myo-inositol-hexakisphosphate 3-phosphohydrolase

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 1999 - 23 November 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

23 July 1999

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Physical appearance: brown liquid
Storage conditions:in a freezer in the dark or, after thawing, in a refrigerator in the dark

Specific details on test material used for the study:

- A correction factor of 5.2 was applied to correct for the purity of the substance.

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 hours after blood collection, lymphocyte cultures were started.
- Sex, age and number of blood donors: healthy adult males
* dose-range finding study: age 39, Average Generation Time (AGT) = 14.2 h
* first cytogenetic assay: age 20, AGT = 13.4 h
* second cytogenetic assay: age 33, AGT = 13.9 h

MEDIA USED
- Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2mM), penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

Dose-range finding test: 100, 333, 1000, 3330 and 5000 µg/mL
Experiment 1: 1000, 3330 and 5000 µg/mL
Experiment 2: 1000, 3330 and 5000 µg/mL

Vehicle / solvent:

- Solvent: Milli-Q water
- Justification for choice of solvent: according to OECD guideline 473.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24 h and 48 h (without S9) and 3 h (with S9)
- Fixation time experiment 1: 24 h
- Fixation time experiment 2: 24 h (24 hour exposure period) and 48 h (3 and 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity (set to maintain): 80-100%
- Temperature (set to maintain): 37.0°C

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL)

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in water and allowed to dry.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P <0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
None of the test concentrations induced a statistically significant (Chi-square test, P<0.05) increase in the number of cells with chromosome aberrations.

ACCEPTABILITY CRITERIA:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range (min=0, max=5 (mean=0.8, std dev.=1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=0, max=5 (mean=0.8, std dev=0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded).
b) The positive control substances should produce a statistically significant (Chi-square test, P<0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
- pH: 6.81 (compared to 7.24 in the solvent control)
- Osmolarity: 291 mOsm/kg (compared to 285 in the solvent control)

EXPERIMENT 1:
- Without S9: a significant increase in the number of cells with chromosome aberrations at 1000 µg/mL was observed. Since higher concentrations did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations, the number of cells with chromosome aberrations was just outside the historical solvent control data range and the background level of the solvent control was zero, the increase is not considered biologically relevant.
- With S9: no statistically or biologically significant increase in the number of cells with chromosome aberrations was observed.

EXPERIMENT 2:
- Without S9: at the 48 hour treatment a statistically significant increase in the number of cells with chromosome aberrations at 3300 µg/mL was observed (gaps excluded). Since the highest tested concentration did not induce a statistically significant increase in the number of cells with chromosome aberrations, the number of cells with chromosome aberrations did not exceed 5% of the number of cells scored and moreover the number of cells with chromosome aberrations is still within the historical solvent control data range, the increase is considered not biologically relevant. Furthermore, 100 cells of the cultures treated with 3300 µg/mL were additionally scored for chromosome aberrations to verify that this increase was irrelevant.
In the 24 hour exposure, no statistically significant increase in the number of cells with chromosme aberrations was observed.
- With S9: no statistically significant increase in the number of cells with chromosme aberrations was observed.

ACCEPTABILITY:
- Results found in the solvent control cultures were within the laboratory historical control data range.
- The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.

Applicant's summary and conclusion

Conclusions:

Based on the results of a chromosome aberration test, performed according to OECD guideline 473 and GLP principles, NPH 54 is not clastogenic in human lymphocytes.