Registration Dossier

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:
1991-01-28 to 1991-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 26, 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Code of Federal Regulations, Titel 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay"
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): RV 777
- Substance type: Reaction mass of dialuminium tricalcium hexaoxide (CAS No. 12042-78-3, EC No. 234-932-6) and calcium hydrogen phosphonate (CAS No. 21056-98-4, EC No. 244-182-1)
- Analytical purity: 90 %
- Lot/batch No.: CH. 659 B
- Physical state: white solid
- Expiration date of the lot/batch: January 1995
- Stability in vehicle: stable for at least 48 hours in H2O, polyethylenglycol, carboxymethylcellulose
- Storage condition of test material: moisture protected, room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf CH-4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks at start of acclimatization
- Weight at study initiation: appr. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: Appr. 18 hours before treatment the animals received no food but water ad libitum.
- Housing: single housing in Makrolon Type I cages with wire mesh top
- Diet: ad libitum, pelleted standard diet Altromin 1324, D-4937 Lage/Lippe
- Water: ad libitum, tap water
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative nontoxicity for the animals
- Concentration of test material in vehicle: 250 mg/ml
- Amount of vehicle (if gavage or dermal): dose volume was 20 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in carboxymethlycellulose (CMS, 1%). At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted. All animals received a single standard dose volume of 20 ml/kg body weight orally.
Duration of treatment / exposure:
single application
Post exposure period:
24, 48 and 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
5000
Basis:
actual ingested
No. of animals per sex per dose:
12 (6 males, 6 females)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral: gavage
- Doses / concentrations: 30 mg/kg body weight dissolved in physiological saline, dose volume: 10 ml/kg body weight

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In three subsequent pre-experiments 4 animals per dose received orally 2000, 3000, 4000, or 5000 mg RV 777/kg body weight. No animal died within 72 hours p.a.. The treated animals expressed toxic reactions (reduced spontaneous activity, eylide closure, apathy). Higher dosing was not attainable: appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml; application volumes higher than 20 ml/kg body weight were not justifiable for the rodents used.

The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major efects on survival within 72 hours.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48, and 72 hours post-treatment with a single dose animals were sacrificed by cervical dislocation.

DETAILS OF SLIDE PREPARATION: After sacrifice, the femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.


METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100 x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.


OTHER: Five animals per sex and group were evaluated. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.

A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biologically ans statistical significance should be considered together.
Statistics:
Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical symptoms
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 3000, 4000 and 5000 mg/kg body weight
- Solubility: Appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml; application volumes higher than 20 ml/kg body weight were not justifiable for the rodents used.
- Clinical signs of toxicity in test animals: in all dose groups reduced spontaneous activity, eylide closure, apathy in all dose groups
- Evidence of cytotoxicity in tissue analyzed: not analysed
- Rationale for dose selection: maximum attainable dose


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article RV 777. The mean values of micronuclei observed after treatment with RV 777 were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: The ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.
- Appropriateness of dose levels and route: maximum attainable dose tested
- Statistical evaluation: no significant increase of micronucleus frequency
- Positive control: Cyclophosphamide (30 mg/kg body weight administered per os) was used as positive control which showed a distinct increase of micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article RV 777 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, RV 777 is considered to be non-mutagenic in this micronucleus assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It can be stated that during the study described and under the experimental conditions reported, the test article RV 777 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, RV 777 is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD Guideline 474, adopted May 26, 1983, EU method B.12, EEC Directive 84/449 and EPA, Code of Federal Regulations, Titel 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, groups of 6 mice per sex and bone marrow harvest time were given a single oral dose of RV 777 (90% a.i.) in carboxymethylcellulose at a dose of 5000 mg/kg body weight. Bone marrow cells were harvested at 24h, 48 h, and 72 h after application.

RV 777 was tested at an adequate dose. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions (reduced spontaneous sctivity, eylide closure, apathy). The ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.The positive controls did induce the appropriate response.  There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474, EU Method B.12 and the EPA Guideline for in vivo mammalian bone marow cytogenetics tests (micronucleus assay).