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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Remarks:
read across on strutural analogue
Adequacy of study:
key study
Study period:
From 2000-03-23 To 2000-10-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline 473 and GLP but is used in read across based on grouping of substances

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
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): Esterol C
- Physical state: light yellow liquid
- Analytical purity: 75.75 %
- Composition of test material, percentage of components: Mixture of methylesters of saturated and unsatured C16 to C20 fatty acids, no data.
- Lot/batch No.: 0006503
- Expiration date of the lot/batch: February 2001
- Storage condition of test material: at room temperature and protected from light

Method

Species / strain
Species / strain / cell type:
lymphocytes: primary cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 containing 20% fetal calf serum, L-glutamine (2mM), penicillin (100 U/ml), streptomycin (100µg/ml) and phytohaemagglutinin.
Metabolic activation:
with and without
Metabolic activation system:
S9 from induced Aroclor 1254 rat liver
Test concentrations with justification for top dose:
- 18.96, 37.93, 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml (in the first experiment)
- 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml (in the second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: no justification in the study report
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C without S9 mix (3µg/ml for 3 hours of treatment, 0.2 µg/ml for continuous treatment), cyclophosphamide with S9 mix (50 µg/ml)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION (first experiment)
- Exposure duration: 3h
- Expression time (cells in growth medium): 17h
- Fixation time (start of exposure up to fixation or harvest of cells): 20h

DURATION (second experiment)
- Exposure duration: 3h (without S9); 20h and 44h (with S9)
- Expression time (cells in growth medium): 17h and 41h (without S9); 20h and 44h (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20h and 44h (with and without S9)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: approximately 1.5 normal cell cycles or approximately 1.5 normal cell cycles and 24 hours later.

NUMBER OF CELLS EVALUATED: 100 metaphase/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: gaps, chromatid and chromosome breaks and exchanges, multipleaberrations and pulverization

OTHER: blind scoring
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural aberrations for at least one of the dose-levels and one of
the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
The comparison was performed using the Chi-square test (p = 0.05)

Results and discussion

Test results
Species / strain:
lymphocytes: primary culture
Metabolic activation:
with and without
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:
Cytotoxicity:
Without S9:
After 3-hour treatment, a slight decrease in the mitotic index was noted at dose-levels of 1213.64 and 2427.27 µg/ml (up to 34%).
After 20- hour treatment, a 35% decrease in the mitotic index was induced at 2427.27 µg/ml.
After 44-hour treatment, a 33-69% decrease in the mitotic index was noted at dose-levels of 1213.64 and 2427.27 µg/ml.

With S9 mix:
No noteworthy decrease in the mitotic index was noted at the 20 hour harvest time

Chromosal aberration analyses:
The test substance did not induce any significant increase in the frequency of cells with chromosome aberrations in both experiments and at both harvest times, with and without S9 mix.
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):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions, the test substance Esterol C does not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

In a GLP mammalian cell cytogenetic assay (chromosome aberration) (Haddouk, 2000), primary lymphocyte cultures were exposed to Esterol C (batch no. 0006503) at concentration of 18.96, 37.93, 75.85, 151.70, 303.41, 606.82, 1213.64 and 2427.27 µg/ml with and without metabolic activation. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background.