Registration Dossier

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:
23 October 2002 to 09 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in accordance to OECD and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: five to eight weeks old
- Weight at study initiation: 25 to 30g
- Assigned to test groups randomly: yes, under following basis: selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflake bedding.
- Diet (e.g. ad libitum): ad libitum(Certified Rat and Mouse Diet 5LF2, International Product Supplies Limited, Wellingborough, Northants UK)
- Water (e.g. ad libitum): ad libitum (mains drinking water)
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness

IN-LIFE DATES: From: animals were 5 to eight weeks at the beginning of the study, plus minimum of seven days acclimatisation, animals were killed 24 to 48 hrs following dosing.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Concentration of test material in vehicle: 0, 7.5, 15 or 30 mg/mL
- Lot/batch no. (if required): lot number: T63
Details on exposure:
All animals were dosed once only at the appropriate dose level by gavage (dose range study) using a metal cannula or with a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.

It was initially considered to be unnecessary to investigate the intraperitoneal route of administration. However, after preliminary range-finding work it was then decided to investigate the intraperitoneal route following discussions with the Sponsor.
Duration of treatment / exposure:
Brief exposure with the test material.
Frequency of treatment:
Once
Post exposure period:
24-48 hrs
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
75 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
7m/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive Control: cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow Erythocytes.
Details of tissue and slide preparation:
Slide Preparation
Immediately following termination (i.e. 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, stained in May-Griinwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.
Evaluation criteria:
Slide Evaluation
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE• blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Statistics:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant 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.

If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a ~(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:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

GLYCYL (GR34000A): MICRONUCLEUS  TEST IN THE MOUSE

 

 

Table1                    Micronucleus Study·Summary of GroupMean Data

 

 

Treatment Group

NumberofPCEwith

Micronuclei per2000PCE

 

PCE/NCE Ratio

 

 

GroupMean

SD

GroupMean

SD

 

1.   Vehicle Control

48-hour Sampling Time

 

2.3

 

1.3

 

0.78

 

0.30

 

2.    Vehicle Control

24-hour Sampling Time

 

1.6

 

1.1

 

0.80

 

0.31

 

3.   Positive Control

24-hour Sampling Time

 

61.6***

 

32.2

 

1.11

 

0.39

4.    GLYCYL (GR34000A)300mg/kg

48-hour Sampling Time

 

2.1

 

0.9

 

0.58

 

0.13

5.   GLYCYL (GR34000A)300mg/kg

24-hour Sampling Time

 

1.9

 

2.3

 

0.64

 

0.21

6.   GLYCYL (GR34000A)150mg/kg

24-hour Sampling Time

 

1.6

 

1.1

 

0.71

 

0.28

7.   GLYCYL (GR34000A)75mg/kg

24-hour Sampling Time

 

1.0

 

0.8

 

0.74

 

0.24

PCE  =Polychromatic erythrocytes

NCE  =Normochromatic erythrocytes

SD =Standarddeviation

***   =P<0.001

Table 2       Micronucleus  Study -Individual and Group Means and Standard Deviations: Vehicle Control (10ml/kg) 48-Hour

Sampling Time


Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

1.VECHICLE CONTROL 10 ml/kg 48 -hour sampling time

1

29

2000

3

0.15

613

2

0.63

2

29

2000

3

0.15

689

0

0.45

3

26

2000

2

0.10

564

0

0.77

4

27

2000

2

0.10

429

0

1.33

5

27

2000

0

0.00

496

0

1.02

6

27

2000

4

0.20

607

0

0.65

7

28

2000

2

0.10

616

1

0.62

Group Mean

27.6

2000

2.3

0.11

573

0.4

0.78

SD

1.1

0

1.3

0.06

86

0.8

0.30

Table 3        Micronucleus  Study -Individual and Group Means and Standard  Deviations: Vehicle Control (10ml/kg) 24-Hour

Sampling Time

Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

2. VEHICLE CONTROL 10 ml/kg 24 -hour sampling time     

8

26

2000

2

0.10

597

1

0.68

9

28

2000

0

0.00

567

0

0.76

10

28

2000

3

0.15

608

1

0.64

11

28

2000

1

0.05

624

0

0.60

12

27

2000

3

0.15

607

0

0.65

13

29

2000

1

0.05

403

0

1.48

14

27

2000

1

0.05

557

0

0.80

Group Mean

27.6

2000

1.6

0.08

566

0.3

0.80

SD

1.0

0

1.1

0.06

76

0.5

0.31

Table 4 Micronucleus  Study - Individual and Group Means and Standard  Deviations: Cyclophosphamide  (50 mg/kg) 24-Hour Sampling Time

Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

3 CYCLOPHOSPHAMIDE 50 mg/kg 24-hour sampling time

15

30

2000

31

1.55

407

1

1.46

16

27

2000

30

1.50

641

0

0.56

17

27

2000

71

3.55

473

0

1.11

18

25

2000

107

5.35

518

0

0.93

19

28

2000

69

3.45

403

0

1.48

Group Mean

27.4

2000

61.6

3.08

488

0.2

1.11

SD

1.8

0

32.2

1.61

98

0.4

0.39

Table 5       Micronucleus  Study -Individual and Group Means and Standard  Deviations: Test Material (300 mg/kg) 48-Hour

Sampling Time

Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

4 Glycyl (GR34000A) 300 mg/kg 48-hour sampling time

20

27

2000

3

0.15

606

0

0.65

21

27

2000

1

0.05

562

0

0.78

22

27

2000

3

0.15

667

1

0.50

23

29

2000

2

0.10

636

0

0.57

24

27

2000

1

0.05

591

0

0.69

25

26

2000

2

0.10

723

1

0.38

26

27

2000

3

0.15

661

1

0.51

Group Mean

27.1

2000

2.1

0.11

635

0.4

0.58

SD

0.9

0

0.9

0.04

54

0.5

0.13

Table 6 Micronucleus  Study - Individual and Group Means and Standard  Deviations: Test Material (300 mg/kg) 24-Hour Sampling Time

Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

5 Glycyl (GR34000A) 300 mg/kg 24-hour sampling time

27

29

2000

1

0.05

596

0

0.68

28

27

2000

3

0.15

505

1

0.98

29

28

2000

0

0.00

612

0

0.63

30

27

2000

0

0.00

696

2

0.44

31

30

2000

6

0.30

717

0

0.39

32

25

2000

0

0.00

557

0

0.80

33

27

2000

3

0.15

653

0

0.53

Group Mean

27.6

2000

1.9

0.09

619

0.4

0.64

SD

1.6

0

2.3

0.11

75

0.8

0.21

Table 7      Micronucleus Study-Individual and Group Means and Standard Deviations: Test Material (150mg/kg) 24-Hour

Sampling Time

Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

6 Glycyl (GR34000A) 150 mg/kg 24-hour sampling time

34

28

2000

1

0.05

689

0

0.45

35

28

2000

3

0.15

468

1

1.14

36

27

2000

3

0.15

609

1

0.64

37

26

2000

1

0.05

610

1

0.64

38

27

2000

0

0.00

499

0

1.00

39

28

2000

1

0.05

572

0

0.75

40

26

2000

2

0.10

745

1

0.34

Group Mean

27.1

2000

1.6

0.08

599

0.6

0.71

SD

0.9

0

1.1

0.06

98

0.5

0.28

Table 8       Micronucleus  Study -Individual and Group Means and Standard Deviations: Test Material (75 mg/kg) 24-Hour

Sampling Time

Treatment Group

Animal Number

Bodyweight (g) When Dosed

Polychromatic Erthyrocytes (PCE)

Normochromatic Erythrocytes (NCE)

PCE/NCE Ratio

Number scored

PCE+MN

%PCE+MN

Number scored

NCE+MN

PCE/NCE Ratio

7 Glycyl (GR34000A) 75 mg/kg 24-hour sampling time

41

29

2000

0

0.00

543

0

0.84

42

27

2000

1

0.05

628

0

0.59

43

29

2000

0

0.00

447

1

1.24

44

30

2000

1

0.05

576

0

0.74

45

27

2000

1

0.05

601

0

0.66

46

25

2000

2

0.10

676

0

0.48

47

29

2000

2

0.10

604

0

0.66

Group Mean

28.0

2000

1.0

0.05

582

0.1

0.74

SD

1.7

0

0.8

0.04

73

0.4

0.24

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

Introduction

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of the EC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods         

A range-finding test was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes.  There was no marked difference in test material toxicity between the sexes; therefore the main study was performed using only male mice.   The micronucleus test was conducted using the intraperitoneal route in groups of seven mice at the dose maximum tolerated dose (MTD) of 300mg/kg with 150 and 75 mg/kg as the two lower dose levels.  Animals were killed 24 or 48 hours later, the bone marrow was extracted, smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Results 

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. Though it should be noted that at both exposure times marked reductions in the PCE/NCE were observed with the test material at 300 mg/kg.  This accompanied with the presence of clinical signs was taken to indicate that systemic absorption had occurred and exposure of the bone marrow achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic  erythrocytes  hence confirming  the sensitivity  of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion   

The test material was considered to be non-genotoxic under the conditions of the test.