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:
Start of experimental phase: 30 June 2015 and End of experimental phase: 17 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-{octahydro-1H-4,7-methanoindene-2,5-diylbis[(2,1-phenylene)oxymethylene]}bis(oxirane)
EC Number:
943-519-6
Cas Number:
2149603-92-7
Molecular formula:
C28H32O4
IUPAC Name:
2,2'-{octahydro-1H-4,7-methanoindene-2,5-diylbis[(2,1-phenylene)oxymethylene]}bis(oxirane)
Test material form:
not specified

Method

Target gene:
The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth. The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured. The E. coli tester strains of the WP2 series are similarly mutant at the tryptophan locus.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/5,6-Benzoflavone induced rat liver, S9.
Test concentrations with justification for top dose:
Preliminary toxicity test: 50.0/158/500/1580 and 5000 μg/plate.

Main assay:
- TA1535/TA98 and TA100 with and without S9: 5000, 2500, 1250, 625, 313 μg/plate.
- TA 1537: 2500, 1250, 625, 313, 156, 78.1, 39.1 μg/plate (with S9 mix)
- TA 1537: 5000, 2500, 1250, 625, 313 μg/plate (without S9 mix)
- WP2 uvrA: 5000, 2500, 1250, 625, 313, 156, 78.1 μg/plate (with S9 mix)
- WP2 uvrA: 5000, 2500, 1250, 625, 313 μg/plate (without S9 mix)
Vehicle / solvent:
Solutions of the test item, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hour and 12 minutes from the initial formulation.

No assay of test item stability, nor its concentration and homogeneity in solvent were undertaken, as not requested by the Sponsor.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in water
Positive control substance:
sodium azide
Remarks:
For TA 1535 and TA100 (at 1 μg/plate), without S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA 1535 and TA100 (at 1 μg/plate), with S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in DMSO
Positive control substance:
9-aminoacridine
Remarks:
For TA 1537 (at 50 μg/plate), without S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA 1537 (at 1 μg/plate), with S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in DMSO
Positive control substance:
2-nitrofluorene
Remarks:
For TA 98 (at 2 μg/plate), without S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA 98 (at 1 μg/plate), with S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in water
Positive control substance:
methylmethanesulfonate
Remarks:
For WP2 uvrA (at 500 μg/plate), without S9 mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Prepared in DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
For WP2 uvrA (at 0 μg/plate), with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
-Preliminary toxicity test: single plate
-Main assay: Three replicate plates
Evaluation criteria:
Criteria for outcome of the assays
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at
the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies
with increasing dose levels.
Statistics:
Regression line
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal
distribution and homoscedasticity assumptions. The regression equation is expressed as:
y= a+ bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation
co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at the highest or two highest dose levels, both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at the highest or two highest dose levels, both in the absence and presence of S9 metabolism.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility:
Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the
bacteria and the S9 metabolic activity. The test item was found to be soluble at 100 mg/mL. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

Toxicity test
Precipitation of the test item was observed, at the end of the incubation period and in all tester strains, at the highest dose level, in the presence of S9 metabolism. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies, was observed at the two highest dose levels with TA1537 and WP2 uvrA tester strains, in the presence of S9 metabolism. Large increases in the number of revertant colonies were observed with TA1535 in the presence of S9 metabolism.



:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results.

Main Test:

Precipitation of the test item was observed with all tester strains, at the highest or two highest dose levels, both in the absence and presence of S9 metabolism.

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies, was observed in TA1537 and WP2 uvrA tester strains, at higher dose levels, in the presence of S9 metabolism. Large increases in the number of the revertant colonies were observed at all concentrations tested, with TA1535 tester strain, in the presence of S9 metabolism. These increasee were greater than twice the concurrent negative control values and fell outside the distribution of the historical control data and so can be considered a clear evidence of mutation induction. Slight increases in revertant colonies were also observed at lower concentrations with TA100 tester strain, in the presence of S9 metabolic activation. The revertant numbers at these concentrations were higher than the upper confidence limit of the historical control range. The sterility of the S9 mix and of the test item solutions was confirmed by

the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in this test following treatment with the positive control items, indicating that the assay system was functioning correctly.

Since a clear positive result was obtained, no further experiment was undertaken.

Evaluation

Results show that mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control data included (confidence interval: mean value +/-2 standard deviations).

The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.

The test item induced dose-related and statistically significant increases based on a “doubling rate” in TA1535 tester strain, in the presence of S9 metabolism.

HISTORICAL CONTROL DATA WITH AND WITHOUT METABOLIC ACTIVATION (2000-2015)

             TA 1535 Plate incorporation
      Negative control     
   Without S9  With S9   Without S9   With S9
 Media  18 17  537  134 
 SD  2.6  2.1  75.6 27.4 
 UCL  23.5  21.1  688.6 189.2 
 LCL  13.2  12.3  386.3 79.5 
 n  328  329  328 329 
 min  13  12  299 85 
 max  26  22  750 241 

             TA 1537 Plate incorporation
      Negative control     
   Without S9  With S9   Without S9   With S9
 Media  18 21  181 116
 SD  2.1  2.7  4.3 19.6
 UCL  21.8  26.8  261.4 155.7
 LCL  13.3  15.8  100.2 77.3
 n  328  328  328 328
 min  11  15  92 75
 max  27  33  407 181

             TA 98 Plate incorporation
      Negative control     
   Without S9  With S9   Without S9   With S9
 Media  31 39  170 535
 SD 3.3  3.7  26.0 153.9
 UCL  38.0  46.3  221.8 842.7
 LCL  24.8  31.5  117.7 227.2
 n  333  332  333 332
 min  23  31 119 247
 max  43  53  259 994

             WP2 uvrA Plate incorporation
      Negative control     
   Without S9  With S9   Without S9   With S9
 Media  28 33  180 200
 SD 3.0  3.4  18.4 34.2
 UCL  34.0  40.2  216.6 268.0
 LCL  22.0  26.7  143.1 131.2
 n  263  263  263 263
 min  21  26 137 117
 max  38  47  240 333

             TA 100 Plate incorporation
      Negative control     
   Without S9  With S9   Without S9   With S9
 Media 142 152 695 1203
 SD 16.2  16.8  140.2 216.3
 UCL  174.1  185.5  975.7 1635.2
 LCL  109.9  118.2  415.0 770.1
 n  332  332  332 332
 min  113  86 445 607
 max  182  196  1081 2257

SD: Standard deviation

UCL: Upper Confidence Limit (mean value +2SD)

LCL: Lower Confidence Limit (mean value -2SD)

n: Number of experiments

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

It is concluded that the test item induces reverse mutation by base substitution in bacteria, in the presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5-6 Benzoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test

The test item was assayed in the toxicity test at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Precipitation of the test item was observed in all tester strains, at the end of the incubation period, at the highest dose level, in the presence of S9 metabolism. Toxicity was observed with TA1537 and WP2 uvrA tester strains at the two highest concentrations tested, in the presence of S9 metabolism. Increases in the number of revertant colonies were observed with TA1535, in the presence of S9 metabolism.

Main Assay:

On the basis of toxicity test results, in Main Assay, using the plate incorporation method, the test item was assayed at the following dose levels:

 Tester strain S9  Dose level (μg/plate)  
 TA1535, TA98, TA100  +/-  5000, 2500, 1250, 625, 313
 TA1537  +  2500, 1250, 625, 313, 156, 78.1, 39.1
 TA1537  -  5000, 2500, 1250, 625, 313
 WP2 uvrA  +  5000, 2500, 1250, 625, 313, 156, 78.1
 WP2 uvrA  -  5000, 2500, 1250, 625, 313

Precipitation of the test item was observed in all tester strains, at the highest or at the two highest dose levels, both in the absence and presence of S9 metabolism.

Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed in TA1537 and WP2 uvrA tester strains, at higher dose levels, in the presence of S9 metabolism. Large increases in the number of the revertant colonies were observed at all concentrations tested, with TA1535 tester strain, in the presence of S9 metabolism. These increases were greater than twice the concurrent negative control values and fell outside the distribution of the historical control data and so can be considered a clear evidence of mutation induction. Based on this result, no further experiment was undertaken.

Conclusion

It is concluded that the test item induces reverse mutation in bacteria, in the presence of S9 metabolism, under the reported experimental conditions. RTC Study