Phosphorothioic acid, O,O-bis(2-methylpropyl) ester, sodium salt (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

First Ames test:

The in vitro genetic toxicity of the substance CT-473 -91, a 50% solution in water, was assessed using S. typhimurium TA100, TA1535, TA98, TA1538 and TA1537 strains, equivalent or similar to OECD 471 guideline and according to GLP principles. All bacterial strains showed negative responses with and without metabolic activation up to concentrations of 10000 µg/plate.

The study was not conducted with E.coli and/or with S. typhimurium TA102, which have an AT base pair at the primary reversion site, as currently required. Based on the results of this study, the substance CT-473 -91, a 50% solution in water, is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Second Ames test:

S-10783, a waxy solid with 17.5% water, was tested in the Salmonella typhymurium reverse mutation assay with TA1535, TA1537, TA100 and TA98 and in the Escherichia coli reverse mutation assay with WP2uvra, in accordance with OECD 471 guideline and GLP principles.

In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

No increase in the number of revertants was observed upon treatment with S-10783up to concentrations of 5000 µg/plate. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that S-10783 is not mutagenic in theSalmonella typhymurium reverse mutation assay and the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 03, 2013 - January 21, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): S10783
- Name: Sodium diisobutyl monothiophoshate
- CAS: 53378-52-2
- Molecular formula: C8H18O3PS.Na
- Molecular weight: 248.26
- Storage condition of test material: not indicated in the study report
- Purity: 70%

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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 by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test/Main study experiment 1: TA100 and WP2uvrA: Without and with S9-mix: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study experiment 1: TA1535, TA1537 and TA98: Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Prior to test initiation, the solubility of the test substance was performed. The test substance was a white translucent solution at a concentration of 50 mg/ml in Milli-Q water. This solution was not usable for the 0.22 µm filter sterilisation. The test substance formed a slightly turbid beige suspension at a concentration of 50 mg/ml in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany). Therefore DMSO was selected as the vehicle for use in the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle 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 independently repeated experiment.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the top dose of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the top dose of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The substance, a waxy solid with 17.5% water, is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

Executive summary:

S-10783, a waxy solid with 17.5% water, was tested in the Salmonella typhymurium reverse mutation assay with TA1535, TA1537, TA100 and TA98 and in the Escherichia coli reverse mutation assay with WP2uvrA, in accordance with OECD 471 guideline and GLP principles.

In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

No increase in the number of revertants was observed upon treatment with S-10783 up to concentrations of 5000 µg/plate. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that S-10783, a waxy solid with 17.5% water, is not mutagenic in the Salmonella typhymurium reverse mutation assay and the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test:

An in vivo micronucleus study with Aero® 6697 Promoter, a 50% solution in water, in the mouse ((24 hours (5/sex/dose) and 48 hours (5/sex for control and 2000 mg/kg bw) sampling, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines.

In the dose groups of 500, 1000 and 2000 mg/kg lethargy and unsteady gait was observed in all animals but the animals recovered within 4.5 hours. One female animal died after treatment with the test substance at the 2000 mg/kg dose level, this animal probably died as result of internal bleeding very occasionally associated with intraperitoneal administration of substances. No adverse clinical signs were obtained for the vehicle or positive control treated animals.

The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time. The test substance did not cause any significant decreases in the proportion of immature erythrocytes. Based on the these results, it is concluded that Aero® 6697 Promoter, a 50% solution in water, is not clastogenic in the micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 06, 1998 - September 05, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1992)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA (1997) Toxic Substances Control Act Test Guidelines; Title 40 Code of Federal Regulations Part 799

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): AERO® 6697 Promoter
- Name: Sodium diisobutyl monothiophoshate
- CAS: 53378-52-2
- Molecular formula: C8H18O3PS.Na
- Molecular weight: 248.26
- Storage condition of test material: not indicated in the study report
- Batch no.: 294

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: no data
- Weight at study initiation: males: 28 -30 g; females: 22 - 24 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Group housed per sex in plastic disposable cages
- Diet: Free access to pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK)
- Water: Free access to tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 52 to 66
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: purified water was used for the dilutions.
- Lot/batch no.: Water for irrigation, obtained from Baxter, batch numbers 98E29B25 and 98F29B28

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions/suspensions of the test substance were freshly prepared on the morning of the test and were diluted to the concentrations in purified water.

Dosing volume administered: 20 mL/kg body weight.

Duration of treatment / exposure:

Test substance and negative control: 24 and 48 hours.
Positive control: 24 hours.

Frequency of treatment:

Single

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Pre test: 2
Main study: vehicle and 2000 mg/kg bw: 10/sex; 500 and 1000 mg/kg bw: 5/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C in purified water (0.6 mg/mL)
- Route of administration: Single by intragastric gavage.
- Doses / concentrations: 12 mg/kg body weight (20 mL/kg body weight)

Tissues and cell types examined:

Measuring the increase in the incidence of micronucleated immature erythrocytes per 2000 polychromatic erythrocytes in mouse bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose for the Micronucleus test was based on a preliminary study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals were weighed and the individual volume to administered was adjusted to the animal's body weight. Sampling of the bone marrow was done 24 (5/sex/dose) and 48 (5/sex for control and 2000 mg/kg bw) hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 mL of pre-filtered foetal calf serum. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. The prepared smears were fixed in methanol. After air-drying the smears were stained for 10 minutes in 10% Giemsa. Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

Evaluation criteria:

A positive response is normally indicated by a statistically siginificant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and or/group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Statistics:

For incidences of micronucleated immature erythrocytes , exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts.)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg
- Solubility: The test substance was diluted in purified water.
- Clinical signs of toxicity in test animals: No clinical signs were observed in the 200 and 500 mg/kg tretament groups. After dosing with 1000 mg/kg lethargy and piloerection was observed in animals but the animals recovered within 3.5 hours. In the dose group of 2000 mg/kg lethargy, piloerection and ptosis was observed in all animals but the animals recovered within 5.5 hours.
- Results showed that a dose of 2000 mg/kg, the limit dose for the micronucleus test, was expected to be tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

RESULTS OF DEFINITIVE STUDY see "attached background material for details on results"
- Induction of micronuclei (for Micronucleus assay): The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time. The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.
- Ratio of PCE/NCE (for Micronucleus assay): The test substance did not cause any significant decreases in the proportion of immature erythrocytes.

- Clinical signs and mortalities: In the dose groups of 500, 1000 and 2000 mg/kg lethargy and unsteady gait was observed in all animals but the animals recovered within 4.5 hours. One female animal died after treatment with the test substance at the 2000 mg/kg dose level. A post mortem examination showed that this animal probably died as result of internal bleeding very occasionally associated with administration of substances by the intraperitoneal injection.

Conclusions:

Interpretation of results: negative
An in vivo micronucleus study with the test material in the mouse (24 hours (5/sex/dose) and 48 hours (5/sex for control and 2000 mg/kg bw) sampling, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. Based on the absence of increased number of micronucleated immature erythrocytes, it is concluded that the test material is not clastogenic in the in vivo micronucleus test.

Executive summary:

An in vivo micronucleus study with Aero® 6697 Promoter, a 50% solution in water, in the mouse ((24 hours (5/sex/dose) and 48 hours (5/sex for control and 2000 mg/kg bw) sampling, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines.

In the dose groups of 500, 1000 and 2000 mg/kg lethargy and unsteady gait was observed in all animals but the animals recovered within 4.5 hours. One female animal died after treatment with the test substance at the 2000 mg/kg dose level, this animal probably died as result of internal bleeding very occasionally associated with intraperitoneal administration of substances. No adverse clinical signs were obtained for the vehicle or positive control treated animals.

The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time. The test substance did not cause any significant decreases in the proportion of immature erythrocytes. Based on the these results, it is concluded that Aero® 6697 Promoter, a 50% solution in water, is not clastogenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Short description of key information:

In the first Ames test, the substance CT-473 -91, a 50% solution in water, was tested with TA100, TA1535, TA98, TA1538 and TA1537 strains, equivalent or similar to OECD 471 guideline and according to GLP principles.

In the second Ames test, S-10783, a waxy solid with 17.5% water, was tested with TA1535, TA1537, TA100 and TA98 and in the Escherichia coli reverse mutation assay with WP2uvrA, in accordance with OECD 471 guideline and GLP principles.

An in vivo micronucleus study with Aero® 6697 Promoter, a 50% solution in water, in the mouse ((24 hours (5/sex/dose) and 48 hours (5/sex for control and 2000 mg/kg bw) sampling, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines.

All three substances have the same main components.

Justification for classification or non-classification

The substance, a 50% solution in water (first Ames test and in vivo micronucleus test) and a waxy solid with 17.5% water (second Ames test), does not have to be classified and has no obligatory labelling requirement for genetic toxicity in accordance with Regulation EC No. 1272/2008.