Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-27 to 2016-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA 712-C-98-247, August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment (plate incorporation): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I (plate incorporation test with and without rat liver S9): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II (pre-incubation test with and without rat liver S9):3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Aqua dest.; negative control =solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.; negative control =solvent control
Positive controls:
yes
Remarks:
S. typhimurium: TA 100, TA 1535 without metabolic activation dissolved in Aqua dest.
Positive control substance:
sodium azide
Positive controls:
yes
Remarks:
S. typhimurium: TA 98, TA 1537 without metabolic activation dissolved in DMSO
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Positive controls:
yes
Remarks:
S. typhimurium: TA 102 without metabolic activation dissolved in Aqua dest.
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Remarks:
S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation dissolved in DMSO
Positive control substance:
other: 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: pre-incubation

DURATION
Experiment I:
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

Experiment II:
- Preincubation period: 60 min at 37°C
- Exposure duration: 60 min at 37°C and at least 48 h in the dark at 37°C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): histidine (overlay agar)

NUMBER OF REPLICATIONS: 3 (in two cases only two plates were evaluated)

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher (with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 316 µg/plate and higher (with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 316 µg/plate and higher (with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
see any other information on results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: The negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015)):

-S9 + S9 (rat liver)
min max min max
TA 98 13 54 13 61
TA 100 49 139 67 162
TA 1535 4 39 4 32
TA 1537 2 35 3 36
TA 102 141 472 91 586


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
- Other observations when applicable: Toxic effects of the test item were noted in all tester strains used in experiment II at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

Any other information on results incl. tables

Pre-experiment:

Substance

Dose

g/plate)

TA 98

Mutation Factor [toxicity]*

TA 100

Mutation Factor [toxicity]*

without S9

with S9

without S9

with S9

Solvent Control

(A. dest)

 

1.0

1.0

1.0

1.0

4-NOPD

10.0

25.8

-

-

-

NaN3

10.0

-

-

6.1

-

2-AA

2.50

-

53.2

-

11.6

Test Item

 

3.16

1.1

0.9

0.9

0.9

10.0

1.1

0.8

0.9

0.8

31.6

0.9

0.7

1.0

1.1

100

1.1

0.7

1.2

1.1

316

1.2

0.8

1.1

1.1

1000

1.3

0.9

1.2

1.1

2500

1.0

1.1

1.2

1.3

5000

1.3

0.8

1.3

1.2

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

 

Experiment I

TA 98 TA 100 TA 1535 TA 1537 TA 102
Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor
Treatment Dose (µg)/plate  -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
A. Dest   22 31 1 1 128 128 1 1 20 17 1 1 7 9 1 1 297 336 1 1
Test item 31.6 20 22 0.9 0.7 128 135 1 1.1 19 26 0.9 1.6 9 8 1.2 0.9 244 363 0.8 1.1
Test item 100 25 21 1.1 0.7 148 143 1.2 1.1 46 28 2.2 1.7 10 6 1.4 0.6 260 403 0.9 1.2
Test item 316 326 26 1.2 0.8 137 135 1.1 1.1 24 36 1.2 2.1 8 6 1 0.6 307 372 1 1.1
Test item 1000 28 28 1.3 0.9 158 141 1.2 1.1 46 28 2.2 1.7 11 10 1.5 1.1 278 327 0.9 1
Test item 2500 22 33 1 1.1 153 165 1.2 1.3 25 26 1.2 1.5 9 9 1.3 1 288 372 1 1.1
Test item 5000 30 24 1.3 0.8 164 148 1.3 1.2 24 26 1.2 1.6 8 7 1 0.7 317 404 1.1 1.2
4-NOPD/NaN3/MMS 10 576 - 25.8 - 776 - 6.1 - 1009 - 49.6 - 91 - 12.4 - 1303 - 4.4 -
2-AA 2.5 - 1632 - 53.2 - 1488 - 11.6 - 211 - 12.7 - 223 - 24.7 - 757 - 2.3

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I.

Experiment II:

TA 98 TA 100 TA 1535 TA 1537 TA 102
Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor Revertant colonies per plate Mutation factor
Treatment Dose (µg)/plate  -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
A. Dest   25 29 1 1 133 95 1 1 18 19 1 1 11 10 1 1 267 255 1 1
Test item 31.6 26 21 1.1 0.7 126 122 1 1.3 25 29 1.4 1.5 12 8 1.1 0.8 298 377 1.1 1.5
Test item 100 22 35 0.9 1.2 149 124 1.1 1.3 27 20 1.5 1 12 8 1.1 0.7 253 338 0.9 1.3
Test item 316 26 19 1 0.7 149 112 1.1 1.2 20 22 1.1 1.2 10 5 0.9 0.5 267 190 1 0.7
Test item 1000 23 29 0.9 1 90 106 0.7 1.1 31 18 1.7 0.9 7 7 0.6 0.7 266 258 1 1
Test item 2500 18 14 0.7 0.5 123 103 0.9 1.1 22 7 1.2 0.4 9 7 0.8 0.7 277 183 1 0.7
Test item 5000 25 3 1 0.1 79 39 0.6 0.4 19 5 1.1 0.3 7 3 0.6 0.3 155 149 0.6 0.6
4-NOPD/NaN3/MMS 10 545 - 22.1 - 890 - 6.7 - 947 - 52.6 - 128 - 11.7 - 1257 - 4.7 -
2-AA 2.5 - 125 - 4.3 - 1032 - 10.9 - 82 - 4.2 - 103 - 9.9 - 710 - 2.8

Due to more sensitive pre-incubation method, in experiment II toxic effects of the test item were seen in most tester strains.

- In tester strain TA 98 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with metabolic activation).

- In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation).

- In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 316 µg/plate and higher (with metabolic activation).

- In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the ability of the test substance to induce gene mutations, a bacterial reverse mutation assay (Ames) according to OECD 471 was performed. Two experiments were ran: the plate incorporation test (experiment I) and the pre-incubation test (experiment II) with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation (S9 ). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in both experiments: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I.

Due to more sensitive pre-incubation method, in experiment II toxic effects of the test item were seen in most tester strains. In tester strain TA 98 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.