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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:
05 January 2018 - 25 January 2018
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 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
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline S2(R1). Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human use. CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Off-white powder
Test item storage: At room temperature
Specific details on test material used for the study:
The test item is toxic to reproduction.

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan 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, reported as part of the first experiment (TA100 and WP2uvrA):
Without and with S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item exhibited limited solubility.

First experiment (TA1535, TA1537 and TA98):
Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate.
The top dose was selected based on the results of the dose-range finding study.

Second experiment (all tester strains):
Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1000 μg/plate.
The top dose was selected based on the results of the first experiment.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: according to guidelines.
Controls
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-aminoanthracene
Remarks:
For details on the positive controls, see table 1 in 'Additional information on materials and methods'
Details on test system and experimental conditions:
The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3 (in both independent experiments)

EXPERIMENTAL DESIGN: 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 Milli-Q water 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

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.

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 laboratory.
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 results
Key result
Species / strain:
other: All tested strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Cytotoxicity: in both experiments, without and with S9, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

- Mutagenicity: in the first experiment, in tester strain WP2uvrA in the presence of S9-mix, the test item induced a 2.2-fold increase
in the number of revertant colonies compared to the solvent control. In the second experiment, in tester strain TA1537 in the absence of S9-mix, the test item induced a 3.0-fold increase in the number of revertant colonies compared to the solvent control. However, both these increases were only observed in one experiment. Furthermore, the increases were within the historical control data range. Therefore, the increases are considered to be not biologically relevant.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the dose range finding test, precipitation was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards. In the first experiment, precipitation was observed at the concentration of 1600 μg/plate at the start of the incubation period and at 512 μg/plate and above at the end of the incubation period.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: reported as the first experiment

HISTORICAL CONTROL DATA: see table 2 and 3 in 'Any other information on results'
- Results of the solvent control and of the positive control were within the historical data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Any other information on results incl. tables

Table 2 Historical data for the solvent control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 - 29

3 - 27

3 – 20

3 – 23

8 - 41

8 - 55

63 - 176

54 - 160

10 – 59

9 - 69

Mean

10

11

6

7

16

23

108

107

25

32

SD

3

4

2

3

5

7

19

20

7

8

n

2356

2336

2264

2235

2319

2360

2341

2336

2075

2078

SD = Standard deviation; n = Number of observations

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

Table 3 Historical data for the positive control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

-

+

-

+

+

-

+

Range

125 -1248

73 - 1206

55 – 1353

54 – 1051

365 - 1995

250 – 1977

439 - 1848

408 –2651

93 – 1951

111 - 1359

Mean

846

219

787

353

1406

887

901

1232

1094

437

SD

146

119

345

162

258

349

168

343

477

149

n

2348

2229

2003

2234

2200

2276

2335

2327

2021

2084

SD = Standard deviation; n = Number of observations

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

Applicant's summary and conclusion

Conclusions:
The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that D6-Noreth is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.