3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 test: negative

OECD 490 test: negative

OECD 487 test: negative

The data is conclusive but do not meet GHS criteria for classification.

No trigger of in vivo testing genetic toxicity.

Link to relevant study records

Reference

Reference 1

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)

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

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 / 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.

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.

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'.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-11 till 2016-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

tk+/- (thymidine kinase) locus in L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

tk+/- (thymidine kinase) locus in L5178Y cells

Additional strain / cell type characteristics:

other: tk+/- (thymidine kinase) locus in L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolising system.

Test concentrations with justification for top dose:

The test item was examined up to 2000 µg/mL (recommended highest concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125 and 62.5 µg/mL,
Assay 1, 3-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125 and 62.5 µg/mL,
Assay 2, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125 and 62.5 µg/mL,
Assay 2, 24-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125 and 62.5 µg/mL.

Vehicle / solvent:

Distilled water was used as vehicle of the test item in this study.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

Conclusions:

The objective of this study was to determine whether the test item induces genotoxicity (point mutations and/or gross chromosomal changes) at the thymidine kinase (tk) locus in L5178Y 3.7.2 C mouse lymphoma cells cultured in vitro in the absence and presence of a rat liver metabolising system. No insolubility and no cytotoxicity were observed. Therefore, concentrations up to the highest recommended concentration were selected in all experiments according to the instructions of the relevant OECD guideline 490 regarding non-cytotoxic test items.
No mutagenic effect of the test item was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-15 till 2016-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

This test was performed in the presence and absence of a rat liver metabolizing system (S9 mix of rats induced with Aroclor 1254).

Test concentrations with justification for top dose:

Since the test item was found freely soluble and non-cytotoxic in the preliminary test, the highest
dose-level selected for the cytogenetic experiment was 2000 μg/mL, according to the criteria
specified in the international regulations.

Vehicle / solvent:

water for
injections

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9 mix Clastogen Mitomycin C (MMC): 1 μg/mL; Without S9 mix Aneugen Colchicine (COL): 0.5 μg/mL; With S9 mix Clastogen Cyclophosphamide (CPA): 6 μg/mL.

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Evaluation criteria:

A cytogenetic experiment was considered valid if the following criteria were met:
. the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone
mitotis),
. the mean frequency of micronucleated cells in the vehicle control should be consistent with
(but not necessary within) control historical data of the Laboratory (Appendix 2). In any case,
this frequency should be ≤ 5‰,
. a statistically significant increase in the frequency of micronucleated cells had to be obtained
in the positive controls over the background frequency of the vehicle control cultures.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated
cultures was compared to that of the vehicle control cultures. This comparison was performed
using the χ2 test, unless treated culture data are lower than or equal to the vehicle control data.
P = 0.05 was used as the lowest level of significance. This statistical analysis was performed
using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies
of micronucleated cells and the dose-levels. This statistical analysis was performed using
SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Micronucleus analysis
For the three experimental conditions, the dose-levels selected for micronucleus analysis were
500, 1000 and 2000 μg/mL, the latter corresponding to the highest recommended dose-level.
Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix,
neither statistically significant nor dose-related increase in the frequency of micronucleated cells
was noted at any dose-levels relative to the vehicle controls (Tables 3, 5 and 7 and Appendix 4).
None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate
cultures above the vehicle control historical range.
These results met the criteria of a negative response.

Conclusions:

Under the experimental conditions of the study, the test item did not induce
any chromosome damage, or damage to the cell division apparatus, in cultured mammalian
somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a
rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no data

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No genetic toxicity was detected in three in vitro studies with the substance (w/wo metabolic activation).

Justification for classification or non-classification

Based on the available data, the substance is not classified for mutagenicity according to Regulation (EC) No 1272/2008.