4-(1-piperidinyl)-2-(E)-butenoicacid hydrochloride

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:

9th September 2011 to 2nd November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1-[(2E)-3-carboxyprop-2-en-1-yl]piperidin-1-ium chloride

EC Number:

690-933-5

Cas Number:

197892-69-6

Molecular formula:

C9H16ClNO2

IUPAC Name:

1-[(2E)-3-carboxyprop-2-en-1-yl]piperidin-1-ium chloride

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI BR mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC).

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 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 36.3 ± 1.67 g and for females 28.8 ± 1.78 g and the range was for males 33 - 40 g and for females 25 - 33 g.
- Assigned to test groups randomly: yes
- Housing: The animals were housed in room number A0.18. 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, UK).
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitum
- Acclimation period: The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.6°C
- Humidity (%): 39 - 78%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle. Due to e.g. cleaning procedures, temporary deviations from the light/dark cycle (with a maximum of 4 hours) and the minimum level for humidity (with max. 1%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.

IN-LIFE DATES: From: 9th September 2011 To: 2nd November 2011

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

physiological saline (B. Braun, Melsungen AG, Germany).

Details on exposure:

The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of PF-00818977-01. The route of administration was chosen to maximize the chance of the test substance reaching the target tissue. The dosing volume was 10 ml/kg body weight. PF-00818977-01 concentrations were prepared on the day of administration.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Male and female animals were dosed once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP) 40mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Isolation of bone marrow
Bone marrow of the groups treated with PF-00818977-01 was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing 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 1000 rpm (216 g) for 5 min.

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 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:

Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: PF-00818977-01 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 1500 mg/kg (the maximum tolerated dose)

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid and that PF-00818977-01 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 1500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

Micronucleus test in bone marrow cells of the mouse with PF-00818977-01.
PF-00818977-01 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 E010012221 of PF-00818977-01 was a white powder with a purity of 99.2%. The test substance was dissolved in physiological saline. In the dose range finding test, one animal per sex was dosed with 2000 mg PF-00818977-01 per kilogram body weight. The animals died within 21 hours after dosing. In total 3 animals per sex were dosed with 1500 mg/kg body weight. The following toxic signs were observed after dosing: ataxia, lethargy, rough coat, hunched posture, closed eyes (2 males, 2 females), slow breathing (1 male, 2 females) and discharge from nose and bill (1 female).
In the main study male and female animals were dosed once via intraperitoneal injection with vehicle or with 1500, 750 and 375 mg PF-00818977-01 per kg body weight. A positive control group was dosed once via intraperitoneal injection with 40 mg cyclophosphamide (CP) per kg body weight. In total 4 treatment groups were used and 2 control groups, each consisting of 5 animals. The animals dosed with 1500 mg PF-00818977-01 per kg body weight showed the following toxic signs after dosing: lethargy, ataxia, rough coat, hunched posture and closed eyes (3 males, 1 female). Within 48 hours the animals showed necrosis at the tail point. Animals dosed with 750 mg per kg body weight showed the following toxic signs: rough coat and hunched posture (2 males, 3 females). No treatment related clinical signs or mortality were noted in animals dosed with 375 mg PF-00818977-01 per kg body weight or control animals receiving vehicle or cyclophosphamide.
Bone marrow of the groups treated with PF-00818977-01 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.
No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with PF-00818977-01.
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.
The groups that were treated with 1500 mg PF-00818977-01 per kg body weight 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. All other treatment groups 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.
It is concluded that PF-00818977-01 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 1500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.