2,4,8,10-tetraoxa-3λ6,9λ6-dithiaspiro[5.5]undecane 3,3,9,9-tetraoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021 April

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,8,10-tetraoxa-3λ⁶,9λ⁶-dithiaspiro[5.5]undecane-3,3,9,9-tetrone

EC Number:

859-869-7

Cas Number:

201419-80-9

Molecular formula:

C5H8O8S2

IUPAC Name:

2,4,8,10-tetraoxa-3λ⁶,9λ⁶-dithiaspiro[5.5]undecane-3,3,9,9-tetrone

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test item: 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane,
3,3,9,9-tetraoxide
CAS No.: 201419-80-9
Lot number: AZ08AVL1
Assay (by GC): 99.9 %
Appearance: crystalline solid, white
Expiration date: 21 September 2021
Storage conditions: room temperature, protected from humidity, well-closed
container
Safety precautions: According to the MSDS

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

5.5 Metabolic Activation System
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic
activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).
5.5.1 Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was
provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
The Quality Control & Production Certificate of each lot of S9 was obtained from the
supplier. The original Quality Control & Production Certificates of rat liver S9 are stored in
the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the
used S9 lots are given in Appendix VIII. The following lots of the S9 were applied:
Lot Number: 4063; Expiry date: February 21, 2021; Protein content: 33.8 mg/mL
(used in the informatory toxicity test);
Lot Number: 4086; Expiry date: April 04, 2021; Protein content: 35.9 mg/mL
(used in the informatory toxicity and confirmatory mutation tests);
Lot Number: 4088; Expiry date: April 11, 2021; Protein content: 34.7 mg/mL
(used in all experimental phases of the study);
Lot Number: 4146; Expiry date: September 19, 2021; Protein content: 38.5 mg/mL
(used in the informatory toxicity and initial mutation tests);
Lot Number: 4335; Expiry date: October 06, 2022; Protein content: 38.6 mg/mL
(used in the confirmatory mutation test).
5.5.2 The S9 Mix (with Rat Liver S9)
Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.
The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix (containing 10 % S9) was kept in an ice bath before it was added to the culture
medium.

5.5.3 Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL
Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction
was performed with a mixture of solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm
membrane filter.

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
Recommended maximum concentration according to the OECD 471 guidance is 5000 µg/plate

Based on the results of the preliminary tests, the test item solutions were prepared from the
test item with dimethyl sulfoxide (DMSO) to obtain six dosing solutions. The maximum test
concentration was 5000 µg/plate (±S9).
For the selected concentration range, the absent cytotoxicity and the noticed solubility
properties of the test item were taken into consideration based on the recommendations in
OECD 471 guideline [6]. The recommended maximum test concentration of 5 mg/plate was
chosen as highest test item concentration for initial and confirmatory mutation tests. As
indicated in section 6.1.2, at this top concentration precipitate was expected on the minimal
glucose agar plates. The 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide concentrations investigated in the initial and confirmatory mutation tests:
±S9: 5000, 2000, 800, 320, 128 and 51.2 µg/plate.
The test solutions were freshly prepared at the beginning of the experiments. At the
preparation of the test item solutions any correction (multiplier) factor was not taken into
consideration, the doses were based on the final formulation as is.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Details on test system and experimental conditions:

Origin of the Bacterial Strains
Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia
coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH,
Rathenau Str. 2;
D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.
The identification codes and expiry dates of the actual applied stock cultures are summarized in the Table 3.

Rationale for test conditions:

Based on the results of the preliminary tests, the test item solutions were prepared from the
test item with dimethyl sulfoxide (DMSO) to obtain six dosing solutions. The maximum test
concentration was 5000 µg/plate (±S9).
For the selected concentration range, the absent cytotoxicity and the noticed solubility
properties of the test item were taken into consideration based on the recommendations in
OECD 471 guideline [6]. The recommended maximum test concentration of 5 mg/plate was
chosen as highest test item concentration for initial and confirmatory mutation tests. As
indicated in section 6.1.2, at this top concentration precipitate was expected on the minimal
glucose agar plates. The 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide concentrations investigated in the initial and confirmatory mutation tests:
±S9: 5000, 2000, 800, 320, 128 and 51.2 µg/plate.
The test solutions were freshly prepared at the beginning of the experiments. At the
preparation of the test item solutions any correction (multiplier) factor was not taken into
consideration, the doses were based on the final formulation as is.

Evaluation criteria:

The colony numbers on the untreated, vehicle control, positive control and the test item
treated plates were counted manually, evaluated by unaided eye, and the mean values,
standard deviations and the mutation rates were calculated:
Mutation Rate = Mean number of revertants at the test item (or control*) treatments / Mean number of revertants of vehicle control

A result is considered positive if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups
occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high
as the reversion rate of the vehicle control,
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of
reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic 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, vehicle control, positive control and the test item
treated plates were counted manually, evaluated by unaided eye, and the mean values,
standard deviations and the mutation rates were calculated:
Mutation Rate = Mean number of revertants at the test item (or control*) treatments / Mean number of revertants of vehicle control

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show 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 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide has no mutagenic activity on the applied bacterial tester strains under the
test conditions used in this study.

Executive summary:

Purpose of the study:
The test item2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxidewas
tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation
Assay.
Bacterial strains, exogenous metabolic activation:
The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella
typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the
tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in
the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of
Phenobarbital/β-naphthoflavone-induced rats.
Experimental phases:
The study included preliminary solubility investigations, a preliminary concentration range
finding test (informatory toxicity test applying the plate incorporation method), an initial
mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation
test).
Vehicle, test item concentrations, rationale for dose selection:
Based on the results of the solubility test and the concentration range finding tests, the test
item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations were
prepared and investigated in the initial and confirmatory mutation tests:
±S9: 5000, 2000, 800, 320, 128 and 51.2 µg/plate.
The selection of the concentration range was based on the solubility and cytotoxicity
properties of the test item obtained in the preliminary experiments and in accordance with
the OECD 471 guideline [6].
At the preparation of the test item stock solutions any correction (multiplier) factor was not
taken into consideration, the doses were based on the final formulation as is.
Solubility, precipitation:
Following the plate incorporation and pre-incubation procedures precipitate was observed
by unaided eye on the plates at the concentration of 5000 µg/plate in the absence and
presence of exogenous metabolic activation (±S9). The obtained precipitate did not disturb
the scoring in any case.
Cytotoxicity results:
In the initial and confirmatory mutation tests, inhibitory effects of the test item on bacterial
growth were not observed in the examined strains. All of the noticed lower revertant colony
numbers (when compared to the revertant colony numbers of the corresponding solvent
control) remained within the range of the biological variability of the applied test system and
the background lawn development was not affected in any case.
Electronic copy 1 of 1
Study Number: 993-471-5828
Bacterial Reverse Mutation Assay with
2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide page 10 of 32
Mutagenicity results:
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with
and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants
that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological
relevant increases (more than 3-fold increase) in induced revertant colonies and the number
of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for
the positive control in all experimental phases, in all tester strains.
No biologically relevant increases were observed in revertant colony numbers of any of the
five test strains following treatment with2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane,
3,3,9,9-tetraoxideat any concentration level, either in the presence or absence of metabolic
activation (S9 Mix) in the performed experiments.
Conclusion:
The reported data of this mutagenicity assay show that under the experimental conditions
applied, the test itemdid not induce gene mutations in the genome of the strains of
Salmonella typhimuriumTA98, TA100, TA1535 and TA1537 and ofEscherichia coli
WP2uvrA.
In conclusion, the test item2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide has no mutagenic activity on the applied bacterial tester strains under the
test conditions used in this study.