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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
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

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
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 (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptan-1-ol
EC Number:
203-897-9
EC Name:
Heptan-1-ol
Cas Number:
111-70-6
Molecular formula:
C7H16O
IUPAC Name:
heptan-1-ol
Details on test material:

- Name of test material (as cited in study report): n-heptanol
- Batch No.098033005
-description: colorless liquid
- container: one aluminium flask
- storage condition: at room temperature and protected from light
- purity: 99.85%
-expiry date: 30 july 2000

Method

Target gene:
not applicable (no a mutation gene assay)
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
0.5 ml of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamin (2mM), penicillin (100U/mL), streptomycin (100µg/ml) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37°C for 48 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
First cytogenetic experiment:
With a treatment volume of 35µl/5.5 ml culture medium, the treatment levels were:
-78.125, 156.25, 312.5, 625, 1250, 2500, 3750, and 5000 µg/ml.

Second cytogenetic experiment:
With a treatment volume of 27.5µl/5.5 ml culture medium, the treatment levels were:
-19.53, 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75µg/ml for the experiment without S9 mix.
-39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml forthe experiment with S9 mix.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture treated with the vehicle
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mytomycin in the absence of S9 mix and cyclophosphamide (CPA) in the presence of S9 mix
Remarks:
Valid
Details on test system and experimental conditions:

Method of application: in medium


Duration:

First experiment:
Lymphocyte cultures were then exposed to the test or control substances, both in the absence and presence of S9 mix, for 3 hours 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 from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.


Second experiment:
. without S9 mix, cells were exposed continuously to the test or control substances.
. with S9 mix, cells were exposed to the test or control substances 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 from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles
and 24 hours later.

Spindle inhibitor (for cytogenetic assay) : colcemid

Stain: Giemsa

Number of cells evaluated: 200 metaphases/dose-level (with 44 to 46 chromosomes), with 100 metaphase/culture whenever possible. Only 50 metaphases/culture were analysed when at 10% cells with structural chromosome aberrations were observed.

All analyses were performed using blind scoring.
The followiing structural aberrations were recorded for each metaphase: gaps, chromatid, and chromosome breaks and exchanges and others (multiple aberrations and pulverizations) and the following numerical aberrations: polyploidy and endoreplication.





Evaluation criteria:
A reproductible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was cosndiered as a postivie result. Reference to historical data of 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 aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was perfomed unsing the X2 test in which p=0.05 was used as the lowest level of significance.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The test substance was freely soluble in the vehicle (DMSO) at 785.715 mg/ml.

In the culture medium, the dose-level of 5000 µg/ml showed a slight to marked emulsion. At this dose-level, the pH was about 7.7 (as for the vehicle control) and the osmolality equal to 324 mOsm/kg H20 (402 mOsm/kg H20 for the vehicle control).

In the mutagenicity experiments, a slight to marked emulsion was noted in the culture medium at dose-levels 1250 µg/ml, at the end of the treatment period.

First cytogenic experiment:
With a treatment volume of 35µg/5.5ml culture medium, the treatment-levels were:
-78.125, 156.25, 312.5, 625, 1250,2500, 3750 and 5000 µg/ml.

Cytotoxicity:
With and without S9 mix, the test substance was totally toxic at dose-levels 625 µg/ml. At the lower dose-levels, a slight to moderate decrease in the mitotic index was induced.

Chromosomal aberration analysis:
With and without S9 mix, the analysis of metaphases was performed at the dose-levels of
78.125, 156.25 and 312.5 µg/ml.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted, after a 3-hour treatment, with and without S9 mix.

Second cytogenetic experiment:
With a treatment volume of 27.5 pl/5.5 ml culture medium, the treatment-levels were:
- 19.53, 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment without
S9 mix,
- 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment with S9 mix.

Cytotoxicity:
With and without S9 mix, the test substance was totally toxic at dose-levels 625 µg/ml. At the lower dose-levels, a slight to moderate decrease in the mitotic index was induced.

Chromosomal aberration analysis:
With and without S9 mix, the analysis of metaphases was performed at the dose-levels of
78.125, 156.25 and 312.5 µg/ml.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted, after a 3-hour treatment, with and without S9 mix.

Second cytogenetic experiment:
With a treatment volume of 27.5 µg/5.5 ml culture medium, the treatment-levels were:
- 19.53, 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment without
S9 mix,
- 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment with S9 mix.

Cytotoxicity:
Without S9 mix, for both treatment period, a slight to severe toxicity was noted at dose-levels
234.38 µg/ml.
With S9 mix, for both harvest time, a slight to marked toxicity was induced at all tested dose-levels.

Chromosomal aberration analysis:
The analysis of metaphases was performed at the following dose-levels:
- 156.25, 234.38 and 312.5 µg/ml for the 20-hour harvest time, with and without S9 mix,
- 234.38 µg/ml for the 44-hour harvest time, without S9 mix,
- 312.5 µg/lml for the 44-hour harvest time, with S9 mix.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted at both harvest times, with and without S9 mix.
The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Remarks on result:
other: No mutagenic

Applicant's summary and conclusion

Conclusions:
Interpretation of results
negative

Under our experimental conditions, the test substance n-heptanol (batch No. 9803005); purity: 99.85%) does not induce chromosome aberrations in cultures human lymphocytes.
Executive summary:

The test substance n-heptanol was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.


First experiment:


Lymphocyte cultures were then exposed to the test or control substances, both in the absence and presence of S9 mix, for 3 hours 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 from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.


Second experiment:


. without S9 mix, cells were exposed continuously to the test or control substances.


. with S9 mix, cells were exposed to the test or control substances 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 from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.


For both experiment, after hypotonic treatment (KCL 0.075M), the cells were fixed in a methanol/acetic mixture (3/1; v/v) spread on glass slides and stained with Giemsa. The test item n-heptanol was dissolved in dimethylsulfoxide (DMSO).


 


The dose-levels of the positive controls were as :


. without S9 mix, mitomycin C: 3 µg/ml (3 hours of treatment) or 0.2 µg/ml (contiuous treatment),


. with S9 mix, cyclophophamide : 50µg/ml


 


The test substance was freely soluble in the vehicle (DMSO) at 785.715 mg/ml.


In the culture medium, the dose-level of 5000 µg/ml showed a slight to marked emulsion.


At this dose-level, the pH was about 7.7 (as for the vehicle control) and the osmolality equal to 324 mOsm/kg H20 (402 mOsm/kg H20 for the vehicle control).


In the mutagenicity experiments, a slight to marked emulsion was noted in the culture medium at dose-levels 1250 µg/ml, at the end of the treatment period.


First cytogenic experiment:


With a treatment volume of 35 µg/5.5ml culture medium, the treatment-levels were:


-78.125, 156.25, 312.5, 625, 1250,2500, 3750 and 5000 µg/ml.


- Cytotoxicity:


With and without S9 mix, the test substance was totally toxic at dose-levels 625 µg/ml. At the lower dose-levels, a slight to moderate decrease in the mitotic index was induced.


-Chromosomal aberration analysis:


With and without S9 mix, the analysis of metaphases was performed at the dose-levels of 78.125, 156.25 and 312.5 µg/ml. No significant increase in the frequency of cells with structural chromosomal aberrations was noted, after a 3-hour treatment, with and without S9 mix.


Second cytogenetic experiment: With a treatment volume of 27.5 pl/5.5 ml culture medium, the treatment-levels were:


- 19.53, 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment without S9 mix,


- 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment with S9 mix.


. Cytotoxicity:


With and without S9 mix, the test substance was totally toxic at dose-levels 625 µg/ml. At the lower dose-levels, a slight to moderate decrease in the mitotic index was induced.


. Chromosomal aberration analysis:


With and without S9 mix, the analysis of metaphases was performed at the dose-levels of 78.125, 156.25 and 312.5 µg/ml. No significant increase in the frequency of cells with structural chromosomal aberrations was noted, after a 3-hour treatment, with and without S9 mix.


. Second cytogenetic experiment:


With a treatment volume of 27.5 µg/5.5 ml culture medium, the treatment-levels were: - 19.53, 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment without S9 mix, - 39.06, 78.125, 156.25, 234.38, 312.5 and 468.75 µg/ml for the experiment with S9 mix. Cytotoxicity: Without S9 mix, for both treatment period, a slight to severe toxicity was noted at dose-levels 234.38 µg/ml. With S9 mix, for both harvest time, a slight to marked toxicity was induced at all tested dose-levels.


Chromosomal aberration analysis:


The analysis of metaphases was performed at the following dose-levels:


- 156.25, 234.38 and 312.5 µg/ml for the 20-hour harvest time, with and without S9 mix,


- 234.38 µg/ml for the 44-hour harvest time, without S9 mix,


- 312.5 µg/lml for the 44-hour harvest time, with S9 mix.


No significant increase in the frequency of cells with structural chromosomal aberrations was noted at both harvest times, with and without S9 mix. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.


 


Under our experimental conditions, the test substance, n-heptanol (batch No. 9803005; purity 99.85%) does not induce chromosome aberrations in cultured human lymphocytes.