Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2017 - 21 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial forward mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical appearance: dark brown viscous liquid
- Storage conditions: at room temperature
Specific details on test material used for the study:
- No correction for purity required
- Stability and solubility in vehicle: not indicated
-The test material is a UVCB

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryprophan gene
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:
Rat liver microsomal enzymes (S9), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98, with and without S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (all strains, with and without S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Solvent used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the solvent is according to OECD guideline 471 and the test substance dissolved in DMSO
Controls
Untreated negative controls:
yes
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-aminoanthracene
Remarks:
See table 1 in 'Any other information on materials and methods incl. table'.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Glucose agar medium contained: 18 g/L purified agar, 20 g/L glucose, 12.5 μg/plate biotin and 15 μg/plate histidine (Salmonella typhimurium strains) or 15 μg/plate tryptophan (Escherichia coli strain)

The mutation assay was performed in two indipendent experiments: in the first, the test item was tested both in the presence and absence of 5% (v/v) S9-mix. In a follow-up experiment, the test item was tested both in the absence and presence of 10% (v/v) S9-mix.

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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 follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the second experiment at 1600 and 5000 μg/plate (without S9)
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:
cytotoxicity
Remarks:
In the second experiment at 1600 and 5000 μg/plate (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
In the second experiment at 1600 and 5000 μg/plate (without S9) and at 5000 μg/plate (with S9)
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:
cytotoxicity
Remarks:
In the second experiment at 1600 and 5000 μg/plate (without S9) and at 5000 μg/plate (with S9)
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
- Precipitation: in the first experiment, no precipitation was observed at the start and at the end of the incubation period. In the second experiment, precipitation was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate, no precipitation was observed at the end of the incubation period.

First experiment:
- Cytotoxicity, as evidenced by a decrease in the number of revertants, a reduction of the bacterial background lawn and/or the presence of or microcolonies, was observed in all tester strains in the presence and absence of S9-mix.
- No increase in the number of revertants was observed.

Second experiment:
- Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies,was observed in the absence S9-mix in all tester strains at 1600 and 5000 μg/plate, except in tester strain WP2uvrA. In the presence of S9-mix, cytotoxicity was only observed in the tester strains TA98 and TA100 at the highest dose level tested.
- In strain TA100 in the absence of S9-mix, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 2800 μg/plate. Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- No increase in the number of revertants was observed.

HISTORICAL CONTROL DATA (see table 2 and 3 in 'Any other information on results incl. tables')
- Positive historical control data: strain-specific control values were within the laboratory historical control data ranges.
- Negative (solvent) historical control data: negative control values were within the laboratory historical control ranges.
These results indicate that the test conditions were adequate and that the metabolic acitivation system functioned properly.

Any other information on results incl. tables

Table 2 Historical data of the solvent control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 - 36

3 - 32

3 – 20

3 – 23

8 - 41

9 - 55

66 - 161

63 - 160

10 – 59

9 - 69

Mean

11

11

6

7

16

23

105

105

25

31

SD

4

4

3

3

5

7

19

20

7

8

n

2057

2039

1950

1931

2023

2083

2027

2033

1739

1745

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Table 3 Historical data of the positive controls

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

-

+

-

+

+

-

+

Range

125 - 1381

78 - 1058

55 – 1324

537 – 1848

408 - 2651

93 – 1951

93 - 1359

55 – 1051

410 – 1995

250 - 1977

Mean

839

220

736

908

1330

1128

422

382

1369

929

SD

153

112

331

178

324

484

151

150

310

345

n

2065

1967

1740

2007

2020

1679

1728

1933

1920

2014

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Applicant's summary and conclusion

Conclusions:
The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that X-19933 salt is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.