O,O,O',O'-tetraoctyl-(branched and linear) S,S'-{methylenebis[imino(3-oxopropane-3,1-diyl)]} bis(phosphorothioate)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 24 June 1998 and 30 July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with an existing guideline with no dificiencies or none of significance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

EC Number:

700-945-5

Molecular formula:

Complex UVCB substance

IUPAC Name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

Test material form:

other: Straw coloured viscous liquid

Details on test material:

Description: Straw coloured viscous liquid

Test animals

Species:

mouse

Strain:

other: Cri:CD-1™(1CR)BR

Sex:

male

Details on test animals or test system and environmental conditions:

Mice were obtained from Charles River (UK) Limited, Margate, Kent. At the start of the main study the mice weighed 22 to 30g and were approximately five to eight weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding. Free access to mains drinking water and food (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study. The animal room was maintained at a temperature of 20 to 22 •C and relative humidity of 52 to 56%. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Arachis oil and cyclophosphamide

Details on exposure:

Groups of 7 male mices were given a single i.p. doses of 500, 1000 and 2000 mg/kg of the test stubstance.

Duration of treatment / exposure:

Animals were killed 24 or 48 hours after treatment.

Frequency of treatment:

Single dose, 10ml volume

Post exposure period:

24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000
Basis:
nominal conc.

No. of animals per sex per dose:

7 males per group

Control animals:

yes, concurrent no treatment

Positive control(s):

cyclophosphamide (5 mice)

Examinations

Tissues and cell types examined:

Bone marrow was extracted, and smear preparations were made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Details of tissue and slide preparation:

Immediately following sacrifice (ie. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re­ suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

Evaluation criteria:

A positive mutagenic response was considered demonstrated when a statistically significant dose-responsive increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was considered demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part Ill (1989). The data was analysed following a square root of (x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Hunched posture, lethargy, pilo-erection, decreased respiratory rate, laboured respiration, ptosis and ataxia was observed in one 2000 mg/kg animal at the end of the 48 hour observation period.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The range finding study showed that there was no sex or route of exposure differences at 1000 and 2000 mg/kg. No mortality was observed. Clinical signs were limited to hunched posture 1 hour following i.p. administration.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of this study this substance does not require classification under EU Regulation (EC) No. 1272/2008 for germ cell mutagenicity.

Executive summary:

The potential of this substance to cause somatic cell mutagenicity was evaluated in an in vivo mouse micronucleus assay (OECD 474). Based on the results of a range finding study groups of mice (7/group) were administered single i.p. doses or 500, 1000, and 2000 mg/kg with a dose volume of 10 ml. The study included a concurrent vehicle (arachis oil) and positive (cyclophosphamide) control groups. Immediately following sacrifice (ie. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re­ suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium. A positive mutagenic response was considered demonstrated when a statistically significant dose-responsive increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was considered demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group. No evidence of a significant increase in the incidence of micronucleated PCEs with the test material compared to the vehicle control was observed. Also, no statistically significant decreases in the PCE/NCE ratios were found in the 24 or 48 hour test material groups. Evidence that absorption was demonstrated by the presence of clinical signs in one animal in the 48 hour, 2000 mg/kg group. The study was determined to be valid based on the marked increase in the frequency of micronucleated PCEs in the positive control group. Based on the results of this study this substance does not require classification under EU Regulation (EC) No. 1272/2008 for germ cell mutagenicity.