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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:
23 September 2009 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; 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:
yes
Remarks:
minor deviations, see below
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Principles of method if other than guideline:
The study was performed in accordance with the study plan No. 36144 MLH and subsequent amendment, with the following deviation from the agreed study plan:
. to prevent any invalidation of an assay because of a possible technical problem, haemolysis or contamination of the positive controls, duplicate cultures of the positive controls have been performed in each experiment. The fixed cells were spread on slides and cells in mitosis were scored. But, the metaphase analyses were not performed since the conditions described above were not met. This deviation was not considered to have compromised the validity or the integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptanal
EC Number:
203-898-4
EC Name:
Heptanal
Cas Number:
111-71-7
Molecular formula:
C7H14O
IUPAC Name:
heptanal
Constituent 2
Reference substance name:
Heptaldehyde
IUPAC Name:
Heptaldehyde
Constituent 3
Reference substance name:
Heptaldehyde P-PH
IUPAC Name:
Heptaldehyde P-PH
Constituent 4
Reference substance name:
n-heptanal
IUPAC Name:
n-heptanal
Details on test material:
- Name of test material (as cited in study report): Heptanal
- Substance type: monoconstituent
- Physical state: colorless liquid
- Molecular weight: 114.8 g/mole
- Analytical purity: 97.47%
- Purity test date: 30 July 2009
- Lot/batch No.: 0905017
- Expiration date of the lot/batch: January 2010 (Since a third experiment had to be performed after the end of the test item shelf life, a new analysis was performed on 11th March 2010 to analyze if this batch could still be used in this study. Since, minor differences were found during this analysis (i.e. purity = 97.47%), the expiry date of the batch was extended until September 2010 and this initial batch has been used for the third experiment)
- Storage conditions of test material: at room temperature, keeping the container tightly closed in a dry and well-ventilated place, away from heat and sources of ignition

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
To prepare each culture, 0.5 mL of heparinized whole blood was added to 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 specified
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:
- 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment, both with and without S9 mix,
- 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 mM for the second experiment, both with and without S9 mix,
- 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the third experiment without S9 mix.
Vehicle / solvent:
- Vehicle used: DMSO
Controlsopen allclose all
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
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
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.

The third experiment was performed as follows: the cells were exposed without S9 mix continuously to the test or control items, until harvest.
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. When the frequency of cells with structural chromosome aberration (excluding gap) was greater than that of the vehicle control, the comparison was done using the chi 2 test, in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results
Species / strain:
lymphocytes: human lymphocytes
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The test item was freely soluble in the vehicle (DMSO) at 229.6 mg/mL.
In the culture medium, the dose-level of 10 mM (corresponding to 1148 µg/mL) showed no precipitate. At this dose-level, the pH was approximately 7.4 (as for the vehicle control) and the osmolality was equal to 366 mOsm/kg H2O (384 mOsm/kg H2O for the vehicle control).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix
Following the 3-hour treatment, a slight to severe decrease in the mitotic index was noted at dose levels = 1.25 mM (33 to 100% decrease).
Following the 20-hour treatment in the second experiment, a slight decrease in the mitotic index was noted at 2.5 mM (40% decrease).
Following the 20-hour treatment in the third experiment, a slight to severe decrease in the mitotic index was noted at dose levels = 0.625 mM (37 to 100% decrease).
Following the 44-hour treatment in the second experiment, no noteworthy decrease in mitotic index was noted at any of the tested dose-levels (up to 2.5 mM).
Following the 44-hour treatment in the third experiment, a severe decrease in the mitotic index was noted at dose levels = 2.5 mM (98 to 100% decrease).

Experiments with S9 mix
At the 20-hour harvest time in the first experiment, a slight to severe decrease in the mitotic index was noted from the lowest selected concentration of 0.078 mM, without any clear evidence of a dose-response relationship (30 to 100% decrease).
At the 20-hour harvest time in the second experiment, a moderate decrease in the mitotic index was noted at dose levels = 1.25 mM (45% decrease).
At the 44-hour harvest time, a slight to moderate decrease in the mitotic index was noted at dose levels = 1.25 mM (28 to 53% decrease).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under our experimental conditions, the test item HEPTANAL (batch No. 0905017) did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a rat metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item HEPTANAL (batch No. 0905017, purity: 98.06%) to induce chromosome aberrations in cultured human lymphocytes.

The study was performed according to the international guidelines (OECD 473 and Commission Directive No. B10) and in compliance with the principles of Good Laboratory Practice.

Methods

The test item was tested in three independent experiments with and/or 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 and third experiments, 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 about 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. The third experiment was performed as follows: cells were exposed continuously to the test or control items in the absence of S9 mix. 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 HEPTANAL 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 and 25 µg/mL.

Results

In the culture medium, the dose-level of 10 mM (corresponding to 1148 µg/mL) 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:

- 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment, both with and without S9 mix,

- 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 mM for the second experiment, both with and without S9 mix,

- 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the third experiment without S9 mix.

 

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

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment, a slight to severe decrease in the mitotic index was noted at dose-levels = 1.25 mM (33 to 100% decrease).

Following the 20-hour treatment in the second experiment, a slight decrease in the mitotic index was noted at 2.5 mM (40% decrease).

Following the 20-hour treatment in the third experiment, a slight to severe decrease in the mitotic index was noted at dose-levels = 0.625 mM (37 to 100% decrease).

Following the 44-hour treatment in the second experiment, no noteworthy decrease in mitotic index was noted at any of the tested dose-levels (up to 2.5 mM).

Following the 44-hour treatment in the third experiment, a severe decrease in the mitotic index was noted at dose-levels = 2.5 mM (98 to 100% decrease).

Metaphase analysis

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

- 0.313, 0.625 and 1.25 mM for the 3-hour treatment, the latter showing a 33% decrease in mitotic index and higher doses being too cytotoxic,

- 0.625, 1.25 and 2.5 mM for the 20-hour treatment in the second experiment, the latter inducing a 40 % decrease in mitotic index and being the highest dose-level tested,

- 0.313, 0.625 and 1.25 mM for the 20-hour treatment in the third experiment, the latter inducing a 62 % decrease in mitotic index,

- 2.5 mM for the 44-hour treatment in the second experiment, the latter being the highest dose-level tested,

- 1.25 mM for the 44-hour treatment in the third experiment, the latter inducing a 10 % decrease in mitotic index and higher dose-levels being too cytotoxic.

Following the 3-hour treatment, no significant increase in the frequency of cells with structural chromosomal aberrations was noted.

Following the 20-hour and 44-hour treatments in the second experiment, no significant increase in the frequency of cells with structural chromosomal aberrations was noted at the dose-levels analysed. But in this experiment, the level of toxicity induced with the highest dose-levels tested at both harvest times did not reach the ideal one defined in the study plan (a reduction in mitotic index greater than 50%). Therefore, a third experiment was performed using the same experimental conditions but a higher range of dose-levels (up to 10 mM). Following the 20-hour and 44-hour treatments in the third experiment, a significant toxicity was reached and no significant increase in the frequency of cells with structural chromosomal aberrations was noted at the dose-levels analysed.

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a slight to severe decrease in the mitotic index was noted from the lowest selected concentration of 0.078 mM, without any clear evidence of a dose-response relationship (30 to 100% decrease).

At the 20-hour harvest time in the second experiment, a moderate decrease in the mitotic index was noted at dose-levels = 1.25 mM (45% decrease).

At the 44-hour harvest time, a slight to moderate decrease in the mitotic index was noted at dose-levels = 1.25 mM (28 to 53% decrease).

Metaphase analysis

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

- 0.313, 0.625 and 1.25 mM for the 20-hour harvest time in the first experiment, the dose-level of 0.313 mM inducing a 56% decrease in mitotic index,

- 0.625, 1.25 and 2.5 mM for the 20-hour harvest time in the second experiment, the latter dose-level inducing a 45% decrease in mitotic index,

- 2.5 mM for the 44-hour harvest time in the second experiment, the latter inducing a 53% decrease in mitotic index. 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 HEPTANAL (batch No. 0905017) did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or absence of metabolizing system.

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