Heptanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are three in vitro GLP guideline studies:

In vitro Gene mutation (Bacterial reverse mutation assay/Ames test): A guideline (OECD 471) study is available.

In vitro mammalian cell gene mutation test: L5178Y mouse lymphoma assay : A guideline (OECD 476) study is available.

In vitro mammalian chromosome aberration test : A GLP guideline OECD (473) study is available.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Not applicable

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

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.

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'.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 october 1980 To 14 November 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 2c Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10 % by volume)
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Homogenate prepared from Fischer 344 adult male rat liver induced by Aroclor 1254.

Test concentrations with justification for top dose:

- 0.781, 37.5, 50, 75 and 100 nl/ml (without metabolic activation)
- 6.25, 100, 150, 200 and 250 nl/ml (with metabolic acitvation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was immiscible with deionised water at 100 µl/ml but dissolved easily in DMSO at the same concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulfonate (without metabolic activation) Dimethylnitrosamine (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: None
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 or 3 days
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 or 13 days

SELECTION AGENT (mutation assays): 100 µg/ml of BrdU

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

See the field below "any other information on materials and methods incl. tables"

Statistics:

No statistics were performed.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

treatment with 125 nl/ml and higher concentrations were completely lethal

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of this test, the test compound did not induced increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells with or without rat liver S9 microsomal activation.

Executive summary:

The test compound was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), equivalent or similar to the OECD n°476 Guideline.

In a mammalian cell gene mutation assay (TK+/-), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test compound in DMSO for 4 hours at concentrations of 0.781, 37.5, 50, 75 and 100 nl/ml (without metabolic activation) and of 6.25, 100, 150, 200 and 250 nl/ml (with metabolic acitvation). Appropriate positive controls were used and showed a statistical increase in mutant colonies. The average cloning efficiencies for the negative controls varied from 72 % without activation to 68 % with activation, which demonstrated good culturing conditions for the assay. The negative control mutant frequencies were normal, and the positive control compounds induced mutant frequencies that were greatly in excess of the negative controls. The EMS positive control was slightly below the usual lower limit (300x 10 ^-6) of the normal range, but this result (which could be due to EMS hydrolysis) was not considered to have adversely affected the assay. The test compound did not induce increases in the mutant frequeny at the TK locus in L5178Y mouse lymphoma cells with or whithout rat liver S9 microsomal activation. Without activation, concentration up to 100 nl/ml became highly toxic without causing mutant frequency increases. With activation, 250 nl/ml was moderately toxic and nonmutagenic, whereas 400 nl/ml was completely lethal. The test material was therefore considered to be inactive in the mouse lymphoma forward mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 2b Guideline study with acceptable restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no assay was performed on strain allowing detection of certain oxidising mutagens, cross-linking agents and hydrazines like E. coli WP2 uvrA, E. coli WP2 uvrA pKM101 or S. typhimurium TA102

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA1535, and TA1537

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with Aroclor 1254

Test concentrations with justification for top dose:

1, 3, 10, 33, 100, 166, 333, 1000, 1666, 3333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

see below details on test system and conditions

Positive control substance:

other: Sodium azide (TA1535 and TA100), 4-nitro-o-phenylenediamine (TA98), and 9-aminoacridine (TA97 and TA1537) without S9. 2-aminoanthracene with S9 (all strains).

Details on test system and experimental conditions:

EXPERIMENTS
- one range-finding study
- one main study

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h


DETERMINATION OF CYTOTOXICITY (preliminary range-finding test)
- Test : in TA100 strain, with or without S9 mix;
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn) (Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lown, or both)

CONTROLS
* Without S9
- 4-nitro-o-phenylenediamine (TA98)
- sodium azide (TA100 and TA1535)
- 9-aminoacridine (TA97 and TA1537).
*With S9
- 2-aminoanthracene (TA98, TA100, TA1535, TA1537, TA15387).

Evaluation criteria:

Evaluations were made at both the individual trial and chemical levels.
Individual trials were judged mutagenic, weakly mutagenic, questionable, or nonmutagenic, depending on the magnitude of the increase in his+ revertants, and the shape of the dose-response. A trial was considered questionable if the dose-response was judged insufficiently high to support a call of weakly mutagenic, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response, and a nonmutagenic or weakly mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic. A chemical was judged mutagenic or weakly mutagenic if it roduced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a weakly mutagenic response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

No statistics were performed.

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535, and TA1537

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:

RANGE-FINDING/SCREENING STUDIES:
no data on results were reported

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the experimental conditions, n-heptanal did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

In a reverse gene mutation assay on bacteria (Zeiger, 1992), strains of Salmonella typhimurium (TA 1535, TA 1537, TA97, TA98 and TA100) were exposed to n-heptanal at concentration of 0, 1, 3, 10, 33, 100, 166, 333, 1000, 1666 and 3333 µg/plate in the presence or absence of mammalian metabolic activation. Within the positive controls performed, all induced the appropriate responses in the corresponding strains. No northworthy increase in the number of revertant colonies was induced in all tested strains with and without metabolic activation. Therefore, n-heptanal does not show any mutagenic activity in the bacterial reverse test. This study is classified as reliable with restriction. It satisfies OECD requirements for the test OECD 471 except in that no strain allowing detection of certain oxidising mutagens, cross-linking agents and hydrazines like E. coli WP2 uvrA, E. coli WP2 uvrA pKM101 or S. typhimurium TA 102 were used here.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Heptanal has been tested for genotoxicity in in vitro studies.

In vitro Gene mutation (Bacterial reverse mutation assay/Ames test): (Zeiger 1992)

In a reverse mutation assays Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537 were exposed to heptanal at concentration ranging from 1 up to 3333 µg/plate with or without S-9 metabolic activation at concentration. No reproducible or dose-related response over solvent was seen.

In vitro mammalian cell gene mutation test : (Myhr 1981)

In a Mouse lymphoma cells (L5178Y) forward mutation assay, mammalian cell were exposed to heptanal, with and without metabolic activation. Concentrations were 0.78 to 100 nl/ml without metabolic activation and 6.25 to 250 ng/ml with metabolic activation. Weak to high toxicity was seen at all doses. No mutagenic activity was seen.

In vitro mammalian chromosome aberration test : (Sarlang 2010)

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 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 test item HEPTANAL was dissolved in dimethylsulfoxide (DMSO).

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.

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.

In conclusion, Heptanal was not mutagenic in these three in vitro assays.

Justification for classification or non-classification

No classification for mutagenicity has to be applied for heptanal according to EU regulation (EC) No 1272/2008 (CLP).

Justification : Negative results are recorded in all three in vitro tests (In vitro Gene mutation, In vitro mammalian cell gene mutation test, In vitro mammalian chromosome aberration test).