N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylmethanimid amide

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:

29th November 2011 to 2nd March 2012

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylimidoformamide

EC Number:

813-618-8

Cas Number:

1269400-04-5

Molecular formula:

C11H12N4O3

IUPAC Name:

N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylimidoformamide

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The NMRI BR mouse was used as the test system because it is a readily available rodent species, which is commonly used for this purpose, and with documented susceptibility to a wide range of toxic substances.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mean body weights were for males 35.8 ± 1.9 g and for females 27.1 ± 1.3 g and the range was for males 32 - 39 g and for females 25 - 30 g in the first main experiment. The mean body weights in the second main test were 33.6 ± 1.5 g and the range was 31 - 37 g.
- Assigned to test groups randomly: Yes
- Housing: The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages (type MIII height: 15 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 20.6°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 14 October, 2011 To: 01 February, 2012

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: corn oil (Sigma, Zwijndrecht, The Netherlands).
The specific gravity of corn oil is 0.9 g/ml. PF-05188294 concentrations were blended to obtain a homogeneous suspension.
The dosing volume was 10 ml/kg body weight.

Duration of treatment / exposure:

The first sampling time was 24 h after treatment and the second sampling time was 48 h after treatment.

Frequency of treatment:

The animals were dosed once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Remarks:

males, second main test, due to unexpected deaths in the first main test

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day

Remarks:

females, first test

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Remarks:

females, first test

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day

Remarks:

females, first test

Doses / concentrations 5

Dose / conc.:

250 mg/kg bw/day

Remarks:

males, first test

Doses / concentrations 6

Dose / conc.:

500 mg/kg bw/day

Remarks:

males, first test

Doses / concentrations 7

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

males, first test

No. of animals per sex per dose:

At least five male and five female mice were used per sampling time in each treatment group

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Baxter B.V., Utrecht, The Netherlands) dissolved in physiological saline (B. Braun, Melsungen AG, Germany) dosed as a single intraperitoneal injection of 40 mg/kg body weight.
The route and frequency of administration and the volume administered of the positive control were the same as those of the test substance.

Examinations

Tissues and cell types examined:

Isolation of bone marrow
Bone marrow of the groups treated with PF-05188294 was sampled 24 or 48 (maximum tolerated dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 and 48 hours after dosing (first and second main study, respectively) and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min.

Details of tissue and slide preparation:

Preparation of bone marrow smears
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated coverslipper

Evaluation criteria:

To prevent bias, all slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated

Statistics:

Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid. The increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of animals treated with PF-05188294 when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 500 and 300 mg/kg, respectively, is considered equivocal under the experimental conditions described in this report.

Executive summary:

Micronucleus test in bone marrow cells of the mouse with PF-05188294.
PF-05188294 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch E010011212 of PF-05188294 was an orange powder with a purity of 99.7%. The test substance was suspended in corn oil.
In the dose range finding test, in total three animals per sex were dosed with 1000 mg PF-05188294 per kilogram body weight. The male animals showed the following toxic signs after dosing: ataxia, tremors, hunched posture, rough coat, quick breathing, ventral recumbency (1 animal) and emaciated appearance (1 animal). Two female animals died within 5 hours after dosing. The other female animal showed the following toxic signs after dosing: ataxia, tremors, hunched posture, rough coat and quick breathing and emaciated apperance. One female animal was dosed with 750 mg PF-05188294 per kilogram body weight and died within 21 hours after dosing. In total three female animals were dosed with 500 mg PF-05188294 per kilogram body weight. Two female animals died within 5 and 45 hours, respectively. The other female animal showed the following toxic signs after dosing: hunched posture, rough coat and quick breathing. In total three female animals were dosed with 300 mg PF-05188294/kg body weight. The animals showed the following toxic signs: lethargy, ataxia, tremors, hunched posture and closed eyes (1 animal).
In the main study male and female animals were dosed via intraperitoneal injection with vehicle or with 1000, 500 and 250 mg PF-05188294 per kg body weight (males) or with 300, 150 and 75 mg PF-05188294 per kg body weight (females). A positive control group was dosed via intraperitoneal injection with 40 mg cyclophosphamide (CP) per kg body weight. In total 8 treatment groups were used and 4 control groups, each consisting of at least 5 animals. Three additional animals per sex were dosed with the maximum tolerated dose, to compensate for possible deaths.
The animals dosed with the maximum tolerated doses showed the following toxic signs after dosing:
lethargy, ataxia and tremors. In addition six male animals and nine female animals had a hunched posture. Twelve out of 13 male animals died within 19 hours after dosing. Animals dosed with the intermediate doses were lethargic and showed ataxia. All male animals and one female also showed tremors and all male animals had a hunched posture. Four male animals also had their eyes closed and one of them also had an increased breathing rate.
Male animals dosed with the low dose were lethargic, showed ataxia and one animal had a rough coat and a hunched posture. Another male animal showed tremors. Two females dosed with the low dose were lethargic. All other animals showed no abnormalities. No treatment related clinical signs or mortality were noted in control animals receiving vehicle or cyclophosphamide.
Bone marrow of the groups treated with PF-05188294 was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.
Due to unexpected deaths in the main study (12 male animals dosed with 1000 mg/kg body weight) an additional study was performed using male animals which were dosed via intraperitoneal injection with vehicle or with 500 mg PF-05188294 per kg body weight. A positive control group was dosed via intraperitoneal injection with 40 mg cyclophosphamide (CP) per kg body weight. In total 1 treatment group was used and 2 control groups, each consisting of at least 5 animals. Three additional animals were dosed with 500 mg/kg body weight, to compensate for possible deaths.

Males dosed with 500 mg/kg body weight showed the following toxic signs: lethargy, ataxia, tremors, hunched posture, rough coat (5 animals) and closed eyes (4 animals). Bone marrow of the groups treated with PF-05188294 and the negative and positive control groups was harvested 48 hours after dosing.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met.
In the first main experiment fluctuations in the number of micronuclei were observed above the historical control data range both in male and female animals. The scorer rescored some slides. The number of micronuclei were within the historical control data range and it appeared that micronuclei were not evenly distributed on the slides. Quality control performed of the slides by a different scorer did not confirm the increase in the number of micronuclei. Although the observations were not dose dependent and the mean number of micronuclei of each dose group was  well within the historical control data range, the increases were statistically significant and confirmed in the second main experiment.
Quality control of the slides of the second main experiment (by a second scorer) did not show an increase in the amount of micronuclei. A third scorer confirmed these findings. For this reason two of the scorers scored two slides in parallel using a dual bridge teaching microscope. Again it appeared that the micronuclei were not evenly distributed on the slides. Rescoring by these two scorers showed an increase in the number of micronuclei above the historical control data range in one of the two slides. Although quality control did not confirm the increase in the number of micronuclei, statistical significant increases were observed in both sexes. Moreover, rescoring by two scorers showed an increase in the number of micronuclei above the historical control data range in one of the two slides. In addition the mean frequency of micronucleated polychromatic erythrocytes in bone marrow of animals treated with 500 mg PF-05188294 per kg body weight with a 48 hour sampling time was above the historical control data range.
Since the increase in the number of micronuclei was not dose dependent and it appeared that the area of scoring had influence on the number of micronuclei, the test results are considered equivocal. This was observed in two sexes and two independent experiments.

Female animals that were treated with 300 (48 hours sampling time), 150 and 75 mg PF-05188294/kg body weight and male animals dosed with 500 mg/kg body weight showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis.
Male animals (all concentrations first main study) and female animals dosed with 300 mg PF-05188294/kg body weight with a 24 hours sampling time and the groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.
It is concluded that this test is valid. The increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of animals treated with PF-05188294 when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 500 and 300 mg/kg, respectively, is considered equivocal under the experimental conditions described in this report.