Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-12-15 to 1999-03-15
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cyanure de benzyl
- Physical state: liquid
- Analytical purity: 99.5 %
- Purity test date: 1998-11-25
- Lot/batch No.: CAL 4990/98
- Expiration date of the lot/batch: January 2000
- Storage condition of test material: dry, cool and well ventilated place
No further data

Method

Target gene:
No relevant
Species / strain
Species / strain / cell type:
lymphocytes: primary culture
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Three experiments:
- Sixteen concentrations from 15.65 µg/mL to 1171 µg/mL with spacing factors less than 0.6.
- Twelve concentrations from 49.46 µg/mL to 1171 µg/mL with spacing factors less than 0.6.
- Fourteen concentrations from 297.6 µg/mL to 1171 µg/mL with spacing factors less than 0.6.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No
No further data
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: and cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Experiment 1: 3 hr +/- S-9
Experiment 2: without S-9 29 hr, with 3 hr
Experiment 3: 3 hr with S-9, no test without S-9
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiments 1, 2 and 3: 20 hr +/- S-9

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 160 out 200 initially scheduled

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, 50 % inhibition

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

No further data
Evaluation criteria:
- Proportion of cells with structural aberrations at one or more concentration exceed the noraml rnage in both replicate and
- a stastically significant increase in the proportion of cells with structural aberrations (ecluding gaps) occurs at such doses
Statistics:
Yes Fisher's esxact test

Results and discussion

Test results
Species / strain:
lymphocytes: prmiary culture of three healthy women
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: no impact
- Precipitation: precipitates were osberved from the 494 µg/mL concentration

COMPARISON WITH HISTORICAL CONTROL DATA: chromosome aberrations were within the normal rangte for the negative controls

No further information
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous with metabolic activation at the highest tested concentration

In the test conditions, the authors concluded that phenylacetonitrile induced chromosome aberrations in cultured hulman peripheral blood lymphocytes. The effect was only observed in the presence of phenobarbitone and beta-naphthoflavone induced S-9 at the highest tested concentration (10mM). Thus, since the highest concentration is at the limit of cytotoxic effects and since at this concentration precipitates were observed, results are considered ambiguous.
Executive summary:

The authors investigated the cytogenetic effects of benzyl cyanide (CAS n° 140 -29 -4) on a culture of human primary peripheral blood lymphocites based on the OECD methodology described in the OECD guideline 473. Three experiments were conducted to assess whether benzyl cyanide at concentrations up to 1171 µg/mL dissolved in DMSO would induce significant chromosomal aberrations with or without metabolic activation (S-9 mix). Aberrations were analyzed, scored and compared to historical levels for the DMSO. Postive controls were also included in the experiments as requested in the OECD guideline. For the experiments 2 and 3, the highest dose was selected based on the first experiment which settled the 10 mM value as a maximum value.

In the test conditions, the authors observed no differences in the experiment 1 were observed compared to the negative controls, structurally and numerically speaking.

In the experiment 2 and 3, the highest tested dose induced significant changes in chromosomal aberrations whenever metabolic activation was applied in both replicates. However, it is reported in all experiments that precipitates are observed in cultures treated at more than 494 µg/mL. Provided the elements above, all validation criteria were fulfilled:

  • No evidence of significant heterogeneity between replicate cultures was obtained in the binomial dispersion test
  • The proportion of cells with structural aberrations (excluding gaps) in negative controls cultures fell within the normal range
  • at least 160 cells out of 200 were analysed at each dose level
  • The positive controls induced statistically significant increases in the number of cells with structural aberrations

However even though these criteria are fulfilled it is not clear whether physical effects (i.e: precipitates) did not increase or decrease anyhow the apparent effects observed (e.g: changes in bioavailability of the product by degradation...). Hence the results presented above are considered ambiguous with metabolic activation. Without metabolic activation, benzyl cyanide is not considered to induce significant chromosomal aberrations.

Altogether this study is very well documented and respects the OECD guideline 473. All validity criteria are fulfilled and thus this study is considered as reliable without restrictions, a Klimisch 1.a study.