[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl]trimethylammonium chloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Study period:

1992-03-17 to 1993-01-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Basic Red 018 Chloride - Similar Substance 02

IUPAC Name:

Basic Red 018 Chloride - Similar Substance 02

Test material form:

other: solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 12 weeks of age
- Weight at study initiation: 28 - 43 g
- Housing: females: in groups of a maximum of three mice; males: individually in Macrolon type I cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in physiological saline solution, stirred with a magnetic mixer during administration and injected intraperitoneally.

Duration of treatment / exposure:

test item dose groups: 16, 24 and 48 hours,
negative/positive control: 24 hours

Frequency of treatment:

once

Post exposure period:

no

Doses / concentrations

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

three test item related experimental dose groups: 16, 24 and 48 hours

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: cyclophosphamide
- Route of administration: intraperitoneal
- Dosis: 20 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow derived erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. For the results please box "Additional information on results".

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
see Table 1 in box "Any other information on materials and methods incl. tables".

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were automatically stained with an Ames HemaTek Slide Stainer and then destained with methanol, rinsed with deionized water and dried. After drying, the slides were covered with xylene and a cover glass.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. Moreover, the ratio of polychromatic to normochromatic erythrocytes was determined (number of normochromatic erythrocytes per 1000 polychromatic ones). Additionally, the number of normochromatic erythrocytes showing micronuclei was also established.

Evaluation 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. 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.

Statistics:

The test item group(s) with the highest 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

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. The following symptoms were recorded for up to 48 hours, starting at 10 mg/kg bw: apathy, roughened fur, red discoloration of hairless parts of skin, staggering gait, spasm, leaping coloured urine. In addition, 3 of 5 animals died in the 75 mg/kg group and all animals died in the 100 mg/kg bw group. Based on the results, 50 mg/kg bw was considered as 1 MTD for the main test.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. No symptoms were recorded for the control groups.
- Mortality: One of forty treated animals died during the test period due to acute toxicity. No animals died in the control group.
- Induction of micronuclei (for Micronucleus assay): no clastogenic effect, see Table 2
- Ratio of PCE/NCE (for Micronucleus assay): slightly changed, see Table 2
- Statistical evaluation: see Table 2

Any other information on results incl. tables

Table 2: Summary of results of micronucleus test with the test item
experimental groups		Number of evaluated poly-chromatic erythrocytes (PCE)		Number of normo-chromatic erythrocytes per 1000 PCE		micronucleated cells per 1000
						normo-chromatic erythrocytes		poly-chromatic erythrocytes
Negative control		10000		811 +/- 208		1.2 +/- 1.6		1.5 +/- 1.1
Test item_16 hours		10000		1321* +/- 319		1.3 +/- 1.0		1.6 +/- 1.0
Test item_24 hours		10000		1088 +/- 549		1.3 +/- 1.6		1.5 +/- 1.0
Test item_48 hours		10000		775 +/- 199		0.4 +/- 0.7		1.3 +/- 1.2
Positive control		10000		557 +/- 231		0.4 +/- 1.3		12.6* +/- 6.9

Concentration: test item: 50 mg/kg bw; positive control: 20 mg/kg bw

* p< 0.01 (tested by non-parametric Wilcoxon ranking test)

Applicant's summary and conclusion

Conclusions:

Non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

Method

In a NMRI mouse bone marrow micronucleus test conducted in accordance with OECD Guideline 474, 5 mice/sex/treatment group were treated intraperitoneally once with the test item (95.6% purity) at doses of 0 and 50 mg/kg bw. The vehicle used was physiological saline. The animals were sacrificed and bone marrow cells were harvested after 16 hours (test item), 24 hours (negative/positive control and test item) and after 48 hours (test item).

After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. The test item was tested at an adequate dose based on a preliminary dose range finding test. The positive control induced the appropriate response.

Results

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (16, 24 and 48 hours).