2-[[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulpho-1-anthryl)amino]cyclohexyl]amino]-6-fluoro-1,3,5-triazin-2-yl]amino]benzene-1,4-disulphonic acid, potassium sodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 13, 1992 to December 14, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals "Genetic Toxicology: Micronucleus Test", No. 474

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449/EEC B.12. Other Effects –Mutagenicity Micronucleus Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulpho-1-anthryl)amino]cyclohexyl]amino]-6-fluoro-1,3,5-triazin-2-yl]amino]benzene-1,4-disulphonic acid, potassium sodium salt

EC Number:

277-179-9

EC Name:

2-[[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulpho-1-anthryl)amino]cyclohexyl]amino]-6-fluoro-1,3,5-triazin-2-yl]amino]benzene-1,4-disulphonic acid, potassium sodium salt

Cas Number:

72987-16-7

Molecular formula:

C29 H26 F N7 O11 S3 . x K . x Na C29H(26-x-y)FK(x)N7Na(y)O11S3

IUPAC Name:

potassium sodium 2-{[4-({4-[(4-amino-9,10-dioxo-3-sulfonato-9,10-dihydroanthracen-1-yl)amino]cyclohexyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}benzene-1,4-disulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The animals used were young adult male and virgin female mice, strain Bor: NMRI (SPF Han), bred and supplied by F. Winkelmann, Borchen.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals initially weighed 28-44 g, and were thus approximate 8 to 12 weeks of age.
The breed's state of health is regularly spot-checked for the major specific pathogens.

On the day of arrival, the health of the animals was appraised before acclimatizing them to the housing conditions for a period of at least one week. Only healthy animals without symptoms were used in the study.
The females were kept in groups of a maximum of three mice in Makrolon type I cages. Males were kept singly in type I cages. Bedding of soft wood granules, type S 8/15 (J. Rettenmaier & Sohne, Fullstoff-Fabriken, 7092 Ellwangen-Holz-muhle) was used. The animals were identified by cage and picric-acid marks.
The wood granules were spot-checked for contaminants at regular intervals.
Husbandry was standardized, with twelve hours of electrical lighting daily (6.00 hours to 18.00 hours, about 500 lux), 22.5-23 °C room temperature, and 38-45% mean relative humidity. EP IT Elb. 2 (engineering department) gives the following settings for the animal room: 22±1.5 °C, 40% to 70% humidity, and air change about ten times per hour.
The animals in this study were kept in Animal Room 447, Building 514. The fixed-formula feed was Altromin 1324 Standard Diet, produced according to specification by Altromin GmbH, Lage. Tap water and feed were available for ad libitum consumption.
The nutritive composition and contaminant content of the standard diet were routinely spot-checked and analysed.
The water was of drinking quality.
The feed was offered in troughs. Water was provided in polycarbonate bottles, 300 ml volume
Cages, bottles and troughs were cleaned by spraying hot water without detergent through power jets. Then deionized water, to which a detergent had been added, was sprayed over the equipment. This promoted spotless drying, particularly of the synthetic material. Finally the equipment was dried with hot air.
Cages, wood granules and water were not changed during the study, due to the short duration of a maximum of two days. The animal room was disinfected twice monthly. Blattanex pesticide was occasionally used.
This definitely had no effect on the test since no pesticide was employed during acclimatization and testing periods.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Physiological saline solution.
A stability test in the vehicle did not detect a relevant change in the percent active ingredient. Reactive Blue 181 is stable in the vehicle at room temperature at concentrations ranging from 1 mg/ml to 50 mg/ml for at least twenty-four hours.

Details on exposure:

C.I. Reactive Blue 181 was suspended in physiological saline solution, stirred with a magnetic mixer during administration.
In all of the groups, the administered volume was 10 ml/kg body weight.

Duration of treatment / exposure:

Single dose administration

Frequency of treatment:

Single injection

Post exposure period:

The femoral marrow of groups treated with C.I. Reactive Blue 181 was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours.

Doses / concentrations

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose (5 male/5 female)

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide was dissolved in deionized water and administered in the same way.

Examinations

Tissues and cell types examined:

Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The number of normochromatic erythrocytes per 1000 polychromatic ones was noted.

Details of tissue and slide preparation:

Preparation of Specimens
Schmid's method was used to produce the smears.
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg.
The femur was separated from muscular tissue.
The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end.
The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel.
A suitable tube was filled with sufficient fetal calf serum.
A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula.
The cannula was pushed into the open end of the marrow cavity.
The femur was then completely immersed in the calf serum and pressed, against the wall of the tube, to prevent its slipping off.
The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.
Finally, the flushing might be repeated from the other end, after it had been opened.
The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder.
The sediment was mixed to produce a homogeneous suspension.
One drop of the viscous suspension was placed on a well cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.
The labelled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.

The Staining of Smears
The smears were stained automatically with an Ames HemaTek Slide stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.

The Covering of Smears
Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. The slides were removed singly (e.g. with tweezers) to be covered.
A small amount of covering agent was taken from a bottle with a suitable object (e.g. glass rod) and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent had dried.

Criteria for dose selection:
The selection of the C.I. Reactive Blue 181 dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 100 mgjkg, 250 mg/kg, 350 mg/kg and 500 mg/kg C.I. Reactive Blue 181. The following symptoms were recorded for up to 48 hours, starting at 100 mg/kg: apathy, reduced motility, roughened fur, blue discoloration of hairless parts of skin, staggering gait, sternal recumbency, spasm and difficulty in breathing. In addition, 5 of 5 animals died in the 350 and 500 mg/kg groups. Based on these results, 250 mg/kg C.I. Reactive Blue 181 was chosen as MTD for this test.

Evaluation
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
It is expedient to establish the ratio of polychromatic to normochromatic erythrocytes for two reasons:
1. Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alteration of this ratio may show that the test compound actually reaches the target.

Therefore, the number of normochromatic erythrocytes per 1000 polychromatic ones was noted. If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A relevant, treatment related alteration of the ratio polychromatic to normochromic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established. This information is useful in two ways. Firstly, it permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated polychromatic erythrocytes, it permits a representation of the time-effect curve for positive substances.
An increase in the number of micronucleated normochromatic erythrocytes, without a preceding increase in micronucleated polychromatic erythrocytes, is irrelevant to the assessment of a clastogenic effect, since normochromatic erythrocytes originate from polychromatic ones. Before an effect can be observed in normochromatic erythrocytes, there must be a much greater increase in micronucleated polychromatic erythrocytes, due to the "dilution effect" of the "old" cells, i.e. normochromatic erythrocytes already present at the start of the test, and this effect would have been observed previously.

Evaluation criteria:

Assessment Criteria
A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

Assay Acceptance Criteria
An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Statistics:

The C.I. Reactive Blue 181 group(s) with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test With respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2 -test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

apathy, roughened fur, blue discoloration of hairless parts of skin, staggering gait, spasm and blue discolored urine. There were no substance induced mortalities.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toleration by the Animals
After single intraperitoneal administration of 250 mg/kg C.I. Reactive Blue 181, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, blue discoloration of hairless parts of skin, staggering gait, spasm and blue discolored urine. Their feeding behavior was normal. There were no substance induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic Evaluation
Concerning the assessment of the clastogenic potential of C.I. Reactive Blue 181 there were no relevant variations in results between males and females. Therefore, they were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with C.I. Reactive Blue 181, being 1000: 556 (1s=110) in the negative control, 1000: 1938 (1s=903) in the
16 hours group, 1000: 1324 (1s=294) in the 24 hours group and 1000: 651 (1s=152) in the 48 hours group. Relevant variations were thus noted.
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 250 mg/kg C.I. Reactive Blue 181, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 1.6/1000 (1s=1.6) in the negative control, and 2.0/1000 (1s=1.6), 2.2/1000 (1s=0.8) and 2.3/1000 (1s=1.2) in the C.I. Reactive Blue 181 groups.
Similarly, there could be no biologically significant variation between the negative control and C.I. Reactive Blue 181 groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.
The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 24.3/1000 (1s=10.8), which represents a biologically relevant increase in comparison in the negative control.
There could not have been biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.
No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [1000 : 578 (1s=192), as against 1000: 556 in the negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

Any other information on results incl. tables

 

Summary of Results of Micronucleus Test with C.I. Reactive Blue 181 (after acute intraperitoneal treatment with 250 mg/kg body weight)

Experimental groups	Number of evaluated polychromatic erythrocytes	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
Negative control	10,000	556 ± 110	1.3 ± 1.1	1.6 ± 1.6
Test substance 16 hours	10,000	1938* ± 903	1.3 ± 1.2	2.0 ± 1.6
Test substance 24 hours	10,000	1324 ± 294	1.1 ± 1.0	2.2 ± 0.8
Test substance 48 hours	10,000	651 ± 152	0.5 ± 0.8	2.3 ± 1.2
Positive control CP 20 mg/kg	10,000	578 ± 192	2.2 ± 1.7	24.3* ± 10.8

*P < 0.01 in non-parametric Wilcoxon ranking test

Results of Micronucleus Test with C.I. Reactive Blue 181

Negative Control

Sacrifice 24 hours after treatment

Random number and sex	Number evaluated polychromatic erythrocytes	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
20 M	1000	731	0	1
22 M	1000	625	1.6	3
33 M	1000	453	2.2	0
36 M	1000	668	3.0	0
49 M	1000	405	0	3
54 F	1000	634	0	0
70 F	1000	534	1.9	2
75 F	1000	600	1.7	1
79 F	1000	454	2.2	5
95 F	1000	460	0	1
Mean 1s	1000	556 110	1.3 1.1	1.6 1.6

M = Male

F = Female

 

Results of Micronucleus Test with C.I. Reactive Blue 181 (after acute intraperitoneal treatment with 250 mg/kg)

Sacrifice 16 hours after treatment

Random number and sex	Number evaluated polychromatic erythrocytes	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
8 M	1000	2590	0.4	1
15 M	1000	2278	2.6	6
30 M	1000	1290	3.1	3
34 M	1000	4099	0.5	1
42 M	1000	1551	0.6	3
57 F	1000	1538	0.7	2
78 F	1000	860	3.5	1
90 F	1000	1923	0.5	1
97 F	1000	1775	0.6	1
98 F	1000	1472	0.7	1
Mean 1s	1000	1938 903	1.3 1.2	2.0 1.6

M = Male

F = Female

 

Results of Micronucleus Test with C.I. Reactive Blue 181 (after acute intraperitoneal treatment with 250 mg/kg)

Sacrifice 24 hours after treatment

Random number and sex	Number evaluated polychromatic erythrocytes	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
17 M	1000	1363	2.2	2
18 M	1000	1216	0.8	2
26 M	1000	989	1.0	2
32 M	1000	1634	0.6	3
43 M	1000	1515	0	3
51 F	1000	1360	1.5	2
59 F	1000	1518	1.3	1
64 F	1000	1757	0.6	1
71 F	1000	912	0	3
96 F	1000	974	3.1	3
Mean 1s	1000	1324 294	1.1 1.0	2.2 0.8

M = Male

F = Female

 

Results of Micronucleus Test with C.I. Reactive Blue 181 (after acute intraperitoneal treatment with 250 mg/kg)

Sacrifice 48 hours after treatment

Random number and sex	Number evaluated polychromatic erythrocytes	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
1 M	1000	568	0	3
11 M	1000	723	0	2
41 M	1000	836	1.2	2
44 M	1000	845	1.2	5
45 M	1000	845	2.4	1
52 F	1000	494	0	2
55 F	1000	466	0	2
68 F	1000	533	0	2
74 F	1000	661	0	1
100 F	1000	535	0	3
Mean 1s	1000	651 152	0.5 0.8	2.3 1.2

M = Male

F = Female

 

Results of Micronucleus Test with C.I. Reactive Blue 181

Positive Control Cyclophosphamide, 20 mg/kg i.p.

Sacrifice 24 hours after treatment

Random number and sex	Number evaluated polychromatic erythrocytes	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
25 M	1000	525	3.8	24
28 M	1000	924	1.1	34
38 M	1000	804	4.0	43
47 M	1000	327	0	33
50 M	1000	450	4.4	26
62 F	1000	651	1.5	19
63 F	1000	731	0	26
89 F	1000	505	2.0	7
93 F	1000	430	2.3	20
94 F	1000	428	2.3	11
Mean 1s	1000	578 192	2.2 1.7	24.3 10.8

M = Male

F = Female

Historical Controls

 

Negative Controls 1988

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 4027465	Corn	679	0.5	1.8
T 1027552	0.5% C	722	0.9	1.2
T 6027638	Lutrol	896	1.7	1.4
T 6027700	Water	1215	0.7	1.8
T 6029483	0.5% T	981	0.8	1.2
T 6029807	0.5% C	792	0.7	0.8
T 7029808	Lutrol	947	0.9	1.3
T 8029809	Lutrol	1097	0.9	1.2
T 0029982	0.5% C	890	0.5	0.9
T 4029887	Water	635	1.1	1.0
T 6029898	Lutrol	831	1.5	1.7
T 7029899	Lutrol	1262	1.0	1.1
T 7030121	0.5% C	830	1.1	1.2
T 7030338	0.5% C	1053	1.1	0.9
T 2040513	Lutrol	847	0.3	1.2

 

Negative Controls 1988

Sacrifice 48 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 4030281	Lutrol	907	1.0	1.7

 

Negative Controls 1989

Sacrifice 24 hours, intraperitoneal treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 8033840	Lutrol	1266	1.4	1.3
T 1033825	0.5% C	850	1.2	1.5
T 3033061	Lutrol	1179	1.3	1.2
T 0032852	0.5% C	574	1.1	1.6
T 2032719	0.5% C	745	1.0	1.5

 

Negative Controls 1990/I

Sacrifice 24 hours, intraperitoneal treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 6033839	Paraf.	1105	1.3	1.7
T 8033877	Corn	1012	1.4	1.5
T 9033878	Corn	879	0.6	1.2
T 8034515	0.5% C	1117	1.7	1.5
T 4034539	Peanut	1183	1.3	1.4
T 3034619	Corn	886	1.8	2.0
T 7034668	0.5% C	856	1.2	2.0
T 0034986	Water	971	14.0	1.4
T 3037012	Water	996	2.3	1.9
T 3037012	Water	1081	1.2	1.7
T 4037103	Water	852	0.8	1.2

 

Negative Controls 1990/II

Sacrifice 24 hours, intraperitoneal treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 4037112	0.5% C	766	1.5	2.3
T 1037209	Water	995	0.9	2.8
T 3037210	0.5% C	903	2.0	2.6
T 5037285	Corn	1147	1.5	0.6
T 0037316	0.5% C	1010	1.2	1.1
T 3038048	Peanut	1265	1.3	1.4
T 1039504	Corn	929	1.3	1.9
T 7039609	0.5% C	1013	1.7	1.1
T 7039636	Water	690	2.1	2.4
T 7038717	Water	779	0.4	1.0

 

Negative Controls 1991

Sacrifice 24 hours, intraperitoneal treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 2039938	0.5% C	576	0.5	1.4
T 9040573	0.5% C	1046	1.8	2.8
T 2040602	0.5% C	1026	1.4	1.6
T 7040670	0.5% C	821	1.7	1.6
T 4040712	0.5% C	745	1.4	1.9
T 2040738	0.5% C	1222	0.9	2.2
T 7039898	Corn	899	0.6	2.3
T 7040021	Corn	947	1.1	1.4
T 1040106	Corn	843	0.2	1.6
T 8040149	Corn	848	2.0	1.6
T 8040400	Corn	669	2.0	2.5
T 4040532	Corn	763	1.4	1.8
T 0040475	Corn	863	1.1	1.9
T 1040575	Corn	901	1.3	1.4
T 7039852	Paraf.	996	1.5	1.7
T 5040317	Water	789	1.9	2.2
T 8040518	Water	896	1.2	2.0

 

Negative Controls 1991

Sacrifice 48 hours, intraperitoneal treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number and Vehicle		Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
			Normochromatic erythrocytes	Polychromatic erythrocytes
T 0039819	0.5% C	809	2.5	1.3
T 8039899	0.5% C	982	1.5	2.2
T 9040401	0.5% C	635	2.1	1.6
T 1040548	Corn	915	2.0	1.6
T 7040652	Corn	1146	1.1	3.4

 

Explanation of abbreviations:

C = Cremophor in water

T = Tylose in water

Peanut = Peanut oil

Paraf. = Parafin oil

Corn = Corn oil

 

Positive Controls 1988

20 mg/kg Cyclophosphamide per os

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
		Normochromatic erythrocytes	Polychromatic erythrocytes
T 4027465	715	1.1	15.6
T 1027552	855	1.2	14.9
T 6027638	829	0.8	20.7
T 6027700	1162	0.4	17.6
T 6029483	974	0.8	15.8
T 6029807	942	1.1	14.2
T 7029808	857	1.0	17.0
T 8029809	678	1.3	14.7
T 0029982	1285	0.7	14.7
T 4029887	779	1.1	16.5
T 6029898	812	1.4	17.5
T 7029899	929	0.9	18.7
T 7030121	921	0.6	14.3
T 4030281	961	0.5	14.3
T 7030338	1276	1.3	15.1
T 2030513	904	0.5	12.1

 

Positive Controls 1989

20 mg/kg Cyclophosphamide intraperitoneal

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
		Normochromatic erythrocytes	Polychromatic erythrocytes
T 8033840	1102	1.5	18.5
T 1033825	612	0.6	22.9
T 3033061	881	1.3	16.1
T 0032852	826	1.3	19.5
T 2032719	697	1.3	16.3

 

Positive Controls 1990/I

20 mg/kg Cyclophosphamide intraperitoneal

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
		Normochromatic erythrocytes	Polychromatic erythrocytes
T 6033839	959	0.7	21.1
T 8033877	744	0.6	15.7
T 9033878	588	1.0	18.4
T 8034515	1417	1.6	15.0
T 4034539	682	2.7	22.2
T 3034619	683	0.9	18.8
T 7034668	776	1.2	24.8
T 0034986	1227	1.0	21.6
T 3037012	747	1.3	16.8
T 4037103	938	1.8	12.4

 

Positive Controls 1990/II

20 mg/kg Cyclophosphamide intraperitoneal

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
		Normochromatic erythrocytes	Polychromatic erythrocytes
T 4037112	842	1.6	13.4
T 1037209	855	1.7	17.8
T 3037210	924	2.4	14.9
T 5037285	993	2.4	19.2
T 0037316	1154	0.8	22.0
T 3038048	945	1.1	17.6
T 7039609	933	1.3	17.7
T 7039636	610	0.8	17.8
T 7039717	774	0.58	12.5

 

Positive Controls 1991/I

20 mg/kg Cyclophosphamide intraperitoneal

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
		Normochromatic erythrocytes	Polychromatic erythrocytes
T 0039819	893	1.5	18.4
T 7039852	715	1.8	19.9
T 7039898	718	0.3	11.3
T 8039899	1113	1.2	20.3
T 7040021	661	0.9	10.2
T 1040106	748	0.5	12.8
T 8040149	707	0.5	15.1
T 2039938	591	1.4	20.4
T 5040317	762	1.6	17.9
T 9040401	728	1.5	16.9
T 8040400	783	1.4	17.4

 

Positive Controls 1991/II

20 mg/kg Cyclophosphamide intraperitoneal

Sacrifice 24 hours after treatment. Results based on 10,000 polychromatic erythrocytes per study

Study Number	Number of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cell per 1000
		Normochromatic erythrocytes	Polychromatic erythrocytes
T 8040518	831	1.5	19.8
T 4040532	675	1.8	15.7
T 0040475	812	1.3	16.1
T 1040548	963	2.2	18.4
T 9040573	996	2.2	18.4
T 2040602	960	2.0	19.4
T 7040652	908	1.2	17.5
T 1040575	879	1.1	12.7
T 7040670	846	1.2	16.1
T 4040712	693	1.6	12.9
T 2040738	1499	0.8	14.0

 

Applicant's summary and conclusion

Conclusions:

No indications of a clastogenic effect of C.I. Reactive Blue 181 were found after a single intraperitoneal treatment with 250 mg/kg.
The substance is not classifiable according to CLP criteria.

Executive summary:

The micronucleus test was employed to investigate C.I. Reactive Blue 181 in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control.

 

The treated animals received a single intraperitoneal administration of either C.I. Reactive Blue 181 or cyclophosphamide. The femoral marrow of groups treated with C.I. Reactive Blue 181 was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of C.I. Reactive Blue 181 and the positive control, cyclophosphamide, were 250 and 20 mg/kg (body weight, respectively).

 

The animals treated with C.I Reactive Blue 181 showed symptoms of toxicity after administration. However, all animals survived until the end of the test.

 

There was an altered ratio between polychromatic and normochromatic erythrocytes.

 

No indications of a clastogenic effect of C.I. Reactive Blue 181 were found after a single intraperitoneal treatment with 250 mg/kg.

 

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.