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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-27 until 2015-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: per guideline study (GLP)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
These facts had no impact on the results or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
EC Number:
221-088-9
EC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
Cas Number:
3001-98-7
Molecular formula:
C7H14O6P2
IUPAC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3lambda5,9lambda5-diphosphaspiro[5.5]undecane-3,9-dione
Test material form:
solid: particulate/powder
Details on test material:
AFLAMMIT® PCO 962

Method

Target gene:
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Date of arrival and origin: 21 April 2015, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Additional strain / cell type characteristics:
other: see "Target gene" above
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
Test concentrations with justification for top dose:
Based on the results of a solubility test, the test item was formulated in Distilled water. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate; concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
In the study, two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals: Distilled water and Dimethyl sulfoxide (DMSO).
True negative controls:
no
Positive controls:
yes
Remarks:
Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test. 4-nitro-1,2-phenylene-diamine (NPD) and Methyl-methanesulfonate (MMS) were selected based on the scientific literature/experience.
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine and 2-aminoanthracene
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or; - a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and
Escherichia coli WP2 uvrA bacterial strains; - the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines [5][6][7], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a
reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual
examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants
per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
6.2. PRELIMINARY CONCENTRATION RANGE FINDING TEST
In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range, the observed minor differences were considered as biological variability of the test system.
No signs of inhibitory, cytotoxic effect were detected on the plates in the preliminary experiment.
No precipitate was detected in the Preliminary Concentration Range Finding Test.
The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.

6.3. INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test pre-incubation method was used.
The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests, each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate with and without metabolic activation. In the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate with and without metabolic activation.
In the Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration without metabolic activation (the mutation factor value was 1.52). However, there was no dose response, the observed mutation factor value was below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
In the Confirmatory Mutation Tests (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535
bacterial strain at 158.1 μg/plate concentration with metabolic activation. The observed mutation factor value was 1.62. However, there was no dose response, the observed mutation factor value was below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range. Furthermore, higher number of revertant colonies compared to the Distilled water control were detected for untreated control (MF: 1.31) also in this strain with metabolic activation. Thus, the observed values were considered as having no real biological relevance.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main test in some other sporadic cases. However, no dosedependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main test at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Test.
Precipitate was not detected in the Initial Mutation Test and Confirmatory Mutation Test.
The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 (Appendix 2) and Appendices 4-5.

6.4. VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated,
negative (solvent) and positive control plates were within the historical control range in all strains.
The examined concentration range was considered to be adequate as the highest examined concentration was the highest recommended concentration (5000 μg/plate). At least five analyzable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test, an Initial Mutation Test, a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation test the plate incorporation method was used. In the
Confirmatory Mutation Test, the pre-incubation method was used.
The reported data of this mutagenicity assay show (see Appendix 2 to 5) that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item had no mutagenic activity in the examined bacterial strains under the test conditions of this
study.