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:
24 February 2011 to 10 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, US and OECD test guidance in compliance with GLP and reported with a valid GLP certificate.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F08-0146

Test animals

Species:
rat
Strain:
other: Wistar RJHan:WI
Sex:
male/female
Details on test animals and environmental conditions:
EXPERIMENTAL ANIMALS

Species and strain: Wistar RJHan:WI rats
Source: Laboratoire Elevage Janvier, B.P. 4105, Route des Chênes Secs, 53940 Le Genest-St-Isle CEDEX France
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals:
Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group
At the completion of the study, the spare animals were returned to CiToxLAB Hungary Ltd. spare colony, as their use was not required (no replacements with spare animals had to be performed)
Age of animals: Young adult rats, approximately 10-11 weeks old at starting and 12-13 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 359 g – 424 g, Females: 231 g - 267 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 7 days (5 days from animal arrival to pre-treatment ophthalmoscopy examination, 7 days from animal arrival to onset of treatment)

Husbandry

Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 525
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 23.8°C
Relative humidity: 30 - 64%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

The temperature and humidity were measured twice daily; no deviations from the target ranges were noted during the study.

Food and water supply

Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).

The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification

Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section.

The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.

Randomization

All parental/adult (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The test item was formulated in distilled, sterile water for injection at 6.25, 25 and 100 mg/mL concentrations without correction for purity, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared and stored refrigerated at 2-8ºC pending use within 7 days. Stability tests (CiToxLAB Hungary Ltd. study code 10/285-316AN and additional evaluation during the current study) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated 1 day stability at room temperature and up to 7 days, while stored refrigerated at 2-8ºC in the dark, when the recovery range was 99%-100%, which lies within the acceptance range of 100 ± 10%.

Vehicle

Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 7530810, 8490910
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, August 2013, September 2013 respectively
Storage: Room temperature
Details on exposure:
Dosing procedure

Main animals

Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume in relation to the body weight (10 mL/kg bw) was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Dosing of both sexes began after an acclimation period (A) of least 7 days after the animal arrival; the animals were dosed for 2 weeks before mating, during the mating/post-mating, and were continued up to and including the day of necropsy.

Males were dosed for at least 28 days (14 days pre-mating, 14 days mating/post-mating period and on the day of necropsy), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.

Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to and including the day of necropsy (at least 4 days post-partum). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum PPD0) for dams or Day 0 post-natal (PND0) for the offspring. Females that proved not to be pregnant were sacrificed as practical, 26 to 28 days after the end of the mating period.



Duration of treatment / exposure:
In the Main Groups, male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postnatal/lactation Day PND 5.
Frequency of treatment:
Once daily, 7 days per week.
Post exposure period:
Main animals were treated with test item up to euthanasia.
Recovery animals were kept for at least 14 days without treatment prior to euthanasia.
Positive control animals were euthanised approximately 24 hours after administration of cyclophosphamide.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 250 and 1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Control animals:
yes, concurrent vehicle
other: Positive control: cyclophosphamide
Positive control(s):
Positive Control Micronucleus Test (MNT) animals
Group 5 animals were mated and females allowed to deliver, similarly to the Main animals. All animals were treated with 20 mg/kg bw/day Cyclophosphamide, administered by intraperitoneal injection approximately 24 h prior to scheduled necropsy (males, on Day 27 for necropsy on Day 28; females, on PND4 for necropsy on PND5).

Positive Control Name: Cyclophosphamide monohydrate
Lot number: 079K1569
Supplier: Sigma-Aldrich Co.
Retest/Expiry date: July 2012
Storage condition: Refrigerated (2-8 °C)
Purpose of use: Positive Control item Group 5


Examinations

Tissues and cell types examined:
Four sets of bone marrow smears for MNT were prepared from the animals, including the Vehicle Control (water) and the Positive Control (Cyclophosphamide) groups. According to the study plan and/or subsequent amendment(s), the bone marrow was collected from the right femur of the rats immediately after euthanasia (the left femur of Main and Recovery group animals was used for routine histopathology, the left femur of Positive Control animals was discarded) and flushed with foetal bovine serum (5 mL) using a syringe and needle.
Details of tissue and slide preparation:
Cells were concentrated by a gentle centrifugation. Smears of the cell pellet were made on standard microscope slides and the slides were then air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for at least 5 minutes and allowed to air-dry.

One set of Giemsa-stained slides was given unique code numbers for blinded evaluation (the code labels covered all unique identification markings on the slides to ensure that they were scored without bias). All slides were blinded; only those of the Control (Gr. 1), Positive Control (Gr. 5) and High dose (Gr. 4) Main animals were sent for evaluation.

At least 2000 polychromatic (immature) erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated (MN) cells, expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic fieldThe proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei was recorded in both types of erythrocytes.

Criteria for Identification of Micronucleated Erythrocytes

A micronucleus is defined in following way:

- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.


Evaluation criteria:
Criteria for a positive response: The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related.
Statistics:
Data were collected by completing a pre-prepared sheet by hand. The data were tabulated using appropriate forms for reporting. The frequencies of micronucleated polychromatic erythrocytes in animals in the test groups were compared to the values found in the corresponding negative control group. Statistical analysis was performed using Kruskal Wallis Non Parametric ANOVA test (level of significance 5%). Statistical analysis of the positive control data was not necessary as all values were higher than any of the corresponding negative control values.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
See Chapter 7.5.1 for details of effects.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No statistical analysis of the frequencies of micronuclei in the animals treated with the high dose in either males or females was appropriate as the average number of micronuclei was lower than the corresponding negative controls in both cases. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, thus, no further slide examination was considered required.

One animal (4002) treated with 1000 mg/kg of the test item gave a value of 13 micronuclei in 2000 PCEs, while animals 5002 and 5010, treated with the positive control cyclophosphamide gave values of 7 and 8 respectively. These animals were further assessed by examining a further 2000 PCEs where possible; for this purpose an additional animal was included (5009) and the slides were recoded to ensure unbiased analysis. The results in the tables include the additional analysis, with the exception of animal 5010, where there were insufficient cells to evaluate a total of 4000 PCEs.

Statistical analysis of the frequencies of micronuclei in the High dose and Control males gave a value of H = 3.314 (n.s.). Statistical analysis of the frequencies of micronuclei in the High dose and Control females gave a value of H = 0.042 (n.s.). The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, thus, no further slide examination was considered to be required.

Although one animal in both males and females treated with the positive control showed fewer than 10 micronuclei/2000PCE, all the values in the positive control groups were greater than any values in the corresponding negative control groups. Statistical analysis gave H = 17.458 and 17.349 respectively (p<0.001), indicating a highly significant response.

Any other information on results incl. tables

TABLE 1: DOSE GROUP - CONTROL MALES

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1001

30

2

358

1002

11

4

360

1003

20

5

397

1004

43

0

344

1005

36

2

387

1006

19

1

401

1007

53

2

383

1008

40

1

293

1009

1

4

403

1010

48

1

363

1011

10

1

367

1012

28

1

363

Mean

 

2.000

368.25

SD

 

1.537

30.39

TABLE 2: DOSE GROUP - HIGH DOSE 1000 mg/kg bw/day MALES

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

4001

32

0

423

4002

55

9.5 (*)

430

4003

15

1

399

4004

26

3

395

4005

60

6

390

4006

5

2

378

4007

45

3

304

4008

49

4

406

4009

23

4

389

4010

38

2

406

4011

33

7

384

4012

7

4

352

Mean

 

3.792

388.00

SD

 

2.658

33.41

TABLE 3: DOSE GROUP - CYCLOPHOSPHAMIDE MALES

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5001

50

11

280

5002

16

10.5 (*)

241

5003

6

18

291

5004

34

27

355

5005

24

11

278

5006

14

27

243

5007

39

13

194

5008

4

19

262

5009

31

29 (*)

297

5010

44

6(*)

290

5011

25

12

218

5012

54

16

285

Mean

 

16.792

269.50

SD

 

7.297

41.88

TABLE 4: DOSE GROUP - CONTROL FEMALES

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1501

78

2

459

1502

63

6

483

1503

99

2

496

1504

94

5

512

1505

119

4

394

1506

73

8

427

1507

82

1

397

1508

104

5

437

1509

114

5

528

1510

70

3

464

1511

108

0

472

1512

88

2

434

Mean

 

3.583

458.58

SD

 

2.314

42.54

TABLE 5: DOSE GROUP - HIGH DOSE 1000 mg/kg bw/day FEMALES

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

4501

115

3

477

4502

67

5

431

4503

101

5

455

4504

110

8

503

4505

120

5

448

4506

75

2

400

4507

80

2

436

4508

95

3

524

4509

64

1

501

4510

91

1

501

4511

86

4

443

4512

106

7

373

Mean

 

3.833

457.67

SD

 

2.250

45.30

TABLE 6: DOSE GROUP - CYCLOPHOSPHAMIDE FEMALES

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000

PCE+NCE

5501

111

26

444

5502

81

35

411

5503

116

12

321

5504

65

65

518

5505

96

26

402

5506

74

34

470

5507

85

16

386

5508

90

16

406

5509

100

52

402

5510

66

32

384

5511

79

9

335

5512

105

37

460

Mean

 

30.000

411.58

SD

 

16.492

55.46

(*) number of micronucleated PCEs determined in a total of 4000 PCEs

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE RED F08-0146 to Wistar rats daily by oral gavage to the High dose Main animals at 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study. There was no evidence of any test item-related genotoxic activity under the conditions of this study.
Executive summary:

The objective of this study was to assess the potential genotoxic effect of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals. 

This study was conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE RED F08-0146 to Wistar rats daily by oral gavage to the High dose Main animals at 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study. There was no evidence of any test item-related genotoxic activity under the conditions of this study.