Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Blue 160 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were tested in the absence and presence of metabolic activation from rat and hamster liver.

Neither relevant toxicity nor precipitation of the test item was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism, in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

It is hence concluded that the test item Reactive Blue 160 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 14 Oct 2016; End of experimental phase: 14 Oct 2016; Study completion: the signature date of Final Report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

The E.coli used for this study was the strain WP2 uvrA.

Details on mammalian cell type (if applicable):

Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement: No Growth onMinimal plates+Biotin; Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates; Growth onMinimal plates+Tryptophan.
- uvrA, uvrB: Sensitivity to UV irradiation.
- rfa: Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from rats pre-treated with Phenobarbital and 5,6-Benzoflavone for plate-incorporation and S9 mix-Prival modification for pre-incubation.

Test concentrations with justification for top dose:

Preliminary toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate (based on solubility test).

Main Assay I:
- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate
-TA100 :+ S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

Main Assay II:
- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate
-TA100 :+ S9: 5000, 2500, 1250, 625, 313,156 and 78.1 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water for injection
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

methylmethanesulfonate

other: 2-aminoanthracene and Trypan Blue

Remarks:

Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Details on test system and experimental conditions:

The preliminary toxicity test and the first experiment were performed using a plate-incorporation method. The second experiment was performed using a pre-incubation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al. 1981); Regression line.

Key result

Species / strain:

other: S.typhyimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item did not induce two-fold increases in the number of revertant colonies, in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Reactive Blue 160 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II. The test item was used as solution in sterile water for injection.

Toxicity test: The test item Reactive Blue 160 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. A slight reduction in revertant colonies was observed with TA100 tester strain at higher dose levels, both in the absence and presence of S9 metabolism. No relevant increases in revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main Assays: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate

-TA100 :+ S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

Slight toxicity was observed with TA100 tester strain at the highest dose level in the absence of S9 metabolism. No relevant increase in revertant numbers, was observed with any tester strain at any dose levels in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed. Based on the chemical structure of the test item (azo-dyes), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The dose-range was slightly modified to take into account the results ofMain Assay I.

The test item was assayed at the following dose levels:

- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate

-TA100 :+ S9: 5000, 2500, 1250, 625, 313,156 and 78.1 µg/plate

Neither relevant toxicity nor precipitation of the test item was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism, in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

Conclusion: It is concluded that the test item Reactive Blue 160 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on an Unscheduled DNA Synthesis Assay, the test substance was not found to be genetically toxic when tested up to a limit dose level of 2000 mg/kg bw oral gavage in male rats. In addition the test substance was tested negative in a mouse micronucleus test at the limit dose of 5000 mg/kg bw oral gavage in male mice..

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM Guideline by Butterworth et al. (1987)

Principles of method if other than guideline:

The method protocol is essentially described in Mirsalis and Butterworth (1980) as modified by Ashby et al. (1985, 1987).

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

other: Alderley Park rat (Alpk:APfSD)

Details on species / strain selection:

Alpk:APfSD rats will be used as CTL had used this strain in toxicological studies for a long time.

Sex:

male

Details on test animals or test system and environmental conditions:

The animals used for this study will be male Alderley Park (Alpk:APfSD) rats weighing 180-280 g at the time of dosing. These animals will be suppilied by the Zeneca Barriered Animal Breeding Unit (BABU), Alderley Park, Macclesfield, Cheshire. Animals will be held in rodent rooms of animal care units on the Alderley Park site, housed under conditions appropriate to their species. Records of environmental conditions are held by the animal units at CTL.

A maximum of five rats will be housed in each cage. The animals will be supplied with food and water ad libitum. Drinking water will be supplied via an automatic watering system and the diet used will be Porton Combined Diet (PCD) supplied by Special Diets Services Ltd, Stepfieldt Witham, Essex. An analysis of each batch is supplied by Special Diets Services Ltd, and is checked by the Unit co-ordinator at BABU for acceptability of levels of possible contaminants which might influence the outcome of a study before the batch is used. Records for the receipt and storage of diet are held by the animal units at CTL. Since the animals will eventually be housed in a fume cupboard, acclimatisation is not considered appropriate.

Immediately prior to use, the animals will be transported in cages with filter lids to the testing laboratory. Whilst in this laboratory, the animals will be maintained according to SOP No CT90-023. The animals will then be weighed, tail numbered and allocated to the relevant treatment group. The animals will be allocated according to the order in which they are removed from the stock cage, ie, the first animal will be No. 1, the second, No 2 etc. The holding cage will be labelled with the Study number, the Project Licence code, the name of the licensee and the respective treatment details. Animals will be housed up to three per cage in a fume cupboard for a total period not exceeding 24 hours.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water

Details on exposure:

Dosage will be carried out according to SOP Nos CT50-137, (Dosing of Rodents by Gavage) and CT30-181, (Procedure for the In Vivo Rat Hepatocyte UDS Assay). The animals will receive a single oral dose, by gavage at a volume of 10 mL/kg bodyweight. During the course of this study, animals will have free access to PCD diet pellets ang water. A total of five animals for each test compound dose level at two separate timepoints will be treated. Clinical observation of the animals will be carried out following dosing and also immediately prior to perfusion. Any clinical signs of toxicity will be noted in the raw data. Of the two positive and two negative control animals in each experiment only one of each will normaily be scored for induction of UDS.

Details on exposure route selection: oral dosage was used since it was considered the most likely way of human exposure and related in vivo effects.

Duration of treatment / exposure:

4 d

Frequency of treatment:

Single dose

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

1 250 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylthydrazine dihydrochloride
- Route of administration: oral gavage
- Doses / concentrations: 30 mg/kg bw

Tissues and cell types examined:

Hepatocytes (rat liver tissue)

Details of tissue and slide preparation:

The treated rats will be killed by the process of deep anaesthesia followed by perfusion and excision of the liver. Hepatocytes will be isolated from the animals by means of a two stage collagenase perfusion technique. Full details of the methodology to be used are in SOP No CT30-181.

Two or sixteen hours after dosage, animals will be anaesthetised, dissected and cannulae inserted into the hepatic portal vein and superior vena cava. Pumped Buffer 1 solution will be used to flush the liver free of blood and to remove calcium from the desmosomes. Buffer 2 will then be introduced, to which will be added calcium chloride and collagenase to cause disaggregation of the tissue into single cells. The liver will then be removed, cut open, and hepatocytes prepared by low speed centrifugation and resuspension in Williams Medium. Hepatocyte cultures, (at least three per animal), are left to attach onto tissue culture coverslips at 37°C.

The medium covering the hepatocyte cultures will be replaced with Williams Incomplete Medium containing 10 µCi/mL [3H]thymidine. After four hours incubation, the hepatocytes will be washed three times with Williams Incomplete Medium containing 0.25 mM unlabelled thymidine. Cells will be left to incubate overnight at 37°C with the same medium. This is a "cold-chase" procedure designed to remove the majority of unincorporated [3H]thymidine from the hepatocyte cultures.

Cultures will be fixed in glacial acetic acid . ethanol (1:3 v/v) and covers lips mounted onto microscope slides. Slides will be coated with photographic emulsion and left for 14 days at 4°C in the dark. The emulsion will be developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.

Slides will be examined microscopically and only the two highest dose levels not showing undue cytotoxicity, plus at least one negative and positive control animal, will be examined for UDS. Under conditions of high toxicity, cells of unusual morphology are formed, and the thymidine uptake mechanisms may consequently be affected giving false negative responses . Slides will be allocated a random code number generated by a computer. Paper labels, bearing the study number and code number, will be placed on the slides to obscure the slide identifiers by a person unconnected with slide scoring.

Coded hepatocyte cultures wi ll be examined for the induction of UDS using a microscope-mounted image analyser l inked to a computer for data analysis. The numoer of silver grains over the nucleus [N] is determined. Then an equivalent area of cytoplasm tangential to the nucleus and with the highest apparent number of silver grains is scored [C]. The difference between these two values [N-C] is the net nuclear grain count. Normally 30 cells will be scored on each of two slides for each animal . The third slide will not normally be read, unless it is considered that a total of 60 cells will not be obtained from the first two slides examined.

Evaluation criteria:

Criteria for a negative (ie non-genotoxic) response:
A negative response is obtained where the mean net nuclear grain count of each test comoound-treated animal is less than 0.

Criteria for a positive (ie genotoxic) response
An individual animal showing mean net nuclear grain counts of 0 or greater is considered to be indicative of the occurrence of a UDS response. A compound will be assigned as an unequivocal genotoxin in this assay if such a response is reproduced.

Marginal Responses
If the group mean net nuclear grain count of the treated animals is below 0 but with an individual animal exceeding 0, interpretation will consider factors as any statistically significant difference between treated and control cultures, dose response relationships, the percentages of cells in repair and the absolute values obtained for the negative control. ResuIts from the independent experiment will be examined to observe whether any effect is reproducible. In such cases, the ultimate designation will be based on experience and scientific judgement.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. The validity criteria were fulfilled.

Each experiment was validated by concurrent control treatments of pats with sterile double deionised water, the vehicle for Procion Blue H-ERD Press Paste and with the carcinogen dimethylhydrazine dihydrochloride. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from positive control-treated animals had mean net nuclear grain counts of greater than +5. These data show that the background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity.

Hepatocytes from Procion Blue H-ERD Press Paste treated animals were assessed for UDS at both dose levels tested. Treatments with Proc ion Blue H-ERD Press Paste in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.

Conclusions:

Based on an Unscheduled DNA Synthesis Assay, the test substance was not found to be genetically toxic when tested up to a limit dose level of 2000 mg/kg bw (oral: gavage; hepatocytes). The test substance did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver. The test completely fulfilled the validity criteria for both positive and negative controls as defined by the standard test protocol.

Executive summary:

Reactive Blue 160 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Alpk:APfSD rats were treated with a single oral dose of Reactive Blue 160 by gavage at dose levels of 1250 or 2000mg/kg bodyweight. The latter dose level is the limit dose level for this assay. Animals were killed and hepatocytes isolated and prepared two and sixteen hours after dosing. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount

of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was

subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with sterile double deionised water, the vehicle for Reactive Blue 160 and with the carcinogen dimethylhydrazine dihydrochloride [DMH.2HC1]. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from DMH.2HC1-treated animals had mean net nuclear grain counts of greater than +5. These data show that the background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity.

Hepatocytes from Reactive Blue 160 treated animals were assessed for UDS at both dose levels tested. Treatments with Reactive Blue 160 in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.

It is concluded that, when tested up to a limit dose level of 2000mg/kg, the test sample of Reactive Blue 160 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in rat liver.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The CD-1 mouse, which will be used in this study, has been shown to be sensitive to several known clastogens and carcinogens

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK
- Age at study initiation: 7-9 wk in Phase I and 6-20 wk in Phase II
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period: at least 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 °C
- Humidity (%): 55±15 %
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were prepared in water
A solution of cyclophosphamide was prepared in physiological saline. All dosing preparations were administered at a volume of 20 mL/kg bw.

Duration of treatment / exposure:

Bone marrow samples were taken 24 hours and 48 hours after treatment

Frequency of treatment:

single treatment

Post exposure period:

Bone marrow samples were taken 24 hours and 48 hours after treatment

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 65 mg/kg bw

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on patterns of lethalities or severe toxicity observed over a 4-d observation period following a single oral dose in a maximum tolerated dose (MTD)-Phase I

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow smears were prepared 24 and 48 h after dosing for the vehicle control and treated animals and 24 h after dosing for the cyclophosphamide treated animals.

DETAILS OF SLIDE PREPARATION: The preparations were stained with polychrome methylene blue and eosin to visualise the various cell types.

METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of polychromatic erythrocytes (PCE) per slide: 1000 PCE
Percentage of polychromatic erythrocytes in the total erythrocyte population: 1000 Erythrocytes

Evaluation criteria:

A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group

Statistics:

- The incidence of micronucleated PCE and percentage PCE in the erythrocyte sample, were considered by ANOVA.
- All analyses were carried out using the GLM procedure in SAS.
- One-sided Student's t-test:

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.

Executive summary:

A study was conducted to assess the potential of the test substance to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice performed according to OECD Guideline 474 in compliance with GLP.

A single oral dose was given to groups of 5 male mice at a dose level of 5000 mg/kg bw, this being the limit dose level for the assay. Bone marrow samples were taken 24 and 48 h after dosing.

No statistically or biologically significant, increases in the incidence of micronucleated PCE, over the vehicle control values, were seen at either of the sampling times investigated. Comparison of the percentage of PCE showed, no statistically significant decreases, compared to the vehicle control values, at either of the sampling times.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item Reactive Blue 160 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) inMain Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II. The test item was used as solution in sterile water for injection.

Toxicity test:The test item Reactive Blue 160 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. A slight reduction in revertant colonies was observed with TA100 tester strain at higher dose levels, both in the absence and presence of S9 metabolism. No relevant increases in revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main Assays:On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate

-TA100 :+ S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

Slight toxicity was observed with TA100 tester strain at the highest dose level in the absence of S9 metabolism. No relevant increase in revertant numbers, was observed with any tester strain at any dose levels in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed. Based on the chemical structure of the test item (azo-dyes), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The dose-range was slightly modified to take into account the results ofMain Assay I.

The test item was assayed at the following dose levels:

- TA1535, TA1537,WP2 uvrA ,TA98: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA100: −S9: 5000, 2500, 1250, 625, and 313 µg/plate

-TA100 :+ S9: 5000, 2500, 1250, 625, 313,156 and 78.1 µg/plate

Neither relevant toxicity nor precipitation of the test item was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism, in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

Conclusion:It is concluded that the test item Reactive Blue 160 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reactive Blue 160 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Alpk:APfSD rats were treated with a single oral dose of Reactive Blue 160 by gavage at dose levels of 1250 or 2000mg/kg bodyweight. The latter dose level is the limit dose level for this assay. Animals were killed and hepatocytes isolated and prepared two and sixteen hours after dosing. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount

of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was

subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with sterile double deionised water, the vehicle for Reactive Blue 160 and with the carcinogen dimethylhydrazine dihydrochloride [DMH.2HC1]. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from DMH.2HC1-treated animals had mean net nuclear grain counts of greater than +5. These data show that the background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity.

Hepatocytes from Reactive Blue 160 treated animals were assessed for UDS at both dose levels tested. Treatments with Reactive Blue 160 in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.

It is concluded that, when tested up to a limit dose level of 2000mg/kg, the test sample of Reactive Blue 160 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in rat liver.

A study was conducted to assess the potential of the test substance to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice performed according to OECD Guideline 474 in compliance with GLP.

A single oral dose was given to groups of 5 male mice at a dose level of 5000 mg/kg bw, this being the limit dose level for the assay. Bone marrow samples were taken 24 and 48 h after dosing.

No statistically or biologically significant, increases in the incidence of micronucleated PCE, over the vehicle control values, were seen at either of the sampling times investigated. Comparison of the percentage of PCE showed, no statistically significant decreases, compared to the vehicle control values, at either of the sampling times.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.