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
Study period:
22 May 2016 - 06 June 2016
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:
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
Type:
impurity
Type:
impurity
Test material form:
solid
Details on test material:
Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container

Method

Target gene:
- S. typhimurium: Histidine gene
- E. 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 with 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: 0.18, 0.55, 1.7, 5.4, 17 and 52 µg/plate
Experiment 2 (all 5 strains):
Without S9-mix: 0.18, 0.55, 1.7, 5.4, 17 and 52 µg/plate
With S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: without S9, ICR-191 2.5 µg/plate in DMSO for TA1537 (direct plate)
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), pre-incubation assay (second experiment)

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

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.
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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix, except in strain TA1537 with S9-mix (direct plate assay).
Vehicle 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:
cytotoxicity
Remarks:
Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix, except in strain WP2uvrA with S9-mix (pre-incubation).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Direct plate assay: Precipitation of the test item was not observed at the start or at the end of the incubation period.
Pre-incubation assay: Precipitation of the test item was not observed at the start or at the end of the incubation period, except in tester strain WP2uvrA, where slight precipitation was observed at the top concentration of 52 μg/plate in the absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 17 μg/plate and above in the absence of S9-mix and at dose levels of 52 μg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 164 μg/plate and above in the absence of S9-mix and at dose levels of 512 μg/plate and above in the presence of S9-mix.

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 - 1381 78 - 1058 55 – 1565 55 – 1112 410 – 2057 263 - 1907
Mean 785 228 653 387 1155 860
SD 167 105 290 143 370 323
n 1684 1662 1448 1536 1646 1686

TA100 WP2uvrA
S9-mix - + - +
Range 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean 892 1404 1263 342
SD 178 327 461 165
n 1650 1677 1370 1410
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

- Negative (solvent/vehicle) historical control data:

TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 4 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 12 – 68 12 - 70
Mean 14 13 7 9 17 25 100 103 26 32
SD 6 5 3 4 5 7 16 18 7 8
n 1662 1677 1548 1547 1662 1703 1659 1691 1421 1424
SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Direct plate assay:
TA1535: without and with S9: 52 µg/plate
TA1537: without S9: 52 µg/plate
TA98: without and with S9: 52 µg/plate
TA100: without S9: 17 µg/plate and above and with S9: 52 µg/plate and above
WP2uvrA: without S9: 164 µg/plate and above and with S9: 512 µg/plate and above
Pre-incubation assay:
TA1535: without S9: 5.4 µg/plate and above and with S9: 52 µg/plate and above
TA1537: without S9: 5.4 µg/plate and above and with S9: 52 µg/plate and above
TA98: without S9: 5.4 µg/plate and above and with S9: 164 µg/plate
TA100: without S9: 5.4 µg/plate and above and with S9: 52 µg/plate and above
WP2uvrA: without S9: 52 µg/plate

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 guideline and GLP principles, the test substance was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles. In the first mutation experiment, the test item was tested tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrAu and up to concentrations of 52 µg/plate in the strains TA1535, TA1537 and TA98 in the direct plate assay. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in all three tester strains in the absence and presence of S9-mix, except in strain TA1537 in the presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 52 and 164 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay in the absence and presence of S9-mix, respectively. The test item precipitated on the plates at the top dose of 164 μg/plate in tester WP2uvrA (absence of S9-mix) only. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix, except in strain WP2uvrA in the presence of S9-mix.

All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. 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 the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.