Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 October 2011 to 6 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Substance name: Reactive Yellow F01-0555

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
Hygienic level: SPF at the supplier; 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 were performed)
Age of animals: Young adult rats, approximately 11-12 weeks old at starting and 13-14 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 320-404 g, Females: 205 g- 259 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 6 days (6 days from animal arrival to pre-treatment ophthalmoscopy examination, 12 days 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: 524
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). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.7-23.8°C
Relative humidity: 34 - 55%
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.

The new-borns (Offspring, F1 Generation) were identified by cutting off digit-tips up to one day after birth.

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 4 days. Stability tests (CiToxLAB Hungary Ltd. study code 11/174-316AN) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated a 1-day stability at room temperature and 4-day stability while stored refrigerated at 2-8ºC, when the recovery range was 100%-103%, which lies within the acceptance range of 100 ± 10%.

Vehicle
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 2170511, 2190511
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, May 2014, 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 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 at least 6 days acclimation (A) and 12 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 dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 26-27 days after the end of the mating period.





Duration of treatment / exposure:
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 6 days mating period, through gestation and up to and including the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 27-28 days after the end of the mating period.

Positive Control 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).

Recovery animals
Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 56.

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 / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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
Positive Control MNT group: 12 male and 12 female rats, 1 group
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

Name: Physiological saline (0.9% NaCl solution)
Lot number: 6880610
Supplier: TEVA Gyógyszergyár ZRT
Retest/Expiry date: June 2013
Storage condition: Room temperature
Purpose of use: Preparation of Positive Control Group 5 solution

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.

2000 polychromatic (immature) erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated (MN) cells. The 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.

The Micronucleus Test is considered acceptable/valid in the conditions of this study, as it met the following criteria:

-the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls fell within the range of historical laboratory control data.
-the positive control item produced biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
-each treated and control group included at least 5 analysable animals.

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.

Criteria for a negative response: The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.

Equivocal response: It may be necessary to perform further investigations or to score additional cells if equivocal results are obtained which do not meet the criteria for a positive or negative response. In this study there were no equivocal results, therefore no additional scoring was required.
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%) except where the frequency in treated animals was less than that in the negative control animals. As some of the positive control frequencies were less than 12 micronuclei in 2000 PCEs, the positive control data were also tested statistically to verify the overall positive response in these groups.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
See Chapter 7.5.1 for details
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.

The frequencies in the high dose animals were compared with the control animals; in the males, H = 0.244 and in the females H = 0.143 which are both substantially below statistical significance. 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. The positive and negative control results were also compared, and both males and females gave a significant response, with values of H = 4.841 (p<0.05) and H = 14.817 (p<0.001) respectively.

Any other information on results incl. tables

The individual and mean data are presented in Tables 1 – 6 below.

  

 

TABLE 1: DOSE GROUP -           CONTROL MALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1001

15

2

448

1002

02

4

414

1003

29

1.5a

464

1004

34

7

401

1005

39

3

374

1006

21

9

469

1007

07

7

440

1008

47

4

422

1009

27

7

434

1010

51

11.5a

521

1011

13

1

429

1012

44

4

465

Mean

 

5.083

440.08

SD

 

3.225

37.78

                                    aNo. of micronuclei assessed in 4000 PCE

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

18

18a

418

4002

25

8

416

4003

43

3

530

4004

05

0

395

4005

41

15a

499

4006

56

2

397

4007

36

3

452

4008

55

1

354

4009

10

1

490

4010

58

4

384

4011

30

8

446

4012

54

3

423

Mean

 

5.500

433.67

SD

 

5.745

51.82

                                   aNo. of micronuclei assessed in 4000 PCE

 

 

TABLE 3: DOSE GROUP - CYCLOPHOSPHAMIDE MALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5001

06

5

315

5002

42

13

315

5003

11

7

367

5004

31

2

325

5005

49

2a

206

5006

04

7

265

5007

48

29

425

5008

17

24

315

5009

24

19a

331

5010

52

18

348

5011

16

16

380

5012

35

23

319

Mean

 

13.750

325.92

SD

 

9.137

55.14

                   aNo. of micronuclei assessed in 4000 PCE

TABLE 4: DOSE GROUP - CONTROL FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1501

100

4

448

1502

106

3

460

1503

115

5

486

1504

75

10

497

1505

111

5

465

1506

90

7

437

1507

63

5

418

1508

118

6

419

1509

78

7

501

1510

84

4

458

1511

66

2

515

1512

94

1

486

Mean

 

4.917

465.83

SD

 

2.429

31.86

 

 

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

65

11

536

4502

98

12

519

4503

120

2

455

4504

77

5

523

4505

113

4

419

4506

83

1

446

4507

103

2

451

4508

88

13

510

4509

69

11

531

4510

107

2

365

4511

93

6

461

4512

116

5

481

Mean

 

6.167

474.75

SD

 

4.407

51.88

 

TABLE 6: DOSE GROUP - CYCLOPHOSPHAMIDE FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5501

70

33

470

5502

108

51

425

5503

117

14

396

5504

87

5

323

5505

112

68

435

5506

64

25

353

5507

102

17

405

5508

76

61

359

5509

82

66

476

5510

92

36

436

5511

71

36

477

5512

97

28

452

Mean

 

36.667

417.25

SD

 

20.821

51.12

 

 

 

Applicant's summary and conclusion

Conclusions:
No induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE YELLOW F01-0555 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.
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.

Male and female rats were treated with the test item at dose levels of 62.5, 250, and 1000 mg/kg bw/day for a minimum of 28 days.

The frequencies of micronuclei in the high dose animals were compared with the control animals in the males and in the females which

are both substantially below statistical significance. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, hence, no further slide examination was considered required.  The positive and negative control results were also compared, and both males and females showed a statistically significant increase in micronuclei.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of the test item to Wistar rats daily by oral gavage 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.