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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2017 - 11 October 2017
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

Constituent 1
Chemical structure
Reference substance name:
4-methyl-1-oxaspiro[5.5]undec-4-ene
Cas Number:
62062-89-9
Molecular formula:
C11-H18O
IUPAC Name:
4-methyl-1-oxaspiro[5.5]undec-4-ene
Constituent 2
Chemical structure
Reference substance name:
4-methyl-1-oxaspiro[5.5]undec-3-ene
Cas Number:
62062-94-6
Molecular formula:
C11H18O
IUPAC Name:
4-methyl-1-oxaspiro[5.5]undec-3-ene
Constituent 3
Chemical structure
Reference substance name:
4-methylidene-1-oxaspiro[5.5]undecane
Cas Number:
62062-84-4
Molecular formula:
C11H18O
IUPAC Name:
4-methylidene-1-oxaspiro[5.5]undecane

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 fanding:
TA 100 and WP2uvrA (without and with S9): 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 5.4, 17, 52, 164, 512 and 1600 μg/plate
- Experiment 2:
TA 1535, TA 1537, TA 98 and TA 100 (without and with S9): 17, 52, 164, 512 and 1600 μg/plate
WP2uvrA (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 2A:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 0.55, 1.7, 5.4, 17 and 52 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
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
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
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
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 minutes
- Exposure duration: 48 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 condition of the bacterial background lawn and the presence of microcolonies were evaluated, macroscopically and/or microscopically by using a dissecting microscope.
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:
at 1600 μg/plate (direct plate assay); at 52 μg/plate and upwards (pre-incubation assay)
Vehicle controls validity:
valid
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:
at 1600 μg/plate (direct plate assay); at 164 μg/plate and upwards (pre-incubation assay)
Vehicle controls validity:
valid
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 1600 μg/plate (direct plate assay); at 164 μg/plate and upwards (pre-incubation assay)
Vehicle controls validity:
valid
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 1600 and 5000 μg/plate (direct plate assay); at 52 μg/plate and upwards (pre-incubation 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:
at 5000 μg/plate (direct plate assay); at 164 μg/plate and upwards (pre-incubation assay)
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 on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards in tester strains TA100 and WP2uvrA and in tester strains TA1537, TA1535 and TA98 at concentration of 1600 μg/plate. At the end of the incubation period precipitation on the plates was observed at 1600 μg/plate in tester strains TA1537, TA1535 and TA98 and at 1600 and 5000 μg/plate in tester strains TA100 and WP2uvrA, except in tester strain TA100 in the presence of S9-mix were precipitation of the test item on the plates was observed at test concentration of 512 μg/plate and above at the
end of the incubation period.
Pre-Incubation Assay: Precipitation of the test item on the plates was only observed in tester strain WP2uvrA at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 and 5000 μg/plate at the end of the incubation period.
Additional pre-incubation assay: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: A moderate reduction of the bacterial background lawn was observed at 5000 μg/plate in tester strain WP2uvrA. In tester strain TA100 an extreme reduction of the bacterial background lawn was observed with microcolonies at test concentrations of 1600 and 5000 μg/plate in the absence and 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 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: In strain TA1537 in the absence of S9-mix, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 5.4 μg/plate. Due to the low mean number of revertant colonies in the solvent control (5), the mean number of 2 revertants is not considered to be a toxic effect.
A moderate reduction of the bacterial background lawn was observed at 5000 μg/plate in tester strain WP2uvrA and at 1600 μg/plate in tester strains TA1537, TA1535 and TA98. In tester strain TA100 an extreme reduction of the bacterial background lawn was observed with microcolonies at test concentrations of 1600 and 5000 μg/plate in the absence and presence of S9-mix.
- Pre-incubation assay: Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, resulting in only 2 or 3 analyzable concentrations per tester strain.
- Additional pre-incubation assay: In the absence of S9-mix, at a test concentration of 52 μg/plate, a slight reduction in the number of revertants was observed in tester strain TA98 and TA100. In tester strain TA100,
a slight reduction in the number of revertants was also observed in the presence of S9-mix at a concentration of 52 μg/plate. A slight reduction of the bacterial background lawn was observed in tester strain TA1535 at 52 μg/plate in the absence of S9-mix. In all other tester strains no cytotoxicity was observed.

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 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 first, 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 test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate in both tester strains. Except in tester strain TA100 in the presence of S9-mix were precipitation on the plates was observed at dose levels of 512 μg/plate and upwards. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Cytotoxicity was observed at dose levels of 1600 and 5000 μg/plate in TA100 and at 5000 μg/plate in WP2uvrA.

In the first mutation experiment, the test item was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the highest tested dose level. The bacterial background lawn was moderately reduced at 1600 μg/plate in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment the test item was tested up to concentrations of 1600 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to concentrations of 5000 μg/plate in tester strain WP2uvrA in the pre-incubation assay. The test item only precipitated on the plates in tester strain WP2uvrA at dose levels of 1600 and 5000 μg/plate. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Due to cytotoxicity, colonies could only be counted from two or three concentrations in all tester strains in the absence and presence of S9-mix in the second mutation experiment. Therefore an additional mutation experiment was performed.

In the additional experiment the test item was tested at concentration ranges of 0.55 to 52 μg/plate in the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item did not precipitate on the plates at these dose levels. In the absence of S9-mix, at a test concentration of 52 μg/plate, a slight reduction in the number of revertants was observed in tester strain TA98 and TA100. In tester strain TA100, a slight reduction in the number of revertants was also observed in the presence of S9-mix at a concentration of 52 μg/plate. A slight reduction of the bacterial background lawn was observed in tester strain TA1535 at 52 μg/plate in the absence of S9-mix.

The test item 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 in all performed mutation experiments.

In conclusion, based on the results of this study it is concluded that Oxaspirane-819 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.