Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 September 2017 - 02 October 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
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:
Physical appearance: white powder
Storage conditions: at room temperature
Specific details on test material used for the study:
- Stability at higher temperatures: maximum temperature approximately 50°C, maximum duration: 120 minutes

Method

Target gene:
- S. typhimurium: Histidine gene
- E. 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 S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
Dose-range finding test (TA100 and WP2uvrA, without and with S9-mix): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 1 (TA1535, TA1537 and TA98, without and with S9-mix): 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2 (TA1535, TA1537, TA98, TA100 and WP2uvrA, without and with S9-mix): 52, 164, 512, 1600 and 5000 µg/plate
The tested dose-range was selected based on the results of the dose-range finding study.
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: DMSO has been accepted and approved by authorities and international guidelines. A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves until the test item had completely dissolved.
Test item concentrations were used within 1 hour after preparation.
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:
See 'any other information on materials and methods'
Details on test system and experimental conditions:
Two individual experiments were performed. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. The dose-range finding study was reported as part of the first experiment.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

PLATE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. 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 nonactivation 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 for 48 ± 4 h.

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: 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.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following 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 the 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.

INTERPRETATION
A test substance 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 substance is considered positive 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.

Results and discussion

Test results
Key result
Species / strain:
other: all strains tested (TA1535, TA1537, TA98, TA100 and WP2uvrA)
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:
PRECIPITATION
In both experiment, precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

CYTOTOXOCITY
In both experiments, no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants was observed at any of the concentrations tested in all tester strains, without and with S9-mix.
- Other: in the pre-incubation assay, in tester strain TA100, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 5000 μg/plate in the absence of S9-mix. However since the reduction in the mean number of revertant colonies was only minor when compared against relevant historical control data and no toxicity was observed in any of the other tester strains in both experiments, this reduction is caused by an incidental fluctuation in the number of revertant colonies and is considered as not biologically relevant.

MUTAGENICITY
In both experiments, no increase in the number of revertants was observed in any of the strains, at any of the doses tested, with and without S9-mix.

HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 2 Historical control 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

Table 3 Historical control data of the positive controls

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

125 - 1381

78 - 1058

55 – 1324

55 – 1051

410 – 1995

250 - 1977

Mean

839

220

736

382

1369

929

SD

153

112

331

150

310

345

n

2065

1967

1740

1933

1920

2014

 

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

537 – 1848

408 - 2651

93 – 1951

93 - 1359

Mean

908

1330

1128

422

SD

178

324

484

151

n

2007

2020

1679

1728

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:
In an AMES test, performed according to OECD 471 guideline and in accordance with GLP principles, HYDROPIP-NR was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.
Executive summary:

An AMES test was performed, according to OECD guideline 471 and GLP principles, to assess the genotoxic properties of HYDROPIP-NR. All bacterial strains showed negative responses up to 5000µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that HYDROPIP-NR is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptability criteria were met, the study was considered valid.