416-730-1

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:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented, meets criteria of corresponding OECD guideline, no information on test substance purity and expiration date,

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

within the test report no guideline is mentioned but the assay meets basic criteria of the OECD guideline 474 "Mammalian Erythrocyte Micronucleus Test"

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 9 weeks
- Weight at study initiation: weight range was 28.2-38.8 and 22.9-31.4 grams for the males and females, respectively
- Assigned to test groups randomly: yes
- Fasting period before study: /
- Housing: five per cage during quarantine, and housed up to five per cage at randomization
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 +/- 6
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: due to solubility and homogeneity
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw, 10ml/kg bw for positive control
- Lot/batch no. (if required): Lot # 4H0080; Prep.06/07/1996; Exp. 12/07/1996; Batch #1

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions for the assay were prepared by making a 250.02 mg/ml stock for the high dose (5000 mg/kg). This was prepared by adding MC to 13.7514 g of the test article up to a volume of 55 ml. The mixture was stirred with a flat-end spatula for ~1 minute and then slowly mixed on a stirplate for ~ 10 minutes. A thick viscous yellow-orange suspension was obtained. Dilutions of this stock were prepared for the 2500 and 1250 mg/kg dose levels. All dosing stocks were placed on magnetic stir plates during the dosing procedure.

DIET PREPARATION
- Rate of preparation of diet (frequency): this is a 1-day assay, information on storage temperature or frequency of preparation is not necessary

Duration of treatment / exposure:

animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow

Frequency of treatment:

once only

Doses / concentrations

Remarks:

Doses / Concentrations:
1250, 2500, and 5000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 /sex/dose/treatment period

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 80 mg/kg bw

Examinations

Tissues and cell types examined:

extraction of bone marrow from femur, analysis of erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
no mortalities or signs of toxicity at dose range finder study, maximum dose for micronucleus assay was estimated to be 5000 mg/kg bw

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction ofthe bone martow

DETAILS OF SLIDE PREPARATION:
The marrow was flushed from the bone and transferred to centrifiige tubes containing 3 - 5 ml bovine serum (one tube for each animal). Following centrifiigation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped using Depex® mounting medium.

METHOD OF ANALYSIS:
The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this Cri:CD-l®(ICR) BR strain is about 0.0 - 0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size ofthe PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

Statistics:

Data are summarized by sex and dose groups for the different time points. Individual animal data are also presented. The analysis of these data was performed using an analysis of variance (Winer, 1971) on either untransformed (when variances are homogeneous) and rank transformed (when variances are heterogeneous) proportions ofcells with micronuclei per animal. If the analysis of variance was significant (p<0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination. The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: In the dose selection study, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1000, 2000, 3000, 4000, and 5000 mg/kg. Six animals (three males and three females) were assigned to each dose group. Animals were observed for three days after
dosing for toxic signs and/or mortality.
- Solubility: test item forms a suspension in 1% CMC
- Clinical signs of toxicity in test animals: no

RESULTS OF DEFINITIVE STUDY
The test article, Cromophtal Yellow HRP, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any ofthe harvest times, except in the females from the 5000 mg/kg dose group in the 24 hour harvest. This is a statistical anomaly rather than a true test article effect and is due to the very low (0.02%) micronucleated PCE's in the vehicle control group); also the value at this time point (0.14%) is well within the historical vehicle control values of 0.0%-0.22%.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes imder the conditions of this assay and is considered negative in the mouse micronucleus assay.