Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2016 - 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP conditions

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:
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 reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
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:
Direct plate:
- Dose-range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Based on the results of experiment 1, the following dose levels were used:
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate
Preincubation:
- Experiment 2:
Based on the results of experiment 1, the following dose levels were used:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(100 μL/plate DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (all strains; with metabolic activation) / ICR191 (TA1537; without metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat) preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation
Evaluation criteria:
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:
Not performed.

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 experiment 2 at the highest tested dose.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 experiment 2 at the highest tested dose.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 experiment 2 at the highest tested dose.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
at the highest tested dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
cytotoxicity
Remarks:
in experiment 2 at the highest tested dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed at the start or at the end of the in cubation period.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 (absence and presence of S9-mix). No increase in the number of revertants was observed upon treatment with Peomosa under any conditions tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:

TA1535 TA1537 TA98
S9-mix - + - + - +
Range 78 - 1932 81 - 1332 62 – 1565 55 – 1112 347 – 1967 261 - 1885
Mean 791 234 662 409 976 821
SD 261 98 206 126 251 298
n 1732 1737 1409 1428 1721 1737

TA100 WP2uvrA
S9-mix - + - +
Range 549 – 1798 640 - 2760 123 – 1958 85 - 1390
Mean 914 1387 1367 261
SD 150 324 276 276
n 1734 1752 1373 1404

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between November 2013 and January 2016.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 5 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 13 – 68 12 - 70
Mean 17 14 7 9 18 26 104 105 28 34
SD 6 5 3 3 6 7 17 17 7 7
n 1644 1716 1425 1443 1707 1730 1725 1739 1368 1404

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between November 2013 and January 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA1537 (absence of S9-mix) at the dose level of 5000 μg/plate. In the other tester strains, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of S9-mix.
Experiment 2:
In all strains, cytotoxicity, as evidenced by decreases in the number of revertants and reductions in the bacterial background lawn, was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. In tester strain TA98 (absence of S9-mix) also a slight reduction in the bacterial background lawn was observed at the dose level of 1600 μg/plate.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance was determined to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC) and its amendments.
Executive summary:

The mutagenic activity of Peomosa was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. The dose levels were selected based on a dose range finding test with strain TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 (absence and presence of S9-mix). In experiment 2 in all strains, cytotoxicity, as evidenced by decreases in the number of revertants and reductions in the bacterial background lawn, was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. In tester strain TA98 (absence of S9-mix) also a slight reduction in the bacterial background lawn was observed at the dose level of 1600 μg/plate. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that Peomosa is not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC)

and its amendments

.