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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-04 to 2012-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: About 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital/β-naphthoflavone treated rats
Test concentrations with justification for top dose:
Experiment I / II without S9-mix: 2.1, 4.2, 8.4, 16.8, 33.5, 67.0, 134.0, 268.0, 536.0, 1072.0 μg/mL
Experiment I with S9-mix: 2.1, 4.2, 8.4, 16.8, 33.5, 67.0, 134.0, 268.0, 536.0, 1072.0 μg/mL
Experiment II with S9-mix: 67.0, 134.0, 268.0, 536.0, 1072.0 μg/mL

In Experiment I in the absence and presence of S9 mix the pH was adjusted to physiological values at the highest applied concentration (1072.0 μg/mL) using small amounts of 2N HCl.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: Soluble in water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Dissolved in deionised water; 0.3 μg/mL; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: griseofulvin
Remarks:
Dissolved in DMSO; 8.0 μg/mL; continuous treatment; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Dissolved in saline; 15 μg/mL; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: Exp.I/II 4 hrs (with S9 mix); Exp.I 4hrs; Exp. II 24 hrs (wihout S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs


STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (proliferation index (PI) was calculated)



Evaluation criteria:
Evaluation of Results
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data and
- either a statistically significant concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and
- no statistically significant concentration-related increase in the number of micronucleated cells is observed.

Statistics:
Statistical significance was confirmed by means of the Chi square test.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix no mutagenicity was observed up to the highest required concentration.
In Experiment II in the absence of S9 mix statistically significant increases (1.25, 2.28, 7.75 and 3.63 % micronucleated cells) were observed at all evaluated concentrations (134.0 - 1072.0 μg/mL). Since the three highest values clearly exceeded the historical control data range of 0.05 - 1.50 % micronucleated cells, Benzylamine is considered mutagenic.


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH value was slightly increased at the highest applied concentration
- Effects of osmolarity: No
- Precipitation: No


COMPARISON WITH HISTORICAL CONTROL DATA:

Mitomycin C (0.3 μg/mL), Griseofulvin (8.0 μg/mL) and CPA (15 μg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.
The solvent control was deionised water (10 % v/v) saline and culture medium MEM.

Exp. I
Number of micronucleated cells: Without S9 mix: preparation interval 24 hrs, treatment 4 hrs
Solv. control (10.0 %): 0.35 – 1.50 %
Positive control MMC (0.03 – 0.1 μg/mL): 0.85 – 18.15 %

In test:
Solv. control (10.0 %): 0.65%
Positive control MMC ( 0.3 μg/mL): 6.15 %
Test item (268 -1072 μg/mL): 0.3 - 0.9 %


Number of micronucleated cells; With S9 mix: preparation interval 24 hrs, treatment 4 hrs
Solv. control (10.0 %): 0.15 – 1.70 %
Positive control CPA (2.5 – 25.0 μg/mL): 0.90 – 38.30 %

In test:
Solv. control (10.0 %): 0.9 %
Positive control CPA (15.0 μg/mL): 9.9 %
Test item (268 -1072 μg/mL): 0.75 - 1.15 %



Exp. II
Number of micronucleated cells: Without S9 mix: preparation interval 24 hrs, treatment 24 hrs
Solv. control (10.0 % ): 0.15 – 1.50 %
Positive control Griseofulvin (6.3 – 25.0 μg/mL): 2.50 – 31.70 %

In test:
Solv. control (10.0 %): 0.35 %
Positive control Griseofulvin (8.0 μg/mL): 5.2 %
Test item (134 - 1072 μg/mL): 1.25 -7.75 %


Number of micronucleated cells; With S9 mix: preparation interval 24 hrs, treatment 4 hrs
Solv. control (10.0 %): 0.15 – 1.70 %
Positive control CPA (2.5 – 25.0 μg/mL): 0.90 – 38.30 %

In test:
Solv. control (10.0 %): 0.95 %
Positive control CPA (15.0 μg/mL): 16.05 %
Test item (268 - 1072 μg/mL): 0.8 - 1.5 %


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence and presence of metabolic activation no cytotoxicity measured as reduced proliferation index was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion