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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
Study period:
09 September to 17 October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken to GLP and internationally recognised guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2010

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:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Phenylphosphonic acid, zinc salt
IUPAC Name:
Phenylphosphonic acid, zinc salt
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Phenylphosphonic acid, zinc salt
- Substance type: Inorganic
- Physical state: White powder
- Analytical purity: > 99%
- Purity test date: no data
- Lot/batch No.: 6381
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable
- Storage condition of test material: Because the test substance had hygroscopicity, it was stored at room temperature (in the test substance storage room, desiccator-4, permissible
range: 10-30°C).

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier.
- Age at study initiation: 6 weeks
- Weight at study initiation: 34.0 - 39.0 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: stainless steel wire cage lid with food hopper and water
- Diet: pellets, MF, Lot No. 080318 and 080716, Oriental Yeast Co., Ltd., ad libitum
- Water: water from Hita City Water Supply (chlorine addition water) via plastic bottles, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25°C (21.7-24.2°C actual measurement)
- Humidity: 40-70% in relative humidity (48.1-59.5% actual measurement),
- Air changes: 10-15 times/hour
- Photoperiod: 12 hours/day light cycle (lighting: 07:00-19:00).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5 w/v% methylcellulose (MC) aqueous solution (0.5 w/v% MC solution)
- Justification for choice of solvent/vehicle: One w/v% MC solution was used as vehicle in "Micronucleus assay with Phenylphosphonic acid, zinc salt in mice" (non-GLP, study code: NCI08MU039) performed in the Biological Research Laboratories, NISSAN CHEMICAL INDUSTRIES, LTD. From the result of comparison of suspension condition of 200 mg/mL test substance formulation between I w/v% MC solution and 0.5 w/v% MC solution, homogeneity was judged to be equivalent. Therefore, 0.5 w/v% MC was selected as vehicle that the testing facility has background data. The test substance formulation at 200 mg/mL in 0.5 w/v% MC was no exothermic reaction nor change in color at room temperature within 4 hours after preparation.
- Concentration of test material in vehicle: 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10 mg/kg bw
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): Lot No. 7J92N, Otsuka Pharmaceutical Factory, Inc.
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance formulation was prepared on each administration day
- Mixing appropriate amounts with (Type of food): rat pellets
- Storage temperature of food: storedat room temperature and used within 1 hour after preparation.
Duration of treatment / exposure:
24 h
Frequency of treatment:
Twice with a 24 h interval
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: Negative control (CMC)
Remarks:
Doses / Concentrations:
2 mg/kg bw/day
Basis:
other: Positive control
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C;
- Justification for choice of positive control(s): MMC was described as the positive control substance in OECD guideline and the testing facility has background data.
- Route of administration: Oral gavage
- Doses / concentrations: 2 mg/kg bw/day

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No animals died, both female and male at 2000 mg/kglday in preliminary test and it was judged that there was no sex difference in toxicity of the test substance. Therefore, only male mice were used and the highest dose of test substance was selected at 2000 mg/kglday and 1000 and 500 mg/kglday, three doses in total, were set diluted with a geometric progression of 2 in micronucleus test. The negative control and the positive control were set as the control groups.

TREATMENT AND SAMPLING TIMES: treatments were given at 0 and 24 h. Specimens were prepared 24 hours after the second administration (24 hours after the single administration in the positive control group).

DETAILS OF SLIDE PREPARATION: Animals were euthanized by a cervical dislocation. The femur was removed and the bone marrow cells were collected with approximately 0.8 mL of a heat-inactived fetal bovine serum into a centrifuge tube, and the tube was centrifuged at 1000 rpm (185 xg) for 5 minutes. A small amount of the cell suspension was smeared on a slide glass. The smears were fully air-dried and fixed with methanol. Then, the smears were stained with 3 vol% Giemsa solution (Sorensen buffer, pH 6.8) and treated with 0.004 w/v% citrate aqueous solution. In all animals that were alive at the time of the preparation of specimens, two specimens were prepared per animal.

METHOD OF ANALYSIS: Slide numbers were allocated randomly to all observed specimens. All specimens were observed in a blinded manner using a biological microscope.
(1) Incidence of micronuclei
Two thousand polychromatic erythrocytes (PCE) per animal (1000 PCE per specimen) were observed and the incidence of the PCE with micronuclei (micronucleated polychromatic erythrocyte: MNPCE) per PCE, abbreviated as MNPCE/PCE, was calculated.
(2) Growth inhibition of bone marrow cells
Two hundred total erythrocytes (TE) per animal (100 TE per specimen) were observed and the ratio of PCE per TE (PCE / TE), was also calculated.
Evaluation criteria:
This study was regarded to be valid when the mean of the MNPCE / PCE in the negative and the positive control groups were within range of the background data in the testing facility (negative control group: mean ± 3 S.D., positive control group:mean ± 3 S.D.).
Statistics:
Judgment of MNPCE / PCE
The Conditional Binomial test (Kastenbaum and Bowman) was performed in order to compare the MNPCE / PCE in the negative control group with that in each test substance group and the positive control group. The Conditional Binomial test was conducted at upper-tailed significance levels of 5% and 1%. The total number of MNPCE in each group was used for the Conditional Binomial test. When the significant increase was obtained at the dose of the test substance, the Cochran-Armitage trend test would be expected to conduct to confirm whether there was a dose dependency. However, the trend test was not conducted since the significant increase was not obtained. When the MNPCE / PCE increased significantly compared to the negative control and a dose dependency was exhibited, the result was judged to be positive and the other cases were judged to be negative.

Judgment of PCE / TE
First, all the data except for the positive control group data were tested by Bartlett's test for homoscedasticity of variance in male and female, respectively. Since the variances were homoscedastic, Williams' test was performed. Since there were no significant differences with Williams' test, Dunnett's test was performed to compare the mean in the negative control group with that in each dosage group.
The data from the negative control group and the positive control group were tested by the F test for homogeneity of variance between the groups. Since the result of the F test was homoscedastic, Student's t test was performed to compare the mean in the negative control group with that in the positive control group. Statistical tool, "StatLight", was used for above tests. Significance levels of tests were 5% for Bartlett's test, both side of 5% for Williams' test, 5% for the F test and both side of 5% and 1% for Dunnett's test and Student's t test, respectively.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It was concluded that PPA-Zn had no potential to induce the micronuclei under the present test conditions.
Three genotoxicity studies were undertaken. An ames, in vitro, which gave a negative result and a chromosome aberrations, in vitro, which gave a positive result. However, the third one of these studies was a Mouse Micronucleus study which gave a negative result and being an in vivo study over-rules the two in vitro studies. Therefore this substance will not be classified for genotoxicity.

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