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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2009 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
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

Constituent 1
Reference substance name:
ACIDE HEPTANOIQUE
IUPAC Name:
ACIDE HEPTANOIQUE
Constituent 2
Chemical structure
Reference substance name:
Heptanoic acid
EC Number:
203-838-7
EC Name:
Heptanoic acid
Cas Number:
111-14-8
Molecular formula:
C7H14O2
IUPAC Name:
heptanoic acid
Details on test material:
- Name of test material (as cited in study report): ACIDE HEPTANOIQUE
- Physical state: colorless liquid
- Molecular weight: 130.18 g/mole
- Analytical purity: 99.33%
- Purity test date: 30 July 2009
- Lot/batch No.: 0904099
- Expiration date of the lot/batch: 30 July 2010
- Storage conditions of test material: at room temperature and protected from humidity

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human lymphocyte
Details on mammalian cell type (if applicable):
5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and phytohemagglutinin (PHA: a mitogen to stimulate lymphocyte division).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 27.5 µL/5.5 mL culture medium, the treatment-levels were as follows:
. 0.16, 0.31, 0.63, 1.25, 2.5, 5, 7.5 and 10 mM for the first experiment, both with and without S9 mix,
. 0.63, 1.25, 2.5, 5, 7.5 and 10 mM for the second experiment, both with and without S9 mix.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
In the first experiment, lymphocyte cultures were then exposed for 3 hours to the test or control items, both in the absence and presence of S9 mix, then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
. without S9 mix, cells were exposed continuously to the test or control items, until harvest,
. with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level, with 100 metaphases/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration 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:
For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the chi 2 test test, in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test resultsopen allclose all
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Following the 20-hour and 44-hour treatments
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the 20-hour harvest time
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix:
Following the 3-hour treatment, no noteworthy decrease in the mitotic index was noted at any of the tested dose-levels.
Following the 20-hour treatment, a slight to severe toxicity was noted at dose levels = 1.25 mM, as shown by a 34 to 86% decrease in the Mitotic Index (MI).
Following the 44-hour treatment, a severe toxicity was noted at dose-levels = 5 mM, as shown by a 100% decrease in MI.

Experiments with S9 mix:
At the 20-hour harvest time in the first experiment, a slight to moderate toxicity was noted at dose-levels = 5 mM as shown by a 37% to 52% decrease in MI.
At the 20-hour harvest time in the second experiment, a moderate decrease in MI was noted at 10 mM (53% decrease).
At the 44-hour harvest time in the second experiment, no noteworthy decrease in the mitotic index was noted at any of the tested dose-levels.

Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under experimental conditions of the test, the test item ACIDE HEPTANOIQUE (batch No. 0904099, purity: > 99.33%) did not induce chromosome aberrations in cultured human lymphocytes, in the presence or in the absence of a rat metabolizing system.
Executive summary:

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

 

The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account.

 

For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for 48 hours.

 

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

 

The second experiment was performed as follows:

.           without S9 mix, cells were exposed continuously to the test or control items until harvest,

.           with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

 

One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

 

The test item ACIDE HEPTANOIQUE was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

.           without S9 mix, Mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

.           with S9 mix, Cyclophosphamide: 12.5 or 25 µg/mL.

 

Results

In the culture medium, the dose-level of 1301.8 µg/mL (corresponding to 10 mM) showed no precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture.

With a treatment volume of 27.5 µL/5.5 mL culture medium, the treatment-levels were as follows:

.           0.16, 0.31, 0.63, 1.25, 2.5, 5, 7.5 and 10 mM for the first experiment, both with and without S9 mix,

.           0.63, 1.25, 2.5, 5, 7.5 and 10 mM for the second experiment, both with and without S9 mix.

 

The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

 

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment no noteworthy decrease in the mitotic index was noted at any of the tested dose-levels.

Following the 20-hour treatment, a slight to severe toxicity was noted at dose-levels = 1.25 mM, as shown by a 34 to 86% decrease in the Mitotic Index (MI).

Following the 44-hour treatment, a severe toxicity was noted at dose-levels = 5 mM, as shown by a 100% decrease in MI.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.           2.5, 5 and 10 mM for the 3-hour treatment, the latter being the highest recommended dose-level,

.           0.63, 1.25 and 2.5 mM for the 20-hour treatment, the latter inducing a 50% decrease in MI,

.           2.5 mM for the 44-hour treatment, the latter inducing a 26% decrease in MI and higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-, 20- as well as the 44-hour treatments.

 

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a slight to moderate toxicity was noted at dose-levels = 5 mM as shown by a 37% to 52% decrease in MI.

At the 20-hour harvest time in the second experiment, a moderate decrease in MI was noted at 10 mM (53% decrease).

At the 44-hour harvest time in the second experiment, no noteworthy decrease in the mitotic index was noted at any of the tested dose-levels.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.           2.5, 5 and 10 mM for the 20-hour harvest time in the first experiment, the latter inducing a 52% decrease in MI and being the highest recommended dose-level,

.           5, 7.5 and 10 mM for the 20-hour harvest time in the second experiment, the latter inducing a 53% decrease in MI and being the highest recommended dose-level,

.           10 mM for the 44-hour harvest time, the latter being the highest recommended dose-level.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

Conclusion

Under our experimental conditions, the test item ACIDE HEPTANOIQUE (batch No. 0904099,purity: > 99.33%) did not induce chromosome aberrations in cultured human lymphocytes, in the presence or in the absence of a rat metabolizing system.