2-Pentanone, 2,2',2''-[O,O',O''-(methylsilylidyne)trioxime]

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

484-460-1

EC Name:

-

Cas Number:

37859-55-5

Molecular formula:

C16H33N3O3Si

IUPAC Name:

(4E,9E)-4,7,10-trimethyl-7-{[(E)-(pentan-2-ylidene)amino]oxy}-6,8-dioxa-5,9-diaza-7-silatrideca-4,9-diene

Constituent 2

Reference substance name:

O,O',O"-(methylsilylidyne)trioxime 2-pentanone

IUPAC Name:

O,O',O"-(methylsilylidyne)trioxime 2-pentanone

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

All animals used on this study were Crl: CD(SD) rats and were sourced from Charles River UK Limited, Margate, Kent, England. Animals
used on the study weighed between:

Preliminary toxicity test: Males weighed between 194g to 205g.
Females weighed between 164g to 181g.
Micronucleus test: Males weighed between 174g to 224g

Animal age on despatch and on Day 1 of dosing were:

Preliminary toxicity test: On despatch Males and females ca 42 days old.
Day 1 Males and females ca 48 days old.
Micronucleus test: On despatch Males ca 42 days old.
Day 1 Males ca 49 days old.

Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the thermostat and relative humidity target ranges set at 19 to 23°C and 40 to 70% respectively. Temperature and humidity were within range throughout the study. The room was
illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance
diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum.

Food, chew blocks and tap water are routinely analysed for quality at source. All animals were given small soft white untreated wood (ASPEN) chew blocks and a red plastic shelter for environmental enrichment, were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Stability and homogeneity of the test substance and of the test substance in the vehicle were not determined in this test and remain the responsibility of the Sponsor. Chemical analysis of dosing formulations for achieved concentration was not performed in this study.
Suspensions of the test substance were prepared in Corn oil obtained from Sigma, batch number MKBF6012V and MKBG9425V.

Cyclophosphamide obtained from Sigma, batch number 120M1253V was used as the positive control compound. A solution was prepared using purified water at a concentration of 2 mg/mL just prior to administration.

All animals were dosed orally by gavage. The vehicle control and test substance groups were dosed using a dose volume of 2 mL/kg. The positive control group were dosed using a dose volume of 10 mL/kg.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

daily for 2 consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males at 125 mg/kg/day
6 males at 250 mg/kg/day
8 males at 500 mg/kg/day (due to toxicity)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (single dose at 20 mg/kg/day, 2 mg/mL)

Examinations

Tissues and cell types examined:

femur bone marrow

Details of tissue and slide preparation:

The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of the femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).

Fixation and slide staining:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Evaluation criteria:

A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes for the treatment group compared with the vehicle control group (p<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995).
A negative result is indicated where individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group 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.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.01).

Statistics:

For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
For micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
Statistical significance was declared at the 1% level for all tests.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 1000 mg/kg bw/day
- Animals: 2 rats per sex and per dose
- Clinical signs of toxicity in test animals:
At 500 mg/kg bw/day: unsteady gait, piloerection, underactive behaviour and hunched posture, hindlimbs (females). All animals survived until scheduled termination on Day 3. No net loss in bodyweight was observed.
At 1000 mg/kg bw/day: Unsteady gait, piloerection, underactive behaviour, splayed hindlimbs (males), prostrate posture, slow breathing, shallow breathing (males), deep breathing (males) and unresponsive behaviour (males), overactive behaviour (females), uncoordinated gait (females) and hunched posture (females). Animals were killed in extremis on Day 1, 1.5 and 2.5 hours post dose due to the severity of the clinical signs observed, consequently the maximum tolerated dose had been exceeded. The post mortem examinations did not find any signs of mis-dosing

RESULTS OF DEFINITIVE STUDY
- Micronucleated polychromatic erythrocyte counts (MPCE): No statistically significant increases in the number of MPCE in male animals. Cyclophosphamide caused a statistically significant increase in the frequency of MPCE (p<0.01) in male animals.
- Micronucleated normochromatic erythrocytes (MNCE): No significant increases in the incidence of MNCE in male animals.
- Proportion of polychromatic erythrocytes (%PCE): No statistically significant decreases in the proportion of PCE in male animals. Cyclophosphamide caused a statistically significant decrease in the proportion of PCE (p<0.01) in male animals.
- Clinical signs: At 250 mg/kg/day underactive behaviour, piloerection and unsteady gait were observed. At 500 mg/kg/day unsteady gait, underactive behaviour, piloerection, hindlimbs splayed, rales/noisy breathing and hunched posture were observed. Bodyweight loss of 15% from Day 2 to Day 3 was observed in one animal administered 500 mg/kg/day test item.

Applicant's summary and conclusion

Conclusions:

OS 1600 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD(SD) rats when administered orally by gavage in this in vivo test procedure.

Executive summary:

An in-vivo micronucleous test in Crl: CD(SD) rats was performed with OS1600 according to OECD Guideline 474. Based on preliminary resutls, 6 -8 male rats per group were were treated with 0 (control), 125, 250 and 500 mg/kg bw orally by gavage (2 mL/kg) on two occasions approximately 24 hours apart. Bone marrow smears were obtained from animals in the vehicle control and in each of the test substance groups 24 hours after administration of the second dose. One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes (MPCE). The proportion of polychromatic erythrocytes (%PCE) was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes (MNCE) was also kept. No statistically significant increases in the frequency of MPCE and no statistically significant decreases in the % PCE were observed at any treatment level, compared to vehicle control values. It was concluded that OS 1600 did not show any evidence of causing an increase in the induction of MPCE or bone marrow cell toxicity in male rats when administered orally by gavage in this in vivo test procedure.