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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 1994 to 05 May 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU & OECD test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue FC 75311

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winklemann GmbH
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 29 - 46g
- Assigned to test groups randomly: yes, under following basis: randomization plan, produced by the Institute of Biometrics, BAYER AG
- Fasting period before study: No data
- Housing: Makrolon Type 1 cages, females 3 to a cage, males 1 to a cage
- Diet (e.g. ad libitum): Altromin 1324, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Humidity (%): 40 - 70
- Air changes (per hr): Ten times
- Photoperiod (hrs dark / hrs light): 500 lux, twelve hours of electrical lighting daily (06:00 to 18:00)

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: historical usage within the laboratory.

Details on exposure:
The test substance was dissolved in physiological saline solution and injected intraperitoneally.

The control substance, cyclophosphamide, was dissolved in deionized water and administered in the same way. The negative control received physiological saline solution by the same method. Negative and positive controls were performed in a separate control study (T 3055643), which was performed parallel to the treatment with Reactive Blue FC 75311 (T 5055645).

In all of the groups, the administered volume was 10 ml/kg body weight.

The selection of the Reactive Blue FC 75311 dose was based on a pilot test, in which groups of five animals, including both(males and females, were intraperitoneally administered 250 mg/kg, 300 mg/kg, 500 mg/kg and 1000 mg/kg Reactive Blue FC 75311. The following symptoms were recorded for up to 96 hours, starting at 250 mg/kg: apathy, roughened; fur, blue discoloration of hairless parts of skin, spasm, difficulty in breathing, eyelids stuck together and blue discoloration of urine. In addition, 1, 3 and 5 of 5 animals died in the 250, 500 and 1000 mg/kg groups.
Based on these results, 300 mg/kg Reactive Blue FC 75311 was chosen as MTD for this test.
Duration of treatment / exposure:
16, 24 and 48 hours
Frequency of treatment:
Once
Post exposure period:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
300 mg/kg body weight
Basis:
other: pilot test
No. of animals per sex per dose:
Negative control 24 hours exposure - 10 animals, 5 males & 5 females
Male: 300 mg/kg; No. of animals: 5; Sacrifice time: 16 hours
Male: 300 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 300 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 300 mg/kg; No. of animals: 5; Sacrifice times: 16 hours
Female: 300 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 300 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Positive control 24 hours exposure - 10 animals, 5 males & 5 females
Control animals:
yes
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):proven cytostatic agent and known clastogen with bifunctional alkylation action
- Route of administration: Intraperitoneally
- Doses / concentrations: 20 mg/kg body weight

Examinations

Tissues and cell types examined:
chromosomes of bone-marrow erythroblasts
Details of tissue and slide preparation:
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 labeled 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 Hema-TeK 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.



Evaluation criteria:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. These were distinguished from artifacts by varying the focus.
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 poly¬chromatic 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.
Statistics:
The Reactive Blue FC 75311 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 groupfigure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micro-nuclei 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 chii-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.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
After single intraperitoneal administration of 300 mg/kg Reactive Blue FC 75311, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, blue discoloration of hairless parts of skin, spasm, shivering, difficulty in breathing, red tears, diarrhoea and blue discolored urine. Their feeding behavior was normal. One of 40 treated animals died during the test period, due to the acute toxicity of 300 mg/kg Reactive Blue FC 75311. No symptoms were recorded for the control groups. No animals died in these groups.

Under microscopic examination, the ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with Reactive Blue FC 75311 for the 16 and 24 hours groups. However an altered ratio was observed for the 48 hours group. These ratios were 1000: 709 (ls=192) in the negative control, 1000: 726 (ls=183) in the 16 hours group', 1000: 682 (ls=182) in the 24 hours group and 1000: lS"45 (ls=555) in the 48 hours group. A relevant variation was thus noted for the 48 hours group.

No biologically important or statistically significant variations existed between the negative control and the groups treated intra-peritoneally with 300 mg/kg Reactive Blue FC 75311, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells> was 1.5/1000 (ls=1.4) in the negative control, and 1.9/1000 (ls=1.4), 0.9/1000 (ls=1.0) and 1.5/1000 (ls=1.3) in the Reactive Blue FC 7 5311 groups.

Similarly, there could be no biologically significant variation between the negative control and Reactive Blue FC 75311 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 16.3/1000 (ls=7.8), which represents a biologically relevant increase in comparison to the negative control.
There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocy¬tes in the positive control since, in conjunction with the cell-cycle duration, cnormochromatic erythrocytes origi¬nated 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: 729 (ls=212J, as against 1000: 709 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 micro-nuclei

Any other information on results incl. tables

Summary of Results of Micronucleus Test With Reactive Blue FC 75311 (after acute intraperitoneal treatment

 

 

 

Micronucleated cells per 1000

experimental groups

number of evaluated polychro­matic erythro­cytes

number of normochromatic erythrocytes per 1000 polychroma­tic ery­throcytes

normo-

chromatic

erythrocytes

polychromatic

erythrocytes

negative control

10,000

709 ± 192

1.0 ±1.l

1.5 ± 1.4

test sub­stance 16 hours

10,000

726 ± 183

1.0 ± 1.2

1.9 ± 1.4

test sub­stance 24 hours

10,000

682 ± 182

1.3 ± 1.8

0.9 ± 1.0

test sub­stance 48 hours

10,000

1345* + 555

1.6 ± 1.1

1.5 ± 1.3

positive control

CP-20 mg/kg

10,000

729 ± 212

1.5 ± 1.4

16.3* ± 7.8

*P < 0.01 in non-parametric wilcoxon ranking test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The micronucleus test was employed to investigate Reactive Blue FC 75311 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 Reactive Blue FC 75311 or cyclophosphamide. The femoral marrow of groups treated with Reactive Blue FC 75311 was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of Reactive Blue FC 75311 and the positive control, cyclophosphamide, were 300 and 20 mg/kg body weight, respectively.
The animals treated with Reactive Blue FC 75311 showed symptoms of toxicity after administration. One of forty animals died before the end of 'the test due to the acute intraperitoneal toxicity of 300 mg/kg Reactive Blue FC 75311.
There was an altered ratio between polychromatic and nor-mochromatic erythrocytes concerning the 48 hours sampling time. No indications of a clastogenic effect of Reactive Blue FC 75311 were found after a single intraperitoneal treatment with 300 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.
In conclusion, there was no indication of a clastogenic effect of an intraperitoneal dose of 300 mg/kg Reactive Blue FC 75311 in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.
Executive summary:

The substances was assessed by intraperitoneal exposure to mice and the bone marrow harvested to determine the clastogenic potential of the substance. The study was conducted using both negative and positive controls, all of which showed expected results. The test substance gave no increase in the ration of polychromatic to normochromatic erythrocytes and is therefore considered to be non-clastogenic.