and 40, respectively
- Separated lymphocyte cultures were used
- Methods for maintenance in cell culture: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added. All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 - 94%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.5 - 37.5°C).

MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 medium, supplemented with heat-inactivated FCS (20% (v/v)), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and heparin (30 U/mL)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw/day) and β-naphtoflavone (100 mg/kg bw/day)
Test concentrations with justification for top dose:
Dose range finding test/Experiment I:
3 h treatment with and without metabolic activation: 3, 10 and 33 µg/mL

Experiment II:
3 h treatment with metabolic activation: 3, 10 and 33 µg/mL
24 h without metabolic activation: 3, 5 and 7 µg/mL
48 h without metabolic activation: 1, 3 and 5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol absolute
- At concentrations of 1.4 mg/mL and below the test substance was dissolved in ethanol. At concentrations of 2 mg/mL and above the test substance was suspended in ethanol.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 3, 24 and 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment I: 3 h treatment: 24 h; 24 h treatment: 24 h; 48 h treatment: 48 h. Experiment II: 3 h treatment: 48 h; 24 h treatment: 24 h; 48 h treatment: 48 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicates each in 2 independent experiments.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in tap water.

NUMBER OF CELLS EVALUATED: Parallel cultures were treated at each concentration; 100 metaphases per culture were scored, 200 in total.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

OTHER
The S9 batch was characterised in a bacterial reverse mutation assay in Salmonella typhimurium tester strain TA98 with the mutagens benzo[a]pyrene and 2-aminoanthracene at concentrations of 5 μg/plate and 1 μg/plate, respectively.
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in Experiment II without metabolic activation a decrease of the mitotic index was noted at 7 µg/mL (24 h exposure time) and at 5 µg/mL (48 h exposure time).
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 33 µg/mL the test substance precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, blood cultures were exposed to the test substance with 0.3, 1, 3, 10, 33, 100 and 333 μg/mL culture medium without metabolic activation for 24 h and 48 h exposure time, respectively. The highest dose level showed an inhibition of the mitotic index of 50% or greater for the 24 h - and 48 h treatment times. Precipitation was observed in the culture medium for the 3 h exposure time cultures. The concentration levels for Experiment II were selected based on the results of the range-finding test (Experiment 1).

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: The data of the negative control falls within the historical range of the control data (please refer to Table 2 under "Any other information on results including tables").

Table 1: Results

Test item Concentration
in µg/mL
Mitotic Index
in %
Number of cells scored The number of cells with aberrations
with gaps without gaps
Exposure period 3h, fixation time 24h, without S9 mix
Ethanol 0.5% (v/v) 100 200 7 7
MMC  0.5 45 68 *** 68 ***
Test substance 3 87 6 6
10 79 8 8
33 P 69 5 5
Exposure period 3h, fixation time 24h, with S9 mix
Ethanol 0.5% (v/v) 100 200 9 9
CP 10 34 55 *** 55 ***
Test substance 3 99 4 4
10 90 8 8
33 P 75 3 3
Exposure period 24h, fixation time 24h, without S9 mix
Ethanol 0.5% (v/v) 100 200 1 1
MMC 0.2 55 35 *** 35 ***
Test substance 3 68 2 2
5 64 1 1
7 48 0 0
Exposure period 48h, fixation time 48h, without S9 mix
Ethanol 0.5% (v/v) 100 200 0 0
MMC 0.1 75 33 *** 33 ***
Test substance 1 86 0 0
3 69 0 0
5 46 0 0
Exposure period 3h, fixation time 48h, with S9 mix
Ethanol 0.5% (v/v) 100 200 0 0
CP 10 - d) 45 *** 45 ***
Test substance 3 94 1 1
10 93 0 0
33 P 91 0 0

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

P: Precipitation

*** :  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

a) CP was fixed after 48 h. Therefore, the mitotic index could not be calculated as percentage of control.

Table 2: Historical Control Data

  Aberrant cells per 100 metaphases
  Gaps included Gaps excluded
  with S9 mix without S9 mix with S9 mix without S9 mix
Range 0 - 6 0 - 6 0 - 5 0 - 5
Mean 0.8 0.8 0.6 0.6
SD 1.1 1.0 0.9 0.9
n 402 587 405 597
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 19 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon; trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial factor (S9 mix), prepared from the livers of male Sprague Dawley rats treated with Aroclor (500 mg/kg bw).
Test concentrations with justification for top dose:
Dose range finding test (TA100): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (5% (v/v) S9 mix) and without metabolic activation
First experiment (TA98, TA 1535, TA1537, WP2uvrA): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (5% (v/v) S9 mix) and without metabolic activation
Second experiment (all strains): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (10% (v/v) S9 mix) and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (extra pure)
- Justification for choice of solvent/vehicle: Due to the reactivity to moisture of the test substance, ethanol was selected as the vehicle. At concentrations of 10 mg/mL and higher the test substance was suspended in ethanol. At concentrations of 3.33 mg/mL and lower the test substance was dissolved in ethanol.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: 2.5 µg/plate for TA1537 (-S9); 2-aminoanthracene: 1, 2.5, 5 or 10 µg/plate for all strains (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER
Justification for using S9-mix prepared from rats treated with different substances:
Since rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone and by a combination of Aroclor are recommended by the OECD guideline and the positive control data were within the laboratory historical range for each tester strain, this deviation of the S9 homogenate had no effect on the results of the study.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Mean values and standard deviations were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction of the revertant colonies at concentrations of 3333 and 5000 µg/plate without metabolic activation; tested up to the limit dose concentration of 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate with and without metabolic activation. This dose range finding test is reported as a part of the first experiment of the mutation test. Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period. To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA100 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance. In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The slight precipitate does not influence automated counting of the plate, the moderate precipitate required the plate to be hand counted.
Experiment I: Precipitation on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and above. Precipitation on the plates was observed at the end of the incubation period at concentration of 333 μg/plate and above, except in tester strain TA98 where precipitation was observed at 1000 μg/plate and above. The precipitation of the test material at the dose level of 333 μg/plate contained a few particles only (please refer to Table 1 under "Any other information on results incl. tables").
Experiment II: Precipitation on the plates was observed at the start of the incubation period at the concentration of 1000 μg/plate and above. Precipitation on the plates was observed at the end of the incubation period at concentrations of 1000 μg/plate and above. Except in tester strain TA1535 (with and without metabolic activation) and TA98 (with metabolic activation) where a few particles of the test material were also observed at the dose level of 333 μg/ml (please refer to Table 2 under "Any other information on results incl. tables").

HISTORICAL CONTROL DATA
- Positive historical control data: The obtained data falls within the historical control data range (please refer to Table 4 under "Any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The obtained data falls within the historical control data range (please refer to Table 3 under "Any other information on results incl. tables").

Table 1: Test results of Experiment I

With or without S9 Mix Test substance concentration Mean number of revertant colonies per plate 
(μg/plate) (average of 3 plates ± Standard deviation)
  Base-pair substitution type Frameshift type
  TA 1535 TA100 WP2uvrA TA98 TA1537
Solvent control 13 ± 5 110 ± 26 23 ± 9 20 ± 6 10 ± 1
3  - 116 ± 19  -  -
10  - 129 ± 22  - -  -
33 13 ± 2 99 ± 14 22 ± 5 22 ± 2 5 ± 1
100 8 ± 3 94 ± 3 22 ± 6 28 ± 14 3 ± 1
333 4 ± 2 SP 92 ± 27 23 ± 3 SP 12 ± 4 3 ± 2 SP
1000 4 ± 1 SP 77 ± 8 19 ± 4 SP 11 ± 2 SP 4 ± 1 SP
3333 3 ± 1 SP 85 ± 11 22 ± 3 SP 8 ± 4 SP 5 ± 1 SP
5000 3 ± 2 SP 93 ± 14 26 ± 1 SP 9 ± 2 SP 4 ± 1 SP

Positive controls,

-S9

Name  SA MMS 4-NQO NF ICR-191
Concentrations (μg/plate) 5 650 10 10 2.5
Mean No. of colonies/plate (average of 3 ± SD) 728 ± 18 885 ± 27 925 ± 347 1067 ± 29 886 ± 10
+ Solvent control 16 ± 5 129 ± 17 25 ± 7 20 ± 5 9 ± 3
+ 3  - 127 ± 9
+ 10  - 146 ± 43 - -
+ 33 12 ± 3 118 ± 32 22 ± 6 26 ± 8 5 ± 1
+ 100 8 ± 1 89 ± 10 26 ± 3 17 ± 2 4 ± 1
+ 333 5 ± 2 SP 83 ± 12 25 ± 7 SP 24 ± 5 3 ± 1 SP
+ 1000 6 ± 1 SP 54 ± 9 25 ± 2 SP 18 ± 3 SP 4 ± 3 SP
+ 3333 5 ± 2 SP 57 ± 7 27 ± 4 SP 18 ± 2 SP 3 ± 1 SP
+ 5000 5 ± 2 SP 58 ± 3 24 ± 5 SP 19 ± 2 SP 2 ± 1 SP
Positive controls, +S9 (5% (v/v)) Name  2AA 2AA 2AA 2AA 2AA
Concentrations (μg/plate) 2.5 1 10 1 2.5
Mean No. of colonies/plate (average of 3 ± SD) 197 ± 6 1009 ± 16 925 ± 347 1009 ± 16 415 ± 36

Solvent control: 0.1 mL ethanol

SA = sodium azide

MMS = methyl methane sulfa

4NQO = 4-nitroquinoline-N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

SP = slight precipitate

Table 2: Test results of Experiment II

With or without S9-Mix Test substance concentration Mean number of revertant colonies per plate 
(μg/plate) (average of 3 plates ± Standard deviation)
  Base-pair substitution type Frameshift type
  TA 1535 TA100 WP2uvrA TA98 TA1537
Solvent control 9 ± 1 120 ± 30 17 ± 4 16 ± 4 6 ± 1
100 4 ± 1 84 ± 5 20 ± 1 16 ± 3 8 ± 4
333 3 ± 1 SP 92 ± 12 18 ± 4 12 ± 4 5 ± 5
1000 3 ± 1 SP 89 ± 7 SP 24 ± 3 SP 16 ± 1 SP 2 ± 1 SP
3333 3 ± 2 SP 94 ± 2 SP 20 ± 4 SP 15 ± 3 SP 2 ± 1 SP
5000 3 ± 1 SP 89 ± 9 SP 19 ± 1 SP 13 ± 1 SP 2 ± 1 SP
Positive controls, –S9 Name  SA MMS 4-NQO NF ICR-191
Concentrations (μg/plate) 5 650 10 10 2.5
Mean No. of colonies/plate (average of 3 ± SD) 844 ± 14 838 ± 15 1232 ± 41 1081 ± 28 797 ± 35
+ Solvent control 11 ± 4 110 ± 2 22 ± 3 15 ± 3 7 ± 4
+ 100 8 ± 5 79 ± 15 18 ± 2 26 ± 8 1 ± 1
+ 333 4 ± 1 SP 93 ± 13 20 ± 2 19 ± 6 SP 5 ± 4
+ 1000 3 ± 2 SP 92 ± 12 SP 16 ± 2 SP 19 ± 7 SP 3 ± 1 SP
+ 3333 4 ± 1 SP 89 ± 10 SP 15 ± 4 SP 17 ± 3 SP 5 ± 1 SP
+ 5000 4 ± 2 SP 69 ± 6 SP 16 ± 6 SP 14 ± 6 MP 1 ± 2 SP
Positive controls, +S9 (10% (v/v)) Name  2AA 2AA 2AA 2AA 2AA
Concentrations (μg/plate) 2.5 2.5 10 1 5
Mean No. of colonies/plate (average of 3 ± SD) 189 ± 18 1341 ± 68 387 ± 21 780 ± 54 267 ± 11

Solvent control: 0.1 mL ethanol

SA = sodium azide

MMS = methyl methane sulfa

4NQO = 4-nitroquinoline-N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

SP = slight precipitate

Table 3: Historical control data: Negative control

  TA 1535 TA1537 TA98 TA100 WP2uvrA
S9 mix - + - + - + - + - +
Range 3 - 25 3 - 31 2 - 19 2 - 16 11 - 49 12 - 59 61 - 195 58 - 179 8 - 45 6 - 46
Mean 10 8 5 5 19 24 106 88 22 23
SD 4 3 2 2 5 7 22 21 7 7
n 1208 1216 1073 1098 1191 1208 1224 1221 1090 1105

SD = Standard deviation

n = Number of observations

Table 4: Historical control data: Positive control

  TA 1535 TA1537 TA98 TA100 WP2uvrA
S9 mix - + - + - + - + - +
Range 24 - 1270 60 - 943 89 - 1086 82 - 677 401 - 1342 225 - 1656 348 - 1417 229 - 1752 138 - 1479 122 - 1248
Mean 887 265 314 374 1016 792 950 1103 1074 372
SD 112 111 146 104 133 286 117 261 212 137
n 1123 1150 1011 1039 1136 1157 1153 1170 1023 1047

SD = Standard deviation

n = Number of observations

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19 Dec 2016 - 10 Feb 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(no statistical analysis performed, evaluation and acceptability criteria differ from the current version of the Guideline, activity of the metabolic activation system not confirmed)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted Jul 2016
Deviations:
yes
Remarks:
(no statistical analysis performed, evaluation and acceptability criteria differ from the current version of the Guideline, activity of the metabolic activation system not confirmed)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation test
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Japanese Collection of Research Bioresources (JCRB) Cell Bank
- Doubling time: Approximately 13 h
- Number of passages if applicable: 10 (dose finding test) and 6 (gene mutation test)
- Methods for maintenance in cell culture if applicable: The cells were cultured in a humidified CO2-incubator (5% CO2, 37 °C)
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media: Eagle's Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (lot no. 1385110, Gibco, Thermo Fisher Scientific), approximately 0.15 g of L-glutamine (Nissui Pharmaceutical) and approximately 10 mL of 10% (w/v) NaHCO3-solution.
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Pre-test for toxicity
With and without metabolic activation: 0.0016, 0.0031, 0.0063, 0.013, 0.025, 0.050 and 0.10 mg/mL (4 h)
Without metabolic activation: 0.0016, 0.0031, 0.0063, 0.013, 0.025, 0.050 and 0.10 mg/mL (24 h)

Mutation experiment I and II
with and without metabolic activation: 0.0063, 0.013, 0.025 and 0.050 mg/mL (4 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test article was degradable in water and acetone, and insoluble in DMSO and ethanol at the concentration required for the test. However, it was possible to make an uniform dosing formulation in ethanol, but not in DMSO. Therefore, ethanol (lot no. DSR0158, purity: 99.5%, Wako Pure Chemical Industries) was used as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: N-nitrosodimethylamine: 2 mg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 300 x 10E04

DURATION
- Exposure duration: 4 h with and without S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency. 6 days after treatment cells were plated for the determination of the mutation frequency in 6-well plates containing 6-thioguanine selective medium. The 6-well plates were incubated for 9 days.
- Selection time (if incubation with a selection agent): 9 days

SELECTION AGENT (mutation assays): 6-thioguanine (5 µg/mL)

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments in 6-well plates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative survival
Evaluation criteria:
When the average mutant frequencies in the test article treatment groups are three times or more than that of the concurrent negative (solvent) control group. When the mutant frequencies in the concurrent negative (solvent) control are zero, mutant frequencies in the test article treatment groups are three times or more than the mean mutant frequency of the negative control (all data) in the historical control data.
The average mutant frequencies in treatment groups are over ranges of negative control (all data) in the historical control data.

The final judgment was based on a comprehensive evaluation that also took into account the biological point of view.

Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
-S9: at 0.025 mg/mL; +S9: at 0.05 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance in the culture medium was seen by the naked eye at the beginning of the treatment period at 0.050 mg/mL or more under the short-term treatment condition and at 0.025 mg/mL or more under the long-term treatment condition. At the end of the treatment period it was seen by the naked eye at 0.025 mg/mL or more in the short-term treatment without S9 mix and the long-term treatment and at 0.050 mg/mL or more in the short-term treatment with S9 mix.

RANGE-FINDING/SCREENING STUDIES: In order to select the doses of the test article for the gene mutation test, the dose finding test was performed by setting 0.10 mg/mL as the highest concentration at which it was able to definitely confirm precipitate. The relative cell confluency was dose-dependently decreased slightly (less than 50%) in the short-term treatment at the highest concentration of 0.10 mg/mL and severely in the long-term treatment in concentrations ≥ 0.0125 mg/mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: For ethyl methanesulfonate (n=31) the mutant frequency was 1047.5 ± 264.8 (range: 587.6 - 1532.9) and for N-nitrosodimethylamine (n=32) the mutant frequency was 601.3 ± 323.7 (range: 228.9 - 1453.0), thus the mutant frequencies of the positive controls of 1180.6 (-S9) and 798.3 (+S9) were within the range of the historical control data.
- Negative (solvent/vehicle) historical control data: For negative control without metabolic activation (n=42) the mutant frequency was 4.4 ± 5.4 (range: 0 - 21.0) and with metabolic activation (n=50) the mutant frequency was 3.6 ± 2.7 (range: 0 - 10.2), thus the mutant frequencies of the solvent control of 2.4 (-S9) and 6.7 (+S9) were within the range of the historical control data.

Table 1: Summary of results with metabolic activation

Concentration [mg/mL] Relative Cloning efficiency [%] Relative Survival [%] Mutation factor
Non-treatment 97.4 91.1 5.7
Vehicle (EtOH) 100 100 2.4
0.0063 101.1 97.1 2.9
0.013 101.1 97.4 5.6
0.025 102.3 95.0 2.9
0.050 98.0 90.5 4.2
EMS 81.6 27,59 1180.6

EMS = Ethyl methanesulfonate

EtOH = Ethanol

Table 2: Summary of results without metabolic activation

Concentration [mg/mL] Relative Cloning efficiency [%] Relative Survival [%] Mutation factor
Non-treatment 106.8 106.7 1.0
Vehicle (EtOH) 100.0 100.0 6.7
0.0063 107.6 105.5 7.5
0.013 104.8 101.7 5.6
0.025 100.4 97.2 7.9
0.050 92.6 88.2 6.0
NDMA 97.3 87.3 798.3

NDMA = N-nitrosodimethylamine

EtOH = Ethanol

Conclusions:
Interpretation of results: negative
Executive summary:

The Gene Mutation was assessed according to the OECD 476 test guideline using V70 Chinese Hamster Lung cells.

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

Genetic toxicity in vivo

Description of key information

The test substance Dioctyloxostannane (DOTO) [CAS# 870-08-6] was examined for its mutagenic potential in a bone marrow micronucleus test in mice. The study consisted of a dose-range finding acute toxicity test carried out with male and female mice and a main micronucleus test with male mice only.

 

Results of the dose range finding acute toxicity test indicated that there were no sex differences in response and that a limit dose of 2000 mg/kg-bw could be tolerated. Male mice were chosen for the main study and doses of 2000, 1000 and 500 mg/kg-bw were adopted.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 November 2003 to 28 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OECD guideline 474, Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, adopted 21 July 1997.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
No further details specified in the study report.
Species:
mouse
Strain:
Swiss
Details on species / strain selection:
Species selected in accordance with test guideline
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals, housing and care
For the dose-range finding acute toxicity test ( 6 males and 6 females) and for the main micronucleus test (37 males), young adult swiss mice, were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany. The animals arrived on 29 October 2003 (dose-range finding acute toxicity test) and 19 November 2003 (main micronucleus test).
The mice were taken in their unopened shipping containers directly to room number 5.2.15 (dose-range finding acute toxicity test and main micronucleus test). After routine serological examination, carried out on the day following arrival, the animals for the dose-range finding acute toxicity test stayed in the same room. The animals for the main micronucleus test were moved to room 5.2.08 The results of this serological examination were satisfactory. The animals were housed in sterilised Macrolon cages (type I and II), fitted with a grid cover of stainless steel and with a bedding of wood shavings (Espen E0Ol). During the quarantine and acclimatization period the animals were observed daily for overt signs of ill health and anomalies. Housing conditions were conventional. For safety reasons, the animals of the positive control group (main micronucleus test only) were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice. The animal rooms were ventilated with about 10 air changes per hour and were maintained at a temperature of 22 +/- 3 ° C and a relative humidity of at least 30% and not exceeding 70% other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Feed and drinking water
With the exception of the fasting period prior to dosing, feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study. Fresh pellet diet was provided once weekly.
The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3). Batch 3142 (expiry date, 12 March 2004) was used for both the dose-range finding acute toxicity test and the main micronucleus test.
Each batch of this diet is analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants.
The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 98/83/EEC), was supplied by N.V. Hydron Midden-Nederland.
Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO in Zeist for a limited number of variables.
Route of administration:
oral: gavage
Vehicle:
Corn-oil was used as vehicle for the test substance.
Details on exposure:
On the day (day-I) before the dosing day (day 0), the test substance was suspended in corn-oil, at a stock concentration of 100 mg/ml. This stock concentration was stirred overnight on a magnetic stirrer. The two lower concentrations of 50 mg/ml and 25 mg/ml were prepared prior to dosing on day 0.
Duration of treatment / exposure:
Single dose / 24-48 hour exposure
Frequency of treatment:
Single dose
Post exposure period:
No post exposure period specified
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Dose range finding: 4 animal (2 males/2females)
Main study: 5 males per dose (24 hour observation time), plus 5 males per group in control and highest dose with 48 hour observation time
Control animals:
yes, concurrent vehicle
other: Positive control
Positive control(s):
A positive control group consisted of 5 males and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg-bw.
Tissues and cell types examined:
Micronuclei are small secondary nuclear structures resulting either from chromosome breakage or from malfunction of the spindle apparatus, which regulates division of the nucleus at cell division. Micronuclei can easily be visualized in erythrocytes because these cells lack a nucleus.
Details of tissue and slide preparation:
Signs of reactions to treatment were recorded from 1- 4 hours after treatment and daily thereafter. All clinical signs, observed during the performance of the study, are recorded in paragraph 3 .1 ( dose-range finding acute toxicity test) and paragraph 3.3 (main micronucleus test).
At the sacrifice time of 24 hours after dosing, 5 mice treated with the vehicle control, 15 mice treated with the test substance Dioctyloxostannane (5 mice of each dose-level) and 5 mice treated with the positive control substance mitomycin C, were killed by cervical dislocation. At the sacrifice time of 48 ~hours after dosing, 5 mice treated with the vehicle control, together with 5 mice treated with the highest dose-level of the test substance Dioctyloxostannane (2000 mg/kg-bw), were killed by cervical dislocation.
From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grtinwald Giemsa solution. The other smear was stored as reserve slide.

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) 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 was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
Evaluation criteria:
A response is considered to be positive if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test substance is 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 is observed, when compared to the mean number of the vehicle controls.
A test substance is considered to be negative in the micronucleus test if it produces no positive response at any of the dose-levels and time points analysed.
The test substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduce the mean number of PE/E or causes systemic toxicity.
Both statistical significance and biological relevance are considered together in the evaluation.
Statistics:
At time point 24 hours after administration, data on MPE and PE were subjected to a One Way Anova with factor group (A, B, C and D).If the Anova yielded a significant effect (p<0.05), it was followed by pooled error variance t-tests or, if variances were not homogeneous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment groups B, C and D. Furthermore, the positive control group E and the negative control group A were compared using pooled error variance t-tests or, if variances were not homogeneous, separate t-tests. In addition a linear trend test (orthogonal contrasts) was applied across groups A, B, C and D.
At time point 48 hours after administration, for treatment groups A and D, data on MPE and PE were subjected to pooled error variance t-tests or, if variances were not homogeneous, 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).

The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose-range finding acute toxicity test
Due to a failed oral administration, female 3 died on the day following administration. No severe clinical signs as a result of the test substance Dioctyloxostannane, or sex differences, were observed in the dose-range finding acute toxicity test. The results of the dose-range finding acute toxicity test were reported to the sponsor. Thereafter, it was decided to perform the main micronucleus test with the same three dose levels of the test substance Dioctyloxostannane (2000, 1000 and 500 mg/kg-bw), administered in the dose-range finding acute toxicity test and with male mice only.

Clinical signs in the main micronucleus test
At 24 hours after administration, all mice of the C group (Dioctyloxostannane; 1000 mg/kg-bw) and D group (Dioctyloxostannane; 2000 mg/kg-bw) showed a hunched posture, piloerection and a wet anal area as a result of diarrhoea caused by the test substance.
At 48 hours, mouse D78 (Dioctyloxostannane; 2000 mg/kg-bw) still showed a hunched posture, piloerection and a wet anal area as a result of diarrhoea.

Statistical analysis of the main micronucleus test results
At both sacrifice times of 24 hours and 48 hours after treatment, the two-way ANOVA did not yield a statistically significant effect for MPE and PE. This indicates that treatment with Dioctyloxostannane, up to 2000 mg/kg-bw (the limit dose level), did not result in genotoxicity or clastogenicity to the bone marrow target cells.
At the sacrifice time of 24 hours, in the positive control group, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the negative control A. This demonstrates the validity of the test system.
The results of this micronucleus test did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male mice, treated orally with the test substance Dioctyloxostannane.

Dose-range finding acute toxicity test

Clinical signs during the dose-range finding acute toxicity test

Test substance: Dioctyloxostannane

Vehicle: corn-oil

Route: by gavage

Dosing volume: 20 ml/kg-bw

Test substance and dose level (mg/kg-bw)

1h after1)administration

4 h after1)administration

24 h after1)administration

48 h after1)administration

Animal number and sex

Dioctyloxostannane 500 mg/kg-bw

 

sl; se; br

sl; se; p; br; †

 

Male 2

Male 4

Female 1

Female 3

Dioctyloxostannane 1000 mg/kg-bw

 

 

 

 

Male 6

Male 8

Female 5

Female 7

Dioctyloxostannane 2000 mg/kg-bw

 

 

 

 

Male 10

Male 12

Female 9

Female 11

1) empty cells, no clinical signs observed

sl: sluggishness

se: slit-eyes

p: piloerection

br: irregular breathing

†: animal died

 

Body weight in the main micronucleus test

Body weights of the animals prior to the start of treatment

Treatment group

Dose level (mg/kg-bw)

Sex

N

Bw mean (g)*

Bw SEM (g)

A / negative control (corn-oil)

0

M

10

34.83

0.56

B / Dioctyloxostannane

500

M

5

34.38

0.43

C / Dioctyloxostannane

1000

M

5

34.12

0.49

D / Dioctyloxostannane

2000

M

10

34.22

0.55

E / Mitomycin C# (positive control)

0.75

M

5

33.70

0.36

#: Sigma; single (10 ml/kg-bw) intraperitoneal injection (vehicle: saline)

M: male

*: individual body weights are stored in the archives

 

Micronucleated Polychromatic Erythrocytes (MPE) in the mice of the main micronucleus test

The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)

Group:

A

Neg. contr. (corn-oil)

Dioctyloxostannane

(dose level in mg/kg-bw)

E

pos. control mitomycin C (0.75 mg/kg-bw)

Sex

† (h)

B

500

C

1000

D

2000

Male

24

2.0 ± 0.7

2.6 ± 1.1

1.8 ± 0.4

1.4 ± 0.5

50.4 ± 10.3***

Male

48

2.2 ± 1.1

-

-

1.4 ± 0.9

-

Means and standard deviations: ***P<0.001 (t-test)l group size: 5

The positive control group E, at time point 24h, differed significantly from the negative control A (P<0.01).

 

Polychromatic Erythrocytes (MPE) in the mice of the main micronucleus test

Group mean numbers of polychromatic erythrocytes (MPE) per 2000 erythrocytes (E)

Group:

A

Neg. contr. (corn-oil)

Dioctyloxostannane

(dose level in mg/kg-bw)

E

pos. control mitomycin C (0.75 mg/kg-bw)

Sex

† (h)

B

500

C

1000

D

2000

Male

24

89.2 ± 15.2

85.2 ± 14.3

91.0 ± 14.4

92.0 ± 18.2

77.0 ± 7.6

Male

48

88.0 ± 10.8

-

-

75.2 ± 5.2

-

 

Historical data for the Micronucleus Test in Swiss Mice (CD-1 strain)

 

Historical negative controls

Historical negative control data (vehicle, 1 or 2 treatment) from 30 studies carried out from 1999-2003. Overview from 20 May 2003.

Vehicle

Route

Micronucleated PE per 1000 PE at 24-72 hours post-treatment, males and females

Mean ± standard deviation

Range of means

Saline, water or PBS#

 

 

Corn-oil

Orla

i.p.

i.v.

 

oral

1.9 ± 1.0

2.1 ± 1.3

2.1 ± 1.0

 

2.6 ± 1.3

1.6-2.2

1.6-2.6

1.2-3.2

 

1.0-4.8

# PBS = phosphate-buffered saline

 

Historical positive controls

Historical positive control data from studies carried out from 1999-2003. Overview from 20 May 2003.

Vehicle

Route

Micronucleated PE per 1000 -2000 PE at 24 hours post-treatment, males and females

Mean ± standard deviation

Range of means

Mitomycin C 1.5 mg/kg-bw

 

Mitomycin C 0.75 mg/kg-bw

i.p.

 

i.p.

49 ± 17

 

36 ± 9

17-95

 

17-70

 

Conclusions:
The data supports the conclusion that, under the conditions used in this study, the test substance Dioctyloxostannane did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.
Executive summary:

The test substance Dioctyloxostannane (DOTO) [CAS# 870-08-6] was examined for its mutagenic potential in a bone marrow micronucleus test in mice. The study consisted of a dose-range finding acute toxicity test carried out with male and female mice and a main micronucleus test with male mice only.

 

Results of the dose range finding acute toxicity test indicated that there were no sex differences in response and that a limit dose of 2000 mg/kg-bw could be tolerated. Male mice were chosen for the main study and doses of 2000, 1000 and 500 mg/kg-bw were adopted.

 

For the main micronucleus test, animals were treated once by gavage with three graded dose levels of the test substance Dioctyloxostannane. The high dose group (D) consisted of 10 males and each animal received a dose of 2000 mg/kg-bw (the limit dose-level). The moderate dose group (C) consisted of 5 males and each animal received a dose of 1000 mg/kg-bw. The low dose group (B) consisted of 5 males and each animal received a dose of 500 mg/kg-bw. The vehicle control group (A) consisted of 10 males and each animal was dosed in a similar way with the corn-oil vehicle only. A positive control group consisted of 5 males and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg-bw. At 24 hours after treatment, 5 animals of each dose-level of the test substance, 5 negative control animals and 5 positive control animals, were euthanized. At 48 hours after treatment, the remaining 5 animals of group D (the high dose group) and the remaining 5 negative control animals, were euthanized. From both femurs of each animal, the bone marrow cells were collected in foetal calf serum and processed into smears for microscopic examination.

 

At both time points, 24 and 48 hours after treatment, the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse. The mean number of MPE per 2000 PE, at dose levels of 2000, 1000 and 500 mg/kg-bw of Dioctyloxostannane, were not statistically significantly different from the vehicle control mean. Therefore, the test substance Dioctyloxostannane, at dose levels up to 2000 mg/kg-bw, was not genotoxic to bone marrow cells in mice.

 

For the mice of the positive control group, the mean number of MPE per 2000 PE was significantly (p<0.001) elevated compared to the mean of the vehicle control mice. This demonstrates the validity of the test system.

 

At 24 and 48 hours after treatment, the mean number of polychromatic erythrocytes (PE) per erythrocyte (E) in mice, at all treatment levels of Dioctyloxostannane, were not statistically significantly different from the mean of the vehicle control mice. Therefore, treatment with Dioctyloxostannane, at dose-levels up to 2000 mg/kg-bw, was not cytotoxic to the bone marrow of mice.

 

The data supports the conclusion that, under the conditions used in this study, the test substance Dioctyloxostannane did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.

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

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD guideline 471 and in compliance with GLP (WoE, 2011). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were exposed to the test substance using the standard plate incorporation method. Test substance concentrations of 3 to 5000 µg/plate were selected for two independent experiments with and without metabolic activation. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. Cytotoxicity was only observed in tester strain TA 98 in the absence of S9 mix, where a moderate reduction of the revertant colonies was observed at test substance concentrations of 3330 and 5000 µg/plate. The test substance precipitated on the plates at dose levels of 333 µg/plate and above. The slight precipitate did not influence the automated counting of the plates, while the moderate precipitate required the plate to be hand counted. However, the precipitation did not affect the counting of the colonies. All positive and negative control values were found to be within the respective historical control ranges. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.

 

In vitro Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 and under GLP conditions with the test substance, in Chinese hamster lung fibroblasts (V79) (WoE, 2017). The cells were treated for 4 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and 5,6-benzoflavone). The test substance was tested up to the precipitation concentration: 0.0063, 0.013, 0.025 and 0.050 mg/mL with and without metabolic activation. N-nitrosodimethylamine and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively. A vehicle control and a negative control (no treatment) were included. A reduced cloning efficiency was not observed with and without metabolic activation. The positive and negative controls were valid and within the range of the historical control data. No biological significant increase in the mutation frequency at the HPRT locus was observed after treatment either in the absence or in the presence of S9-mix. Therefore, it was concluded that the test substance was not mutagenic in Chinese hamster lung fibroblasts (V79) under the experimental conditions described.

 

In vitro Cytogenicity in mammalian cells

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured peripheral human lymphocytes performed according to OECD guideline 473 and GLP (WoE, 2012). The test substance was dissolved in ethanol and two independent experiments were performed.

In the first experiment, the test substance was tested up to 33 μg/mL for a 3-h exposure time with a 24 -h fixation time in the absence and presence of metabolic activation (S9 mix). The test substance precipitated in the culture medium at this dose level. In the second experiment, the test substance was tested up to 7 μg/mL for a 24 -h continuous exposure time with a 24 -h fixation time and up to 5 μg/mL for a 48-h continuous exposure time with a 48-h fixation time in the absence of S9 mix. Appropriate toxicity was reached at these dose levels. In the presence of S9 mix the test substance was tested up to 33 μg/mL for a 3-h exposure time with a 48-h fixation time. The test substance precipitated in the culture medium at this dose level. The observed precipitation did not affect the validity of the results. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Although one of the solvent controls in the first experiment in the presence of S9 mix was just above the historical control data range, the group mean was within the historical control data range. Therefore this outlier was not considered relevant for the outcome of the study. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly.

The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix, in either of the two independently repeated experiments. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9 mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In conlcusion, the test substance is not clastogenic in human lymphocytes under the experimental conditions described.

Conclusion for genetic toxicity

The available data show that the test substance is not mutagenic in bacteria and mammalian cells (Chinese hamster lung fibroblasts) in vitro, and not clastogenic in cultured peripheral human lymphocytes.

Justification for classification or non-classification

The available data on genetic toxicity with the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.