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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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ultrapure water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
no
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (first experiment); preincubation (second experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies (with TA98 and TA100 with and without S9)

Evaluation criteria:
reproducible increase of revertant colonies per plate (factor >=2 for TA100 and factor >= 3 for TA98m TA1535, TA1537 and WP2)
Statistics:
none

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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
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
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
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:
E. coli WP2 uvr A
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not available

RANGE-FINDING/SCREENING STUDIES: no precipitation and no cytotoxicity observed up to 5000 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): not available

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Any other information on results incl. tables

In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation.

pH of the test substance stock solutions and the pH of the overlay (at highest concentration of 50 mg/mL) was measured as 9.39 and 9.18, respectively. The acidic solutions had only a slight effect on the buffered overlay system that did not impair the validity of the experiment and the results.

Detailed test results:

Initial experiment (plate incorporation) without and with metabolic activation

 control/

mutation rate per test strain: without metabolic activation        

mutation rate per test strain: with metabolic activation   

 concentration (µg/plate)

 TA98

TA100

TA1535 

TA1537 

WP2uvrA 

 TA98

TA100 

TA1535 

TA1537 

WP2uvrA 

 untreated control

 0.98

0.81

0.96 

1.63

1.24 

0.94 

1.17 

1.09 

1.00 

1.00 

 DMSO control

 1.00

 -

 -

1.00

-

 1.00

1.00 

1.00 

1.00 

1.00 

 ultrapure water control

 1.00

1.00

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

test substance: 5000

 1.02

 0.90

 1.36

 0.94

 1.07

 1.02

 1.02

 1.31

 0.88

 1.27

test substance: 1600

1.21

 0.87

 1.39

 0.88

 0.91

 1.26

 0.99

 1.19

 1.50

 1.07

test substance: 500

 1.00

 0.98

 1.54

 1.31

 1.11

 0.98

 0.98

 1.22

 1.00

1.26

test substance: 160

 0.85

 1.07

 1.36

 0.94

 1.30

 1.07

 1.06

 1.22

 1.25

 1.30

test substance: 50

 1.02

 0.97

 1.18

 0.88

 0.93

0.97

 1.10

 0.94

 1.38

 0.96

test substance: 16

 1.04

 0.91

 0.93

 0.81

 1.13

 0.91

 1.10

 0.88

 0.94

 1.32

 NPD (4 µg)

 27.12

 

 

 

 

 

 

 

 

 

 SAZ (2 µg)

 

 14.08

 104.93

 

 

 

 

 

 

 

9AA (50 µg) 

 

 

 

 112.17

 

 

 

 

 

 

 MMS (2 µL)

 

 

 

 34.30

 

 

 

 

 

 2AA (2 µg)

 

 

 

 

 

 52.66

 9.19

 19.93

 22.04

 

 2AA (50 µg)

 

 

 

 

 

 

 

 

 

 13.50

Confirmatory experiment (pre-incubation) without and with metabolic activation

 control/

mutation rate per test strain: without metabolic activation        

mutation rate per test strain: with metabolic activation   

 concentration (µg/plate)

 TA98

TA100

TA1535 

TA1537 

WP2uvrA 

 TA98

TA100 

TA1535 

TA1537 

WP2uvrA 

 untreated control

 1.09

0.86 

1.00 

1.47 

1.04 

1.02 

0.97 

1.04 

1.18 

0.75 

 DMSO control

 1.00

 -

 -

1.00

-

 1.00

1.00 

1.00 

1.00 

1.00 

 ultrapure water control

 1.00

1.00

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

1.00 

test substance: 5000

 1.33

 1.14

 0.78

 1.60

 1.36

 0.78

 0.84

 1.11

1.24

 1.00

test substance: 1600

 1.07

 1.01

 1.05

 1.40

 1.56

 0.88

 0.85

 0.96

 1.29

 0.92

test substance: 500

 1.23

 0.95

 0.95

 1.60

 1.45

 0.78

 0.88

 0.96

 0.82

0.83

test substance: 160

 1.35

 1.10

 0.95

 0.80

 1.40

 1.02

0.88

 1.00

 0.94

 0.74

test substance: 50

 1.37

 0.99

 1.34

 1.40

 1.15

 0.87

 0.83

 0.86

 1.06

 0.74

test substance: 16

 1.05

 1.09

 0.88

 1.40

 1.25

 0.77

 0.95

 1.36

 1.06

 0.71

 NPD (4 µg)

 22.03

 

 

 

 

 

 

 

 

 

 SAZ (2 µg)

 

 9.87

 78.49

 

 

 

 

 

 

 

9AA (50 µg) 

 

 

 

 112.25

 

 

 

 

 

 

 MMS (2 µL)

 

 

 

 

 52.36

 

 

 

 

 

 2AA (2 µg)

 

 

 

 

 

 40.73

 11.83

 12.07

 15.16

 

 2AA (50 µg)

 

 

 

 

 

 

 

 

 

 4.59

Applicant's summary and conclusion

Executive summary:

The mutagenic potential of the substance was investigated in a bacterial reverse mutation assay according to OECD TG 471 under GLP. The substance was soluble and non-cytotoxic up to 5000 µg/plate, i.e. the recommended maximum concentration. The substance was tested in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation (S9).

In a first experiment, six concentration (16, 50, 160, 500, 1600 and 5000 µg/plate) were tested for 48 hours in the plate incorporation method. In the second experiment the same concentrations were tested (48 h) in the pre-incubation method. The number of colonies were evaluated by eye.

While the positive controls were clearly mutagenic, the substance did not increase the number of colonies in any strain in any experiment. Therefore, the substance is considered to be not mutagenic.