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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

n-heptanol-1 was found to be negative in the in vitro gene mutations assays on bacteria (Ames test) and mammalian cells (mouse lymphoma assay and chromosome aberration test), both in the presence and absence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

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)
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
Target gene:
not applicable (no a mutation gene assay)
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
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.
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
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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay
Target gene:
Five strain of bacteria salmonella typhimurium: TA 1535, TA1537, TA98, TA 100 and TA 102.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:

The concentrations are:
.312,5; 625; 1250; 2500 and 5000 µg/plate for the strain 102 in the first assay.
. 156.25; 312.5; 625; 1250; and 2500 µg/plate



Vehicle / solvent:
Dimethyldisulfide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mytomicin
Details on test system and experimental conditions:
All experiment were performed according to the direct plate incorporation method except for the second and third tests with S9 mix, which were perfomed according to the preincubation method (60 minutes, 37°C).
Evaluation criteria:
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
Statistics:
not applicable
Species / strain:
S. typhimurium, other: TA1535, TA 1537, TA98, TA100 et TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under our experimental conditions, the test susbtance n-heptanol (batch No. 9803005, purity: 99.5%) does not show mutagenic activity in the bacterial reverse mutation test with salmonella typhimurium.
Executive summary:

The objective of this study was to evaluate the potential of the test substance  n-HEPTANO L (batchNo.9803005,purity:99.85%)  to induce reverse mutation in Salmonella typhimurium.

 

  A preliminary toxicity test was performed to define the dose-levels of n-HEPTANOL  to be used for the mutagenicity study . The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9mix, prepared from alive rmicrosomal fraction(S9fraction) of rats induced with Aroclor1254. A third experiment was performed with S9mix.

 

All experiments were performed according to the direct plate incorporation method except for the second and third tests with S9 mix,which were performed according to the preincubation method(60minutes,37°C).

 

Five strains of bacteria Salmonella typhimurium:TA1535, TA1537, TA98, TA lOO and TA102 were used. Each  strain  was exposed  to five  dose-levels  of the test substance (three plates/dose-levet).After 48 to 72 hours of incubation at 37°C,the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test substance n-HEPTANOL  was dissolved in dimethylsulfoxide  (DMSO). The dose-levels of the positive controls were as follows :

without S9 mix:

g/plateofsodiumazide(NaN3):TA1535andTA100strains,

50µg/plate of 9-Aminoacridine(9AA):TA1537strain,

0.5 µg/plate of 2-Nitrofluorene(2NF):TA98strain,

.0.5 µg/plate of Mitomycin C (MMC):TA102strain.

 

with S9 mix:

. 2 µg/plate of 2-Anthramine (2AM): TA1535,TA1537,TA98andTAlOOstrains,

. 10µg/plate of 2-Anthramine (2AM): TA102strain.

 

 

 

Results

 

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level for the main test was based

 on the level of toxicity, according to the criteria specified in the international guidelines.


 

Experiments without S9 mix:

The selected treatment-levels were as follows:

.312.5,625,1250,2500 and 5000 µg/plate, for the TA102 strain in the first experiment,

.156.25,312.5,625,1250 and 2500 µg/plate, for the all tester strains except for the TA102 strain in the first experiment as well as for the TA98 and TA102 strains in the second experiment,

. 78.125,156.25, 312.5,625 and 1250 µg/plate, for the TA1535,TA1537 and TA100 strains in the second experiment.

 

A slight to strong toxicity was noted,depending on the tester strain and the dose-levels.

 

The test substance did not induce any noteworthy increase in the number of revertants,in both experiments,in any of the five strains.

 

Experiments with S9 mix:

The selected treatment-levels were as follows:

. 312.5,625,1250, 2500 and 5000 µg/plate, for the TA102strain in the first experiment,

.156.25,312.5,625, 1250 and 2500µg/plate, for the all tester strains except for the TA102 strain in the first experiment as well as for the TA1537 and TA 102 strains in the second experiment,

78.125,156.25,312.5,625 and 1250 µg/plate, for the TA1535, TA98 and TA 100 strains in

these cond experiment as weil as for the TAl02 strain in the third experiment,

39.06,78.125,156.25,312.5and 625µg/plate, for all tested strains,except for the TA102 strain, in the third experiment.

 

A slight to strong toxicity was induced,depending on the tester strain,the dose-level and the experimental conditions.

 

The test substance did not induce any noteworthy increase in the number of revertants, in all the experiments, in any of the five strains.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study

  was therefore considered valid.

 

 Under our experimental conditions, the test substance n-heptanol (batch N. 9803005, purity: 99.5% ) does not show mutagenic activity in the bacterial reverse mutation test with salmonella typhimurium.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February 2011 - ............................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
minor deviation from study plan but not from guideline
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
minor deviation from study plan but not from guideline
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
0.156, 0.313, 0.625, 1.25, 2.5, 3.75 and 5 mM for both experiments (3- and 24-hour treatments).

Experiments with S9 mix (3-hour treatment):
- 0.156, 0.313, 0.625, 1.25, 2.5, 3.75 and 5 mM for the first experiment,
- 0.313, 0.625, 1.25, 2.5, 3, 3.75 and 5 mM for the second experiment.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: the test item was freely soluble in the vehicle (DMSO) at 232.7 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9) and cyclophosphamide (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): Trifluorothimidine medium

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
Evaluation criteria:
The acceptance criteria are defined according to IWGT recommendations (Moore et al., 2006; Moore et al., 2007).

The following criteria were considered to evaluate the validity of the study:
Criteria for the vehicle controls:
- the cloning efficiency (CE2) of the vehicle controls at the end of the expression time should be between 0.65 and 1.2,
- the mutation frequency of the vehicle controls should fall within the normal range of 50 x 10 6 -170 x 10 6,
- the suspension growth of the vehicle controls should be between 8 and 32 for the 3-hour treatment period, and between 32 and 180 for the
24-hour treatment period.

Criteria for the positive controls:
- the increase above the vehicle control mutation frequency (IMF) should be at least 300 x 10-6, the increase in the small colony mutation frequency
accounting for at least 40%,
- or the increase in the small colony mutation frequency should be at least 150 x 10-6 above that seen in the concurrent vehicle control.

In addition, the upper limit of cytotoxicity observed in the positive control culture should have an Adj. RTG greater than 10%.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(with and without S9)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(with and without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
To assess the cytotoxicity of the test item, six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

ADDITIONAL INFORMATION: see executive summary
Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

The test item did not show any mutagenic activity in the mouse lymphoma assay, in the presence or absence of a rat metabolizing system.
Executive summary:

Methods

 

After a preliminary toxicity test, N-HEPTANOL, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG), adjusted relative suspension growth (Adj. RSG) and cloning efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The test item was dissolved in dimethylsulfoxide (DMSO).

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

. without S9 mix: Methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL, (3-hour treatment) or 5 µg/mL (24-hour treatment),

. with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

 

Results

 

The cloning efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level retained for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in the adjusted relative total growth (Adj. RTG)).

 

Experiments without S9 mix

Using a treatment volume of 0.5% in culture medium, the selected dose-levels were 0.156, 0.313, 0.625, 1.25, 2.5, 3.75 and 5 mM for both experiments (3- and 24-hour treatments).

At the end of the treatment periods, a slight emulsion was noted in the culture medium at 5 mM.


Cytotoxicity

Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels = 1.25 mM, as shown by a 29-100% decrease in Adj. RTG.

Following the 24-hour treatment, a moderate to severe toxicity was induced at dose-levels = 0.313 mM, as shown by a 41-100% decrease in Adj. RTG.

 

Mutagenicity

Following the 3-hour treatment, no noteworthy increase in the mutation frequency was observed at dose-levels up to 2.5 mM, which showed a 75% decrease in Adj. RTG. At higher dose-levels, a severe toxicity was noted, preventing the evaluation of the mutation frequency.

Following the 24-hour treatment, no noteworthy increase in the mutation frequency was noted at dose-levels up to 1.25 mM, which showed a 96% decrease in Adj. RTG.

 

Experiments with S9 mix

Using a treatment volume of 0.5% in culture medium, the selected dose-levels were as follows:

.           0.156, 0.313, 0.625, 1.25, 2.5, 3.75 and 5 mM for the first experiment,

.           0.313, 0.625, 1.25, 2.5, 3, 3.75 and 5 mM for the second experiment.

At the end of the 3-hour treatment periods, a slight emulsion was noted in the culture medium at 5 mM.

 

Cytotoxicity

In the first experiment, a severe toxicity was induced at dose-levels = 3.75 mM, as shown by a 98-100% decrease in Adj. RTG.

In the second experiment, a moderate to severe toxicity was induced at dose-levels= 2.5 mM, as shown by a 43-100% decrease in Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency was noted in comparison to the vehicle control in either experiment.

Conclusion

 

Under the experimental conditions of this study, the test item, N-HEPTANOL, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Additional information

 


In vitro Studies


 


Ames test :


n-heptanol-1 was negative for mutagenicity in a standard bacterial mutagenicity test with and without metabolic activation (Haddouk, 2000). This study followed OECD test guideline 471. Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. N- heptanol did not induce mutagenicity in Salmonella bacterial strains with or without metabolic activation.The positive controls responded as expected. 


 


Mouse Lymphoma assay


n-heptanol-1 was negative in a in vivo mammalian cells (mouse lymphoma assay) (Sarlang, 2011). The test was performed under OECD 476 guideline. Under the experimental conditions of this study, the test item, n-heptanol, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or absence of a rat metabolizing system.


 


Mammalian Chromosome Aberration test


n-Heptanol-1 was negative in a in vitro mammalian chromosome aberration test (Griffon, 2000). The study was performed according to OECD 473 guideline. 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.


No significant increase in the frequency of cells with structural chromosomal aberrations was noted at both harvest times, with and without S9 mix. Under these experimental conditions, the test substance, n-heptanol (batch No. 9803005; purity 99.85%) does not induce chromosome aberrations in cultured human lymphocytes.




Justification for classification or non-classification

None of the available test results in vitro showed that substance treatment induced mutagenic effects. The susbtance should not be classified for genotoxicity according to EU Directive 67/584/EEC and EU regulation (EC) No 1272/2008 (CLP).