COAGULANT 122 (SOLID)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-04-25 to 1995-07-18

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

Name of test material (as cited in study report): Coagulant 122 (solid)
Substance type: Clear slightly viscous yelow liquid
Physical state: 80.4% solid
Bathc number: 9435262-848
Storate condition of test material: 4°C in the dark. Handled and store under an inert atmosfere.

Test animals

Species:

other: Speific Pathogen Free CD-1 outbred mice

Strain:

Swiss

Sex:

male/female

Administration / exposure

Route of administration:

other: The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, orally by intragastric gavage.

Vehicle:

Purified water

Duration of treatment / exposure:

24 hours with positive control after dosing
24 or 48 hours with negative control and test substance groups

Post exposure period:

At the end of the observation period surviving animals were killed and discarded.

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight.

No. of animals per sex per dose:

Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours Male: 400 mg/kg; No. of animals: 5; Sacrifice time: 24 hours Male: 800 mg/kg; No. of animals: 5; Sacrifice time: 24 hours Male: 1600 mg/kg; No. of animals: 5; Sacrifice time: 24 hours Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours Male: 400 mg/kg; No. of animals: 5; Sacrifice time: 48 hours Male: 800 mg/kg; No. of animals: 5; Sacrifice time: 48 hours Male: 1600 mg/kg; No. of animals: 5; Sacrifice time: 48 hours Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours Female: 400 mg/kg; No. of animals: 5; Sacrifice times: 24 hours Female: 800 mg/kg; No. of animals: 5; Sacrifice times: 24 hours Female: 1600 mg/kg; No. of animals: 5; Sacrifice times: 24 hours Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours Female: 400 mg/kg; No. of animals: 5; Sacrifice times: 48 hours Female: 800 mg/kg; No. of animals: 5; Sacrifice times: 48 hours Female: 1600 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes

Examinations

Tissues and cell types examined:

Femur bone marrow: proximal epiphysis

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Doses producing toxicity:
In the preliminary test there were 2/4 mortalities at 2000
mg/kg. In addition, in the preliminary and main tests
pilo-erection, ptosis and lethargy were observed at 1600
mg/kg. In the main test there was no significant change in
the ratio of polychromatic to normochromatic erythrocytes.

Observations:
There were no statistically significant increases in the
number of micronucleated erythrocytes at any sampling time.

Any other information on results incl. tables

Preliminary toxicity test:

The detail of any toxic reactions observed are given in Appendix 2a (phase I) and Appendix 2b (phase II) (see attached document).

From the results obtained, the maximum tolerated dose was estimated to be approximately 1600 mg/kg. Dose levels of 400, 800, and 1600 mg/kg were therefore chosen for use in the micronucleus test.

Micronucleus test:

The results of the micronucleus test on Coagulant 122 (solid) at the 24 and 48 hour sampling times are presented in Tables 2 and 3 respectively. Table 1 (in the attached document) gives a summary of the results and the results of statistical analysis. Appendix 4 (in the attached document) summaries the vehicle control micronucleated polychromatic erythrocyte counts obtained in previous, unrelated experiments.

Clinical signs and mortalities

No mortalities were obtained in the micronucleus test.

Clinical signs for animals treated with Coagulant 122 (solid) are detailed in Appendix 3 (in the attached document). No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.

Micronucleated polychromatic erythrocyte counts (mnp)

Coagulant 122 (solid) did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at either sampling [P<0.001 using Wilcoxon's sum of ranks test (Hollander and Wolfe 1973, Langley 1979].

Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.

Micronuclead normochromatic erythrocytes. (mnn)

Coagulant 122 (solid) did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at either sampling time.

Ratio of polychromatic to normochromatic erythrocytes (p/n)

Coagulant 122 (solid) failed to cause any signficant decreases in the ratio of polychromatic to normochromatic erythrocytes [P>0.001 using Wilconox´s sum of ranks test (Hollanders and Wolfe 1973, Langley 1979].

Mitomycin C did not cause any statistically signficant decreases in the ration [P>0.01]- it should be noted that even very cytotoxic compounds such as mitomycin C do not always produce a substantial decrease in this ratio as early as the 24 hour sampling time because of the lag caused by erythrocyte maturation (see EVALUATION CRITERIA).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Since the test substance did not cause any substantial increase in the incidence of micronucleated polychromatic erythrocytes or any substantial decrease in the p/n ratio, it is concluded that Coagulant 122 (solid) did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administrered by intraperitoneal injection in this in vivo test procedure.

Executive summary:

In vivo mammalian cell gene mutation test :

This study was designed to assess the potential induction of micro nuclei by Coagulant 122 (solid) in bone marrow cells of mice. Mice were treated with a single acute intraperitoneal administration of the test substance by injection at a dosage of 400, 800, 1600 mg/Kg. A preliminary toxicity test had previously shown 1600 mg/kg to be approximately the maximum tolerated dosage.

The test substance and negative control were administrated by intraperitoneal injection. The negative control group received the vehicle, purified water. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Bone marow smears were obtained from five male and five female animals in the negative control and test substance groups at 2 sampling times; these being 24 or 48 hours after dosing. Bone marrow smears were obtained from the positive control group 24hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

Mice treated with Coagulant 122 (solid) did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes at either sampling time.

There was no significant decrease in the ratio of polychromatic to normochoromatic erythrocytes after treatment of the animals with Coagulant 122 (solid).

The positive control compound, mitomycin C, produced large, highly significant increase in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded that Coagulant 122 (solid) has not shown any evidence of causing chromosome damage in this in vivo test.