Reaction mass of heptachromium tricarbide and trichromium dicarbide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

3 September 1991 to 23 October 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across to chromium(III) oxide. The release of chromium from chromium carbide is very similar to the release from chromium metal and chromium(III) oxide and therefore the results obtained with these substances can readily be used in the assessment of trichromium dicarbide. OECD guideline study (except for one deviation). The reliablity score of 1 is given although read-across is applied. This is motivated due to the similarity in the release of chromium from chromium(II) oxide and chromium carbide.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(no analytical investigations on the stability of the compound of the vehicle were performed)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Version / remarks:

PB 84-233295 HG-Chromo-Micronuc

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Chromium(III) oxide

IUPAC Name:

Chromium(III) oxide

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Strain Bor:NMRI (SPF Han), supplied by F. Winkelmann, Borchen. 30 female and 30 male mice in total.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 29-42 g
- Assigned to test groups randomly: yes (by a randomization pland produced by the Institute of Biometrics, BAYER AG, Wuppertal)
- Fasting period before study: no data
- Housing: Females were kept in groups of maximum three mice, and males were kept singly in Makrolon type I cages. Bedding of soft wood granules, type S 8/15 (J. Rettenmaier & Söhne, Füllstoff-Fabriken, Ellwangen-Holzmühle, Germany) was used. The animals were identified by cage and picric-acid marks.
- Diet (e.g. ad libitum): Altromin 1324 Standard Diet, produced according to the specification by Alromin GmbH (ad libitum)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23 degrees Celsius
- Humidity (%): 47-54% mean relative humidity
- Air changes (per hr): about ten times per hours
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: Sacrifice 16, 24 and 48 hours after the administration. Negative and positive controls were sacrificed after 24 hours.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 500 mg/ml
Due to the chemical characteristics of the compound and according to an order of the sponsor a stability test in the vehicle was not performed.

Details on exposure:

The test substance was suspended in corn oil and injected intraperitoneally. The dose was 10000mg/kg body weight, injection volume 20 ml/kg bodyweight (= 500 mg/ml). The dose was selected based on a pilot test, in which groups of five animals were given the test substance i.p. at concentrations of 5000 mg/kg and 10000 mg/kg bodyweight. None of the animals died during the follow up period of 48 h.

Duration of treatment / exposure:

Single injection.

Frequency of treatment:

One single injection.

Post exposure period:

The test animals were divided into three groups, which were observed for 16, 24 or 48 hours, before sacrificed. Positive and negative control animals were sacrificed 24 hours after the injection.

Doses / concentrations

Remarks:

Doses / Concentrations:
10 g/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females treated with test substance per group for each sacrifice time (16, 24 or 48 h after injection), total number 15 males and 15 females. Negative controls: 5 males and 5 females. Positive controls: 5 males and 5 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes. 5 male and 5 female mice injected i.p. with 20 mg/kg cyclophosphamide (dissolved in deionized water) in a volume of 10 ml/kg.

Examinations

Tissues and cell types examined:

Erythrocytes (smears produced from at least one intact femur from each sacrificed animal). Normally, 1000 polychromatic erythrocytes were counted per animal. The number of normochromatic erythrocytes per 1000 ploychromatic ones were noted. Coated slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appeared as stained chromatin particles in the anucleated erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose was 10000mg/kg body weight, injection volume 20 ml/kg bodyweight (= 500 mg/ml). The dose was selected based on a pilot test, in which groups of five animals were given the test substance i.p. at concentrations of 5000 mg/kg and 10000 mg/kg bodyweight. None of the animals died during the follow up period of 48 h.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The test substance was suspended in corn oil and injected intraperitoneally. The test animals were divided into three groups, which were observed for 16, 24 or 48 hours, before sacrificed. Positive and negative controls were sacrificed 24 hours after the injection. The femoral marrow was prepared immediately after sacrifice.


DETAILS OF SLIDE PREPARATION: Smears were produced according to Schmid's method (Schmid, W. Mutation Res 31, 9-15, 1975). At least one intact femur was prepared from each scrificed animal. The femur was separated from muscular tissue, and the lower-leg stump was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened, making visible a small opening in the bone-marrow channel. The femur was the completely immersed with in calf serum (by injection), the contents were flushed several times and the bone marrow was passed into the serum as a fine suspension. The supernatant was removed after centrifugation, and the sediment was mixed to produce a homogenous suspension. One drop of the suspension was spread on a slide, and dried overnight. The smears were stained automatically with an Ames Hema-Tek Slide Stainer (Miles Company), after which the slides were "destained" with methanol, rinsed with water and left to dry. Next the slides were transferred to a holder, immersed into acuvette filled with xylene (10 minutes) and removed. A small amount of covering agent was then applied to the coated side of the slide and covered with a glass. The slides were evaluated after the covering agent had dried.


METHOD OF ANALYSIS: The coated slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes, and can be distinguished from the artifacts by varying the focus. Normally 1000 polychromatic erythrocytes were calculated per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The establishment of the polychromatic:normochromatic ratio makes it possible to identify individual animals with pathological bone-marrow dpressions, which can then be excluded from the evaluation. An alteration of the polychromatic:normochromatic ratio also provides evidence that the test substance actually has reached the target.


OTHER:

Evaluation criteria:

If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, without similar effects among other animals in the same group, then the case was regarded as pathological and unrelated to the treatment, and the animal was be omitted from the evaluation. A treatment-related alteration of the ratio may be concluded if it is clearly lower for the majority of the animals in the treated group than in the control group.
The data on normochromatic erythrocytes showing micronuclei permits the detection of individuals already subject to damage before the start of the test. Combined with the data on numbers of micronucleated polychromatic erythrocytes it gives an indication 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 clastogenic effects, due to the fact that normochromatic erythrocytes originate from polychromatic ones.
A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative controls.
A test was regarded negative if no relevant or significant increase in the rate of polychromatic erythrocytes with micronuclei was observed at any time. Tests showing significant increases in that rate, which however were within the range of negative controls (accoring to the laboratory's experience), were also considered as negative.
An unsignificant increase of micronucleated polychromatic erythrocytes above the range of historical negative controls was considered equivocal. In this case, a second test had to be performed at the most sensitive interval.

Statistics:

The treatment group with the highest mean and the positive control were tested 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 <5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes with micronuclei was examined if the rate of polychromatic erythrocytes with micronuclei was already markedly increased. The one-sided chi2-test was utilized for the comparision of the group with the highest mean with the negative controls. The criteraia for statistically significant variations were p<0.05 and a treatment group figure higher than that of the negative control.
Standard deviations were calculated for all means.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

(apathy, stretching of body, roughened fur, staggering gait, spasm and difficulty in breathing)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg and 10000 mg/kg
- Solubility: no data
- Clinical signs of toxicity in test animals: apathy, stretching of body, roughened fur, staggering gait, spasm
- Evidence of cytotoxicity in tissue analyzed: no signs



RESULTS OF DEFINITIVE STUDY
- Treated animals showed the following toxicity symptoms until sacrifice: apathy, stretching of body, roughened fur, staggering gait, spasm and difficulty in breathing. The feeding behaviour was normal and there were no substance-induced mortlities. No symptoms were observed among the control animals.
- Induction of micronuclei (for Micronucleus assay): No significant differences in incidence of micronucleated polychromatic or normochromatic erythrocytes were observed between treatment groups and negative controls
- Ratio of PCE/NCE (for Micronucleus assay): A clear decrease in the decrease in the ratio of polychromatic to normochromatic erythrocytes was observed after treatment with the test substance. However, this ratio is not indicating any clastogenic effects, but suggests a toxic effect in the bone marrow, which may hamper the detection of micronuclei
- Appropriateness of dose levels and route: The positive control (cyclophosphamide) had a clear clastogenic effect, shown by the increased number of micronucleated polychromatic erythrocytes. The polychromatic:normochromatic erythrocyte ratio was not changed in the positive control group.
- Statistical evaluation: The test gropu with the highest mean (provided this superceded the negative control mean) and the positive control group were checked by Wilcoxon's non-parametric rank sum test with the respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. p<0.05 was considered as significant 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 increased. The group with the highest mean was then compared with the negative control using the one-sided chi2-test. p<0.045 was considered as signifant if the treatment group figure was higher than that of the negative control.

Any other information on results incl. tables

No signs of induction of micronuclei were observed in bone marrow polychromatic erythrocytes. A clear decrease in the ratio of polychromatic to normochromatic erythrocytes was observed, which suggests a toxic effect in the bone marrow, and this may hamper the detection of micronuclei.

CHROMIUM(III) OXIDE MICRONUCLEUS TEST ON THE MOUSE
Table 1
Toleration by the animals (exposure to 10000 mg/kg chromium(III) oxide i.p.)
Observed symtoms
Apathy
Stretching of body
Roughened fur
Staggering gait
Spasm and difficulty in breathing
Feeding behaviour normal
No deaths

CHROMIUM(III) OXIDE MICRONUCLEUS TEST ON THE MOUSE
Table 2
Toleration by the animals (exposure to 10000 mg/kg chromium(III) oxide i.p.)
Animal	No of evaluated polychromatic ethrocytes	No of normochromatic erythrocytes per 1000 polychromatic erythrocytes	Micronucleated cells per 1000 normochromatic erythrocytes	Micronucleated cells per 1000 polychromatic erythrocytes
neg control	10000	763 ± 326	1.4 ± 2.0	1.8 ± 2.0
test substance 16 h	10000	2082 ± 843 *	1.0 ± 0.9	1.1 ± 1.0
test substance 24 h	10000	1537 ± 427	1.3 ± 1.1	1.9 ± 1.0
test substance 48 h	10000	1497 ± 287	1.3 ± 0.9	0.6 ± 0.7
positive control (CP 20 mg/kg)	10000	675 ± 214	1.8 ± 2.0	15.7 ± 241 *
	* p<0.01 non-parametric Wilcoxon ranking test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
There were no indications of clastogenic effects after one intraperitoneal dose of 10000mg/kg of the test substance in mice, as detected by number of micronucleated polychromatic erythrocytes.

Executive summary:

A standard micronucleus test was carried out in mice exposed to chromium(III) oxide. Male and female NMRI mice were given a single high dose (10 g/kg body weight) of chromium(III) oxide intraperitoneally, and bone marrow samples were collected 16, 24 and 48 h after administration of the test substance. No signs of induction of micronuclei in bone marrow polychromatic erythrocytes were observed at any of the time points. However, the high dose caused some toxic symptoms. A clear decrease in the ratio of polychromatic to normochromatic erythrocytes was observed. The ratio was decreased by 63%, 50% and 51% at the 16 h, 24 h and 48 h sampling times, respectively. Such a decrease indicates a cytotoxic effect in the bone marrow, and this may hamper the detection of micronuclei.