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

Genetic toxicity: in vivo

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium O,O-diisobutyl phosphorothioate

EC Number:

610-992-2

Cas Number:

53378-52-2

Molecular formula:

C8 H18 O3 P S . Na

IUPAC Name:

Sodium O,O-diisobutyl phosphorothioate

Test material form:

other: grease-like solid

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

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)

Examinations

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.)

Results and discussion

Test results

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.

Any other information on results incl. tables

- 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.

Applicant's summary and conclusion

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.