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 Dec 2017 - 04 Jan 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
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

1
Reference substance name:
Gum benzoin, Siam
EC Number:
232-556-7
EC Name:
Gum benzoin, Siam
Cas Number:
9000-72-0
IUPAC Name:
Benzoin resinoid obtained from the resin of Styrax tonkinensis by extraction with solvent
Test material form:
solid

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, dose range finding:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 2:
All strains (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: a solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide.
Controlsopen allclose all
Untreated negative controls:
yes
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: ICR-191: 2.5 μg/plate in DMSO for TA1537 (direct plate assay)
Remarks:
Without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA) in DMSO, 1 μg/plate for TA100 (direct plate assay) and for TA98, 2.5 μg/plate for TA1535 and TA1537, 5 μg/plate for TA100 (pre-incubation assay), 15 μg/plate for WP2uvrA
Remarks:
With S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 ± 2 minutes
- Exposure duration: 48 ± 4 hours

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

NUMBER OF CELLS EVALUATED: 10^9 bacteria per mL

DETERMINATION OF CYTOTOXICITY
- Method: 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. The condition of the bacterial background lawn was evaluated, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
INTERPRETATION OF RESULTS
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.

ACCEPTABILITY CRITERIA
- 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.
- 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.
- 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.
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:
no cytotoxicity
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:
no cytotoxicity
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:
at 5000 μg/plate (direct plate assay)
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:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Direct Plate Assay: Precipitation of the substance on the plates was observed at the start and the end of the incubation period at concentrations of 1600 μg/plate and upwards in all tester strains.
Pre-Incubation Assay: Precipitation of the substance on the plates was observed at the start of the incubation period at the concentration of 512 μg/plate and upwards. In the absence of S9-mix, at the end of the incubation period, precipitation of the test item on the plates was observed at dose levels of 164 μg/plate and upwards. In the presence of S9-mix, at the end of the incubation period, precipitation of the test item on the plates was observed at dose levels of 1600 and 5000 μg/plate, except for tester strain TA100 where precipitate was observed at dose levels of 512 μg/plate and upwards.

RANGE-FINDING/SCREENING STUDIES: In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix at the dose level of 1600 μg/plate. However, since the increase was just above the historical control database, less than two-fold and no dose relation was observed, this increase was not considered to be biologically relevant.

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 125 – 1248 73 – 1206 55 – 1353 54 – 1051 365 – 1995 250 – 1977
Mean 846 219 787 353 1406 887
SD 146 119 345 162 258 349
n 2348 2229 2003 2234 2200 2276

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1848 408 - 2651 93 – 1951 111 - 1359
Mean 901 1232 1094 437
SD 168 343 477 149
n 2335 2327 2021 2085

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Oct 2015 and Oct 2017.

- Negative (solvent/vehicle) historical control data:
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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Direct plate assay: cytotoxicity was only observed in tester strain TA98 in the absence and presence of S9-mix at the highest concentration (5000 μg/plate) as evidenced by a decrease in the number of revertants.
- Pre-incubation assay: no reduction of the bacterial background lawn observed and no biologically relevant decrease in the number of revertants up to and including concentrations of 1600 μg/plate. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, neither the number of revertants of this dose level could not be determined.

In both assays, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed in tester strain TA1537 (presence of S9). However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD TG 471 guideline and according to GLP principles.

The test was performed in two independent experiments, first a direct plate assay and second a pre-incubation assay, in the absence and presence of S9-mix. Adequate negative and positive controls were included. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The substance precipitated on the plates at and above 1600 μg/plate in both tester strains. In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The substance precipitated on the plates at and above 1600 μg/plate. Cytotoxicity was observed in tester strain TA98 (with and without S9) at the highest dose. No biologically relevant increase in the number of revertants was observed in any of the tester strains, with and without S9. In the second mutation experiment, the substance was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates in the absence of S9 in all tester strains at and above 164 μg/plate. In the presence of S9, precipitation was observed in all tester strains at dose levels of 1600 and 5000 μg/plate, except for TA100 where precipitate was observed at and above 512 μg/plate. No cytotoxicity was observed in any of the tester strains in the absence and presence of S9-mix. However, since the substance precipitated heavily at 5000 μg/plate in all tester strains, the number of revertants of this dose level could not be determined. 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. In conclusion, 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.