Reactive Blue F08-0170

Genetic toxicity: in vivo

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:

30 November 2010 to 25 January 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996

Deviations:

no

Test guideline 5

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

Test material information

Constituent 1

Details on test material:

Name: Reactive Blue F08-0170

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 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: 389 g – 457 g, Females: 230g - 269 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: 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). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.5-24.5°C
Relative humidity: 32 - 66%
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, 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 (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 2.5, 15 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. Stability tests (LAB study code 10/252-316AN) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated up to 4 days stability at room temperature and up to 7 days, while stored refrigerated at 2-8ºC, when the recovery range was 95%-106%, which lies within the acceptance range of 100 ± 10%.

Vehicle
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 7530810
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, August 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 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.

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.

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:
25, 150 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):

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.

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:

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:

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.

Statistical analysis of the frequencies of micronuclei in the High dose and Control females gave a value of H = 0.1632 (n.s.). The average frequency in the High dose males was the same as the Control males, so no statistical analysis was appropriate. 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.

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	171	3	396
1002	176	2	260
1003	160	5	416
1004	183	2	240
1005	150	5	344
1006	185	3	281
1007	154	4	429
1008	164	2	393
1009	180	9	369
1010	167	5	263
1011	153	0	335
1012	159	6	243
Mean		3.833	330.75
SD		2.368	70.53

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	184	2	324
4002	157	8	327
4003	175	6	308
4004	161	7	452
4005	166	4	437
4006	170	4	365
4007	156	1	444
4008	163	2	399
4009	178	1	451
4010	152	3	356
4011	172	5	288
4012	182	3	528
Mean		3.833	389.92
SD		2.290	73.27

TABLE 3: DOSE GROUP -           CYCLOPHOSPHAMIDE MALES

 

Animal code	Slide code	Micronucleated PCE/2000 PCE	PCE/1000 PCE+NCE
5001	169	31	208
5002	174	11	209
5003	162	13	203
5004	151	12	188
5005	173	35	395
5006	155	12	237
5007	179	36	125
5008	165	14	124
5009	181	43	215
5010	168	11	104
5011	158	11	207
5012	177	34	351
Mean		21.917	213.83
SD		12.573	85.86

TABLE 4: DOSE GROUP -           CONTROL FEMALES

 

Animal code	Slide code	Micronucleated PCE/2000 PCE	PCE/1000 PCE+NCE
1501	189	4	636
1502	201	0	420
1503	192	3	580
1504	197	3	401
1505	204	9	440
1506	207	3	500
1507	187	3	496
1508	210	3	542
1509	216	0	377
1510	196	4	376
1511	219	0	449
1512	213	7	659
Mean		3.250	489.67
SD		2.701	97.04

 

 

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	206	5	613
4502	212	1	530
4503	221	8	505
4504	191	7	665
4505	214	2	417
4506	199	3	412
4507	203	1	469
4508	217	3	460
4509	188	4	597
4510	208	3	496
4511	194	2	418
4512	200	6	565
Mean		3.750	512.25
SD		2.301	83.50

 

TABLE 6: DOSE GROUP -           CYCLOPHOSPHAMIDE FEMALES

 

Animal code	Slide code	Micronucleated PCE/2000 PCE	PCE/1000 PCE+NCE
5501	202	53	407
5502	209	78	457
5503	186	20	445
5504	205	42	450
5505	193	55	333
5506	218	44	415
5507	195	46	316
5508	220	41	260
5509	190	40	276
5510	211	29	323
5511	198	34	325
5512	215	60	471
Mean		45.167	373.17
SD		15.171	75.39

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 Blue F08-0170 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 Blue F08-0170 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.