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:
28 April 2011 to 23 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD, EU & US EPA test guidance in compliance with GLP and reported with a valid GLP certificate.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other 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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F03-0318

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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 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 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: 366 g – 407 g, Females: 219 g - 249 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)
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524, 505
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: 19.9 – 24.2°C
Relative humidity: 33 - 69%
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. 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:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 8490910, 0110111
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: September 2013, January 2014 respectively
Storage: Room temperature
Details on exposure:
Formulation and analysis of formulation
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 11/008-316AN 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 91%-105%, which lies within the acceptance range of 100 ± 10%.

Test item formulations were analyzed for concentration and homogeneity in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyze (which was collected in replicates as practical) and one set as a back-up for any confirmatory analyses. Similarly, one sample was taken in duplicate from the vehicle Control Group 1 solution for concentration measurements.

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.
Duration of treatment / exposure:
Dosing procedure

Main animals: Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), 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 the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was 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.

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 PPD4 for necropsy on PPD5).
Frequency of treatment:
Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis.
Post exposure period:
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.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw/Day
Basis:
nominal conc.
No. of animals per sex per dose:
Number of animals: 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
Positive control(s):
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.

Examinations

Tissues and cell types examined:
Micronucleated Erythrocytes
Details of tissue and slide preparation:
The mammalian in vivo micronucleus test (MNT) is used for detection of the damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled from bone marrow of animals, usually rodents. The purpose of MNT is to identify substances that cause cytogenetic damage which results in formation of micronuclei containing lagging chromosome fragments or whole chromosomes. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. The purpose of this test as part of the current study was to determine whether the test item caused genotoxic effects resulting in the formation of micronuclei in erythrocytes as sampled from the bone marrow of treated animals.

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. 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.

Two sets of slides were stained with 10% Giemsa solution for 25 minutes. Slides were thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, cover slips were mounted on them.

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; as no potential treatment-related effect was noted, no additional evaluation will be conducted. 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 field. 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.
Evaluation criteria:
Criteria for Identification of Micronucleated Erythrocytes:
- 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.
Statistics:
Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The frequencies in the high dose animals were lower than the control animals in both the males and females, therefore 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 and no further slide examination at lower dose levels 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 = 17.447 (p<0.001) and H = 15.737 (p<0.001) respectively. In summary, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reactive Red F03-0318 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.

Any other information on results incl. tables

Measured concentrations of the dosing solutions

Analytical occasion

Nominal concentration mg/mL

Measured concentrations with the 95% confidence intervals, mg/mL

Measure concentration in the percentage of the nominal

29 April 2011

(first week of treatment)

6.25

6.18±0.52

99

25

25.7±0.66

103

100

102±2.83

102

20 May 2011

(mid term)

6.25

6.09±0.19

97

25

25.6±1.19

102

100

107±1.03

107

09 June 2011

(last week of treatment)

6.25

6.07±0.12

97

25

23.9±1.23

95

100

99.8±5.38

100

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, 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:

This study has been performed in accordance with the Study Plan, Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 474, "Mammalian Erythrocyte Micronucleus Test." adopted 21stJuly 1997, Commission Regulation (EC) No 440/2008 of 30 May 2008, B.12. Mutagenicity –In vivoMammalian Erythrocyte Micronucleus Test, EPA Health Effects Test Guidelines, OPPTS 870.5395 "Mammalian Erythrocyte Micronucleus Test" August 1998 and the Principles of Good Laboratory Practice.

 

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the possible toxic effects of the test item following repeated daily administration by oral gavage to Wistar rats. Reversibility of any treatment-related changes was evaluated following a 14-day Recovery period. The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum. In addition, the test item was evaluated for genotoxic effects by examining the induction of micronuclei in bone marrow erythrocytes of treated and Control animals.

 

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 postpartum/lactation Day PPD5.

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. 

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 PPD4 for necropsy on PPD5.

 

No mortality or systemic adverse effects occurred during the study. 

 

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment or Recovery periods. No test item related effects or toxicologically significant changes were observed in the landing foot splay or grip strength tests.

 

No induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE RED F03-0318 to Wistar rats daily by oral gavage to the High dose Main animals at 1000 mg/kg bw/day.

 

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for REACTIVE RED F03-0318 for parental/adult systemic toxicity is considered to be 62.5 mg/kg bw/day. There was no evidence of any test item-related genotoxic activity under the conditions of this study.