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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-22 to 2017-09-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid, white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Method

Target gene:
TA98: his D3052
TA100 and TA1535: his G 46
TA1537: his C 3076
E. coli WP2 uvrA: trp-
Species / strainopen allclose all
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:
S9-mix (containing approx. 10 % S9): sodium-ortho-phosphate buffer (100 mM, pH 7.4); NADP (4 mM); glucose-6-phosphate (5 mM); MgCl2 (8 mM); KCl (33 mM)
Test concentrations with justification for top dose:
Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment 2: 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (without metabolic activation) & 2-aminoanthracene (with metabolic activation))
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-experiment/Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

EXPERIMENTAL PERFROMANCE
Experiment 1:
- the following materials were mixed in a test tube and poured onto the selective agar plates: 100 μL test solution at each dose level (solvent or positive control), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL bacteria suspension, and 2000 μL overlay agar
- after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Experiment 2:
- 100 μL test solution (solvent or positive control), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- after pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.
- mixture was poured on minimal agar plates.
- after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

After incubation, revertant colonies were counted.

In parallel to each test a sterile control of the test item was performed. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. This pre-experiment was conducted as plate incorporation test.
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
Rationale for test conditions:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment 1. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in experiment 1 in strain TA 1537 with S9 mix at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitated in the overlay agar in the test tubes at 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in experiment 1 in strain TA 1537 with S9 mix at 5000 μg/plate. No further toxic effects were observed in the remaining test groups.

EXPERIMENT 1 & EXPERIMENT 2:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with cesium tetrafluoroaluminate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Please also refer for results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Historical data

Strain

 

without S9 mix

with S9 mix

 

 

Mean

SD

Min

Max

Mean

SD

Min

Max

 

Solvent control

12

2.5

6

25

12

2.5

7

26

TA 1535

Untreated control

12

3.1

6

28

12

2.9

7

26

 

Positive control

1130

143.1

334

1816

388

58.2

176

668

 

Solvent control

10

2.2

6

19

13

3.5

7

30

TA1537

Untreated control

11

2.7

5

21

14

4.0

7

31

 

Positive control

82

12.7

43

157

191

60.8

83

434

 

Solvent control

25

4.4

13

43

34

6.2

15

58

TA 98

Untreated control

27

4.9

12

43

37

6.5

11

57

 

Positive control

378

73.7

211

627

3949

771.8

360

6586

 

Solvent control

156

26.0

78

209

148

32.3

73

208

TA 100

Untreated control

176

23.6

79

217

172

25.4

85

218

 

Positive control

1966

293.2

498

2767

3798

830.4

536

6076

 

Solvent control

41

5.6

27

63

50

6.8

28

72

WP2uvrA

Untreated control

42

5.8

30

63

52

6.8

36

88

 

Positive control

798

362.7

319

4732

378

112.6

167

1265

Mean = mean value of revertants/plate
SD = standard deviation

Min = minimal value/Max = maximal value

Applicant's summary and conclusion

Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.