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:
25 January 2011 to 24 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

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:
micronucleus assay

Test material

Constituent 1
Details on test material:
Name: Reactive Orange F08-0314

Test animals

Species:
rat
Strain:
other: Wistar RJHan:WI
Sex:
male/female
Details on test animals or test system and environmental conditions:
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 LAB Research 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: 397 g – 463 g, Females: 229 g- 273 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 7 days (7 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: 19.6-23.6°C
Relative humidity: 31 - 62%
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 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 LAB Research 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.
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 (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:
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:
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 LAB Research Ltd. Formulations were prepared and stored refrigerated at 2-8ºC pending use within 7 days.

Rationale for dose selection and route of administration

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of an acute oral toxicity study (LAB study code 10/296-001P) and a repeated dose range finding study in the rat (LAB study code 10/296-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The oral route was selected as it is a possible route of exposure to the test item in humans.

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 7 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.
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 / concentrations
Remarks:
Doses / Concentrations:
0 (control), 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
Positive Control MNT group: 12 male and 12 female rats, 1 group
Control animals:
yes, concurrent vehicle
other: Positive control: cyclophosphamide
Positive control(s):
A Positive Control group for the Mammalian Erythrocyte Micronucleus Test (MNT) was added. The animals were mated and females allowed to deliver, similarly to the Main animals, then treated once with 20 mg/kg bw 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.

For the Positive Control (MNT) Group 5, Cyclophosphamide was dissolved in 0.9% NaCl solution shortly before administration, to obtain a dose concentration of 10 mg/mL for a dose volume of 2 mL/kg; no dose formulation analysis was performed.

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: 3390210
Supplier: TEVA Gyógyszergyár ZRT
Retest/Expiry date: February 2013
Storage condition: Room temperature

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. 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.

A 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%). 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:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The data were examined for treatment-related effects, and to determine whether further treatment doses would need to be examined. The results of the evaluation of the High dose Main animals indicated that no further analysis was necessary, as no potentially test item related effects were noted at 1000 mg/kg bw/day.

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.

4/12 of the positive control males had fewer than 10 micronuclei; however, statistical comparison between the negative and positive male controls gave a value of H = 16.691 (p<0.001). Although one female treated with the negative control had a value of 11, the lowest positive control value was 16 and the average frequency was 47. Statistical comparison between the negative and positive female controls gave a value of H = 17.384 (p<0.001), indicating a strong response in both the positive control groups.

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

23

5

430

1002

50

0

346

1003

13

4

364

1004

46

5

375

1005

17

5

354

1006

1

1

319

1007

27

4

380

1008

30

2

387

1009

6

3

342

1010

39

2

352

1011

36

4

409

1012

57

3

332

Mean

 

3.167

365.83

SD

 

1.642

32.12

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

20

5

435

4002

25

1

356

4003

10

2

421

4004

41

3

440

4005

14

2

397

4006

43

1

336

4007

56

1

390

4008

4

3

395

4009

54

3

377

4010

32

4

434

4011

48

1

272

4012

37

5

424

Mean

 

2.583

389.75

SD

 

1.505

49.37

TABLE 3: DOSE GROUP - CYCLOPHOSPHAMIDE MALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5001

15

21

290

5002

33

5

216

5003

5

9

353

5004

42

16

185

5005

59

19

280

5006

24

6

179

5007

55

6

231

5008

9

20

368

5009

40

12

238

5010

49

15

261

5011

19

29

338

5012

28

13

256

Mean

 

14.25

266.25

SD

 

7.25

62.30

TABLE 4: DOSE GROUP - CONTROL FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1501

89

4

502

1502

103

6

482

1503

73

7

483

1504

120

7

528

1505

78

11

536

1506

115

3

542

1507

95

2

438

1508

63

10

500

1509

108

2

508

1510

84

2

501

1511

66

2

465

1512

101

3

466

Mean

 

4.917

495.92

SD

 

3.232

30.97

 


 

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

92

4

428

4502

70

4

521

4503

98

1

409

4504

75

4

435

4505

106

3

449

4506

81

4

458

4507

87

8

535

4508

112

3

555

4509

65

5

531

4510

104

1

451

4511

79

4

517

4512

118

3

443

Mean

 

3.667

477.67

SD

 

1.826

50.14

 

TABLE 6: DOSE GROUP - CYCLOPHOSPHAMIDE FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5501

80

16

394

5502

105

44

416

5503

117

59

538

5504

119

16

442

5505

64

33

493

5506

111

47

410

5507

74

58

476

5508

97

32

462

5509

99

24

405

5510

69

97

436

5511

91

102

455

5512

86

36

522

Mean

 

47.00

459.08

SD

 

28.271

43.70

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reactive Orange F08-0314 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.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reactive Orange F08-0314 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.