2-{2-[(2R,4R,6R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl]ethyl}-1H-isoindole-1,3(2H)-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

A bacterial reverse mutation test (Ames test) was performed (OECD 471 and EEC B13/14) to assess the genotoxic potential of the test substance. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

An in-vivo micronucleus assay was performed to assess the clastogenic potential of the test substance. It was concluded that the test substance is not clastogenic in the micronucleus test under the experimental conditions used.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 January 2008 - 13 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study report includes a certificate of GLP compliance and study was conducted in accordance with OECD and EEC guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

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: Within 20% of the sex mean (Group means in the range 34.8 - 36.4 g).
- Assigned to test groups randomly: yes, under following basis: animals allocated to treatment groups as they came to hand from the delivery boxes.
- Fasting period before study: Not specified
- Housing: The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages (type Mll height: 14 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France) . Paper bedding was provided as nest material (Enviro-dri, TecniLab-BMI BV, Someren, The Netherlands).
- Diet (e.g. ad libitum): The animals were provided with standard pelleted laboratory animal diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH , Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3ºC (Actual range 19.5 - 22.0ºC).
- Humidity (%): 30 - 70% (actual range 47 - 72%).
- Air changes (per hr): approximately 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day.


IN-LIFE DATES: From: 07 January 2008 To: 13 February 2008

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Not specified
- Concentration of test material in vehicle: 200, 100, and 50 mg/mL (dose levels 2000, 1000, and 500 mg/kg body weight dosed at 10mL/kg).
- Lot/batch no. (if required): Not specified. Supplier - Roth, Karlsruhe, Germany.
- Purity: Not specified.

Details on exposure:

Test item dosed by intraperitoneal injection.

Duration of treatment / exposure:

Single.

Frequency of treatment:

N/A

Post exposure period:

24 hours (Groups A, B, D, and E); 48 hours (Groups C and F (positive control group)).

Remarks:

Doses / Concentrations:
0, 50, 100, and 200 mg/mL.
Basis:
nominal conc.

No. of animals per sex per dose:

Dose range finding study: 3 males and 3 females.
Mirconucleus main test: Five male mice used per treatment group (six groups in total).

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): known to demonstrate toxic effects on erythropoiesis
- Route of administration: Intraperitoneal
- Doses / concentrations: 50 mg/kg body wieght.

Tissues and cell types examined:

Bone marrow tissue was harvested and the erythrocytes were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test.
One dose group, comprising of 3 males and 3 females received a single dose of Lacto!. The study duration was three days. During this period mortality and physical condition were recorded at least once a day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

Isolation of bone marrow
Bone marrow of the groups treated with Lactol was sampled 24 or 48 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 (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.

DETAILS OF 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 serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether (Merck, Darmstadt, Germany) and cleaned with a tissue) and 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 (Merck) 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 (Miles, Bayer Nederland BV). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

METHOD OF ANALYSIS:
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 100x 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 1000x. 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.

OTHER:

Evaluation criteria:

Ratio of polychromatic to normochromatic erythrocytes was measures and assessed.

Statistics:

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, onesided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

During the first three hours of dosing, all animals showed signs of systemic toxicity (see below for details). One animal died within 22 hours of dosing.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a dose range finding study 6 animals (group A: 3 males and 3 females) were dosed intraperitoneally. All animals showed the following toxic signs within 1 hour after dosing: lethargy and hunched posture. Five animals also had a rough coat (3 males, 2 females). Within 4 hours after dosing all animals were still lethargic and had a hunched posture. Two males and two females had a rough coat, one male and two females had their eyes closed. Within 23 hours after dosing two males and two females still had a hunched posture. One of these male animals also had a rough coat. One male and one female animal had recovered from the treatment. Within 47 hours after dosing all animals had recovered from the treatment. Since there were no
substantial differences between sexes in toxic ity only male animals were used in the main test.


RESULTS OF DEFINITIVE STUDY
Mortality and systemic toxic signs
The animals of the groups treated with 1000 and 500 mg Lactol/kg body weight and the animals of the negative and positive control groups showed no abnormalities. The following clinical observations were made in the groups treated with 2000 mg Lactol/kg body weight:
During the first three hours after dosing all animals were lethargic, had a rough coat and a hunched posture. Seven animals also had their eyes closed. Within 22 hours after dosing two animals had a rough coat, one had a hunched posture and one had a hunched posture and a rough coat. One animal died and five had recovered from the treatment.
Within 42 hours after dosing all animals had recovered from the treatment.

Micronucleated polychromatic erythrocytes
The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of polychromatic to normochromatic erythrocytes are presented in Table 1. The individual data are described in Appendix II (refer to figure 1). The mean number of micronucleated polychromatic erythrocytes scored in Lactol treated groups were compared with the corresponding solvent control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of Lactol treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes (Appendix III, refer to figure 1). Hence, the acceptability criteria of the test were met.

Ratio polychromatic to normochromatic erythrocytes
The animals of the groups, which were treated with Lactol showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Table 1: Mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic

erythrocytes and ratio of polychromatic / normochromatic erythrocytes

Group	Treatment	Dose (mg/kg body weight)	Sampling time (hours)	Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes (mean ± S.D.)^(1)	Ratio polychromatic / normochromatic erythrocytes (mean ± S.D.)^(1)
Males
A	Solvent Control	0	24	2.4 ± 0.9	1.02 ± 0.02
B	Lactol	2000	24	2.0 ± 1.6	0.96 ± 0.04
C	Lactol	2000	48	2.8 ± 1.3^(2)	1.01 ± 0.03
D	Lactol	1000	24	1.8 ± 1.1	0.98 ± 0.07
E	Lactol	500	24	2.4 ± 2.1	1.01 ± 0.04
F	CP	50	48	33.6 ± 8.8^(3)	0.56 ± 0.21

Solvent Control = Corn oil

CP = Cyclophosphamide (+ve control)

(1)       Five animals per treatment group

(2)       Four animals per treatment group

(3)       Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that this test is valid and that Lactol is not clastogenic in the micronucleus test under the experimental conditions described in this report.

Executive summary:

An in-vivo micronucleus assay was performed by NOTOX B.V., The Netherlands on behalf of Pfizer Ireland Pharmaceuticals, Ltd to assess the clastogenic potential of the test substance, Lactol. The study was performed to GLP, according to OECD 474 and EU Method B12 Test Guidelines. Five male mice were dosed in each of six treatment groups (a vehicle / negative control group, a positive control group, and dose groups at 500, 1000, and 2000 mg/kg body weight; two groups were dosed at 2000 mg/kg bodyweight) by intraperitoneal injection. Bone marrow was harvested 24 hours (for the negative control group, and dose groups at 500, 1000, and one group at 2000 mg/kg per day) or after 48 hours (for the positive control group and the second 2000 mg/kg bodyweight group) and the number of micronucleated polychromatic erythrocytes relative to the number of normochromatic erythrocytes assessed.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with Lactol. It was concluded that Lactol is not clastogenic in the mirconucleus test under the experimental conditions used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

A bacterial reverse mutation test (Ames test) was performed (OECD 471 and EEC B13/14) to assess the genotoxic potential of the test substance. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98, and TA100) and one strain of Escherichia coli (WP2uvrA) were tested against at least five concentrations of the test substance both in the presence and in the absence of metabolic activation (S9-mix). All bacterial strains showed negative responses over the entire dose range, i.e, no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

An in-vivo micronucleus assay was performed to assess the clastogenic potential of the test substance. The study was performed according to OECD 474 and EU Method B12 Test Guidelines. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test substance. It was concluded that the test substance is not clastogenic in the micronucleus test under the experimental conditions used.

Justification for selection of genetic toxicity endpoint

Study report includes a certificate of GLP compliance and study was conducted in accordance with OECD and EEC guidelines. This in vivo study was chosen over that in vitro study, as the higher level study.

Justification for classification or non-classification

According to the results of the tests and the Official Journal of the European Communities, Annex VI, 'General Classification and Labelling Requirements for Dangerous Substances and Preparations' the substance is not classified on the basis of its mutagenic potential.