Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
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:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
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

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study:
TA1535, TA1537 and TA98; Without and with S9-mix: 5.4, 17, 52, 164, 512, and 1600 µg/plate

Experiment 2: pre-incubation assay
Without and with S9-mix: 5.4, 17, 52, 164 and 512 µg/plate

The test item was tested up to a dose level which showed precipitation on the plates.
Vehicle:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be in DMSO and DMSO is accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9: 2.5 µg/plate in DMSO for TA1537 (direct plate)
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 2.5 µg/plate in DMSO for TA1537 (pre-incubation)
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO with S9
Remarks:
2.5 μg/plate TA1535/TA1537, 1 μg/plate TA98/TA100(direct plate), 5 μg/plate TA100(pre-incubation), 15 μg/plate WP2uvrA
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

Pre-incubation experiment:
0.1 ml of a dilution of the test item in DMSO and 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains were pre-incubated for 30 minutes by 70 rpm at 37°C, with 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).


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

NUMBER OF CELLS EVALUATED: 10E8 per plate

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.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Rationale for test conditions:
To obtain more information about the possible mutagenicity of Norethyl, a pre-incubation experiment was performed in the absence and presence of S9-mix.
Evaluation 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 WIL Research Europe.
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.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 512 μg/plate
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding test: Precipitation of Norethyl on the plates was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards. Except for strain TA100 in the absence of S9-mix, where precipitation was already observed at 164 μg/plate at the end of the incubation period.
First mutation experiment: Precipitation of Norethyl on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 164 μg/plate and above at the end of the incubation period.
Pre-incubation experiment: Precipitation of Norethyl on the plates was observed at the start and at the end of the incubation period at concentrations of 164 μg/plate and upwards.

RANGE-FINDING/SCREENING STUDIES:
No reduction of the bacterial background lawn revertants was observed. No biologically relevant decrease in the number of revertants was observed up to the dose level of 512 μg/plate, except for tester strain TA100 where a slight reduction of the number of revertant colonies was observed in the presence of S9-mix at the dose level of 512 μg/plate. Since Norethyl precipitated heavily on the plates at the test item concentration of 1600 and 5000 μg/plate (dose range finding test) in tester strains TA100 (presence of S9-mix) and WP2uvrA (absence and presence of S9-mix), the number of revertants of these dose levels could not be determined.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative control values were within the laboratory historical control data ranges.
- The 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, except the responses for TA100 (absence of S9-mix in the dose range finding) and TA98 and WP2uvrA (absence of S9-mix in the second mutation experiment). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the values were more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate counts of the positive controls had no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD guideline and GLP principles, Norethyl was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed with Norethyl according to OECD guideline and GLP principles.

In the dose range finding study, Norethyl was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards, except in tester strain TA100 (absence of S9-mix) where precipitation was observed at 164 μg/plate already. Since Norethyl precipitated heavily on the plates at the test item concentrations of 1600 and 5000 μg/plate in tester strains TA100 (presence of S9-mix) and WP2uvrA (absence and presence of S9-mix), the number of revertants of these dose levels could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the presence of S9-mix at the dose level of 512 μg/plate. In the first mutation experiment, Norethyl was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, Norethyl was tested up to concentrations of 512 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Norethyl did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Norethyl is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.