Alkali salt of substituted aryl sulfonyl triazinyl amino hydroxy sulfonyl aryl diazo naphthalene sulfonate

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:

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 for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

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:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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 Red F08-0146

Test animals

Species:

rat

Strain:

other: Wistar RJHan:WI

Sex:

male/female

Details on test animals or test system 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.