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EC number: 277-179-9 | CAS number: 72987-16-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 1 993
- Report date:
- 1993
Materials and methods
Test guidelineopen allclose all
- 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
- 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
- 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
Constituent 1
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.
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