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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames test (OECD 471): Heptanoic acid has been tested for genotoxicity in twoin vitrogene mutation studies in bacteria (Richard 1989; Zeiger 1992). There was no evidence of mutagenicity when heptanoic acid was incubated using established strains ofSalmonella typhimurium, TA 100, TA 1535, TA 1537, and TA 1538 with or without S-9 metabolic activation at concentration up to 5000 or 10000 µg/plate, respectively (Richard 1989). A modification of the standard Ames assay involving preincubation was also negative for a homologous series of 8 carboxylic acids, among them was heptanoic acid, at concentrations of up to 10000 µg/plate (Zeiger 1992).

Mouse lymphoma assay (OECD 476):Heptanoic acid has also been tested for genotoxicity in anin vitromammalian cell gene mutation test in L5178Y TK +/- mouse lymphoma cells. Results did not show any mutagenic activity in this mouse lymphoma assay either in presence or in absence of a rat metabolizing system.

Chromosome Aberration Test in Human lymphocyte cells (OECD 473): Moreover, during an in vitro mammalian chromosome aberration test with human lymphocytes, heptanoic acid did not induce chromosome aberrations in lymphocytes, in the presence or in the absence of a rat metabolizing system.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 15 June 1989 To 25 July 1989
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:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
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:
Range-finding study:
10, 33, 67, 100, 333, 667, 1000, 3333, 6667 and 10000 µg/plate with and without S9
Main study:
- 667, 1000, 3333, 6667 and 10000 µg/plate with S9
- 100, 333, 1000, 2500 and 5000 µg/plate without S9
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: 2-aminoanthracene with S9 for TA98, TA100, TA1535, TA1537, TA15387, 2-nitrofluorene without S9 for TA98 and TA1538, sodium azide without S9 for TA100 and TA1535, ICR-191 without S9 for TA1537.
Details on test system and experimental conditions:
EXPERIMENTS
- one range-finding study
- one main study

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- 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; 10 dose-levels
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn) (The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope)

CONTROLS
* Without S9
- 1 µg/plate of 2-nitrofluorene for TA98 and TA1538
- 1 µg/plate of sodium azide for TA100 and TA1535
- 2 µg/plate of ICR-191 for TA1537.
*With S9
- 0.5 µg/plate of 2-aminoanthracene for TA98, TA100, TA1535, TA1537, TA15387,
Evaluation criteria:
For a test article to be evaluated as positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
No statistics were performed.
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
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:
The results of the dose-range finding study conducted in the presence and absence of microsomal enzymes indicate toxicity to the test system (TA100 strain) without S9 at 3333, 6667 and 10000 µg/plate.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the experimental conditions, n-heptanoic acid did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

In a Salmonella/Mammalian-Microsome plate incorporation mutagenicity assay (Microbiological Associates INC., 1989), n-heptanoic acid was tested to evaluate its mutagenic potential. N-heptanoic acid was tested for its ability to induce back mutations at selected loci of several strains of Salmonella typhimurium in the presence and absence of microsomal enzymes derived from Aroclor 1254 induced rat liver. This test was similar to the international guidelines (OECD 471) and performed in compliance with the Principles of Good Laboratory Practice. The tester strains used in this study were TA98, TA100, TA1535, TA1537 and TA1538. Bacterias were exposed to the test item at five dose-levels (three plates/dose-level) selected from a preliminary toxicity test (667, 1000, 3333, 6667 and 10000 µg/plate with S9 and 100, 333, 1000, 2500 and 5000 µg/plate without S9). After 48 h of incubation at 37°C, the revertant colonies were scored. No positive responses were observed with any of the tester strains in the presence or absence of microsomal enzymes. Positive controls induced the appropriate responses in the corresponding strains.

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
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
Target gene:
Not applicable
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).
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.
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'.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
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 (2mM), 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
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.313, 0.625, 1.25, 2.5, 5 and 10 mM.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: cyclophosphamide
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): trifluorothymidine


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result which should fulfill the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
. and a dose-related trend is demonstrated by a statiscally significant trend test.

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, are not considered as positive result.
A test item is determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if:
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Vehicle solubility: 260.36 mg/mL
- Precipitation: no precipitate at the final dose-level of 1301.8 µg/mL (which corresponds to 10 mM) ; pH of 7.6 and osmolality of 371 mOsm/kg H2O


RANGE-FINDING/SCREENING STUDIES:
To assess the cytotoxicity of the test item, at least six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
A treatment of 3 hours (with and without S9 mix) and 24 hours (without S9 mix) was performed using a final concentration and conditions as described below for the mutagenicity experiment. At the end of treatment, cells were washed and then cell concentrations were adjusted to 2 x 105 cells/mL and cultured for 2 days as for the mutagenicity experiment. Two days after the end of the treatment, cultures were adjusted in order to seed an average of 1.6 cells per well in the 96 well microtiter plates.
Approximately one week after incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted on the plates.
Since the test item was freely soluble and non-severely toxic in the preliminary test, the highest dose-level selected for the main test was 10 mM, according to the criteria specified in the international guidelines.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Using a treatment volume of 100 µL/20 mL, the selected dose-levels for the main experiments with and without S9 mix were 0.313, 0.625, 1.25, 2.5, 5 and 10 mM.

Experiments without S9 mix
Following the 3-hour treatment, no noteworthy toxicity was noted at any of the tested dose-levels, as shown by the absence of decrease in Adj. RTG.
Following the 24-hour treatment, a moderate to severe toxicity was induced at dose levels = 5 mM, as shown by a 53-91% decrease in Adj. RTG.

Experiments with S9 mix
In the first experiment, a slight to moderate toxicity, without any clear evidence of a dose relationship, was noted at the dose-levels of 2.5 and 5 mM, as shown by a 39-50% decrease in Adj. RTG.
In the second experiment, no noteworthy toxicity was noted at any of the tested dose-levels as shown by the absence of decrease in Adj. RTG.

Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under our experimental conditions, the test item ACIDE HEPTANOIQUE (batch No. 0904099, purity: > 99%) did not show any mutagenic activity in the mouse lymphoma assay either in presence or in absence of a rat metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item ACIDE HEPTANOIQUE (batch No. 0904099, purity: 99.33%) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. The study was performed according to the international guidelines (OCDE and Commission Directive B17) and in compliance with the Principles of Good Laboratory Practice.

Methods

After a preliminary toxicity test, ACIDE HEPTANOIQUE was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5 x 106(3-hour treatment) or 0.15 x 106(24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. For the 24-hour treatment, the flasks were gently shaken at least once. Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. The test item was dissolved in dimethylsulfoxide (DMSO).

Results

The cloning efficiencies CE2and the mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid. Since the test item was freely soluble and non-severely toxic in the preliminary test, the highest dose-level selected for the main test was 10 mM, according to the criteria specified in the international guidelines.

Using a treatment volume of 100 µL/20 mL, the selected dose-levels for the main experiments

with and without S9 mix were 0.313, 0.625, 1.25, 2.5, 5 and 10 mM.

Experiments without S9 mix

Cytotoxicity:Following the 3-hour treatment, no noteworthy toxicity was noted any of the tested dose-levels, as shown by the absence of decrease in Adj. RTG. Following the 24-hour treatment, a moderate to severe toxicity was induced at dose-levels = 5 mM, as shown by a 53-91% decrease in Adj. RTG.

Mutagenicity:Following the 3-hour and 24-hour treatments, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Experiments with S9 mix

Cytotoxicity:In the first experiment, a slight to moderate toxicity, without any clear evidence of a dose relationship, was noted at the dose-levels of 2.5 and 5 mM, as shown by a 39-50% decrease in Adj. RTG. In the second experiment, no noteworthy toxicity was noted at any of the tested dose-levels as shown by the absence of decrease in Adj. RTG.

Mutagenicity:In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Conclusion

Under our experimental conditions, the test item ACIDE HEPTANOIQUE

(batch No. 0904099, purity: 99.33%) did not show any mutagenic activity in the mouse lymphoma

assay either in presence or in absence of a rat metabolizing system.

Conclusion

Under our experimental conditions, the test item ACIDE HEPTANOIQUE did not show any mutagenic activity in the mouse lymphoma assay either in presence or in absence of a rat metabolizing system.

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:
10, 33, 100, 333, 1000, 1666, 3333, 6666 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-heptanoic acid 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-heptanoic acid at concentration of 0, 10, 33, 100, 333, 1000, 1666, 3333 and 6666 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-heptanoic acid 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)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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

Justification : negative results in Ames test, in mouse lymphoma assay and in chromosome aberration test.