Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

gene mutation in bacterial cells

S. typhimurium TA97a, TA98, TA100, TA1535 and E. coli(WP2) Ames test (OECD 471, GLP), with and without metabolic activation: negative

S. typhimurium TA1535/pSK1002, umu, with metabolic activation: positive

Cytogenicity in mammalian cells

V79 cell, chromosome aberration test (OECD 473, GLP), with and without metabolic activation: negative

human hepatocytes, UDS, without metabolic activation: negative

rat hepatocytes, UDS, without metabolic activation: positive

CHO-K1, DNA-damage (single strand breaks), without metabolic activation: positive

gene mutation in mammalian cells

V79 Chinese hamster lung cells, without metabolic activation, phosphoribosyltransferase locus and Na/K ATPase locus: positive

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
not specified
GLP compliance:
no
Type of assay:
other: unscheduled DNA synthesis (UDS), in vitro DNA damagae and/or repair study
Species / strain / cell type:
hepatocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human hepatocyte suspensions were prepared from apparently healthy fragments of human liver discarded during the course of prescribed surgery essentially according to Strom et al. (1982). As checked by both macroscopic and histological examinations, these fragments were devoid of appreciable alterations.
- Sex, age and number of blood donors if applicable: Case 1 was a 56-year-old male who underwent surgery for intrahepatic lithiasis; case 2 was a 56-year-old female who underwent surgery for a hepatic metastasis of a colon adenocarcinoma. The proportion of viable hepatocytes after perfusion was 65 % in case 1 and 83 % in case 2.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Isolated human hepatocytes were suspended in Williams E medium supplemented with 10 % calf serum and gentamicin (50 µg/ml), and plated at a concentration of 1 × 10E6 in 35-mm Petri dishes coated with rat tail collagen
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
3, 10, 30, 100 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 3 h
- Exposure duration: 20 h
- Expression time (cells in growth medium): no expression time

STAIN (for cytogenetic assays): 10 µCi / mL [methyl-3H]thymidine

NUMBER OF REPLICATIONS: 2 independent assays

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Silver grains over the nucleus minus the grains over a randomly chosen equal-sized area in the cytoplasm were defined as net grains per nucleus. Cytoplasmic labeling was also considered in order to assess a possible effect on mitochondrial DNA.
Key result
Species / strain:
hepatocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentrations ranging from 3 to 100 mM produced a dose-dependent reduction in the fraction of viable trypan blue-excluding cells in human hepatocytes
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
NDMA (5 mM), used as a positive control, induced UDS in hepatocyte primary cultures from all human donors, thus demonstrating their metabolic competence
Additional information on results:
Any significant increase of net nuclear grains was absent in human hepatocytes and the number of cells in repair was always below 10 %.

Table 1: Percent survival of rat and human hepatocytes exposed for 20 h to hexanal as evaluated with the trypan blue exclusion test

Treatment

Dose(mM)

Percent survivalb

Control

 

92

97

Hexanal

3

88

93

 

10

80

95

 

30

77

94

 

100

 

 

NDMA

5

80

93

bData obtained from two human donors.

Table 2

UDS inhuman hepatocytes after 20-h exposure to hexanala

Treatment

conditions

NUC

(mean ± SD)

CYT

(mean ± SD)

NNG

(mean ± SD)

%Repair

Hexanal

3mM

11.01±2.65

10.10±2.12

0.91±1.78

 

10mM

11.40±4.61

10.40±3.45

1.00±2.95

 

30mM

10.22±3.77

9.16±2.95

1.06±2.59

NDMA

5mM

24.64±5.38

6.51±3.94

18.13±4.95 *

aNUC, nuclear grain count; CYT, cytoplasmic grain count; NNG, net nuclear grains.Counts of 200 cells from four autoradiographs were carried out for each dose level. Grain counts include cells with no nuclear labeling encountered in the 50 cells counted for each slide. The percentage repair is the percentage of cells with net nuclear labeling 5 grains.

* Statistically significant (p < 0.001) compared to controlsas assessed by Student's t-test (two-tailed).

Conclusions:
After 20 h exposure, human hepatocytes failed to induce UDS.
Executive summary:

Hexanal was examined for cytotoxicity similar to OECD Guideline 482, as evaluated by the trypan blue exclusion test, and for genotoxicity, as evaluated by the induction of unscheduled DNA synthesis (UDS), in primary cultures of human hepatocytes. After 20 h exposure, human hepatocytes failed to induce UDS. These results seem to suggest that at the concentrations which are presumably attained after ingestion with food or generated by lipid peroxidation processes hexanal is presumably unable to induce genotoxic effects in the human liver.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
either hypoxanthine-guanine phosphoribosyltransferase locus as resistance to 6-thioguanine or at the Na/K ATPase locus as resistance to ouabain.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: NIH, Coriell Institute for Medical Research (Camden, NJ)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 3, 10, 30 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: N-nitroso-N-methylurea
Details on test system and experimental conditions:
DURATION
- Exposure duration: 60 min
- Expression time (cells in growth medium): 6 d (6-thioguanine assay), 2 d (ouabain assay)

SELECTION AGENT (mutation assays):
6-thioguanine (hypoxanthine-guanine phosphoribosyltransferase locus) or ouabain (Na/K ATPase locus)
Evaluation criteria:
Mutagenic potency (D3C), defined as the concentration (mM) of aldehyde yielding a mutant frequency three times higher than the spontaneous mutant frequency,was calculated, according to Bradley et al. (1981), from the formula

D3C = 3CD/Y

where C is the spontaneous mutant frequency (mutants per 106 survivors), and Y the observed mutants per 106 survivors in cultures treated with the dose D (mM). In practice D3C was calculated for all the concentrations tested and averaged.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table1: Mutagenicity of hexanal in Chinese hamster V79 cells

Compound

Concentration

(mM)

Mutation frequency/relative survivala

TGr

OUAr

Hexanal

0

6.4±0.4/(0.84 ±0.09)

0.9 ± 0.4/(0.88 ± 0.05)

 

3

7.8±1.0/ 0.78±0.06

5.5 ± 2.5**/0.81 ± 0.17

 

10

15.3±2.4/ 0.59±0.09

9.0 ± 4.0**/ 0.52± 0.19

 

30

31.7±4.5** / 0.46±0.08

 

EMS

0

 

1.4 ± 0.8 /(0.81 ± 0.08)

 

8

 

18.1 ± 4.6**/ 0.69 ± 0.05

 

16

 

28.4 ± 7.0**/ 0.56 ± 0 14

MNU

0

15.1±0.3/(0.89 ±0.04)

 

 

0.3

38.1±5.3**/0.84 ±0.04

 

 

1.0

106.8±33.3**/ 0.64±0.12

 

The mutation frequency represents the number of mutant colonies per106 viable cells. The relative survival was calculated from the ratio (cells in treated cultures/fraction of viable cells in control cultures); the fraction of viable cells in controls is indicated in parentheses. The value of both mutation frequency and relative survival represents the mean ± SD of data obtained from at least three independent experiments

*P< 0.05;**P< 0.01 versus controls; the significance level was determined according to Kastenbaum and Bowman (1970).

Table 2: Mutagenic potencies of hexanal

D3Ca

 

TGr

OUAr

Hexanal

12.7

2.3

MNU

0.39

 

EMS

 

2.13

aConcentration (mM) that increases mutation frequency 3-fold above spontaneous background, calculated as indicated in Materials and methods.

Conclusions:
Hexanal was found to induce a dose-dependent increase in the frequency of both TG and OUA clones at concentrations ranging from 3 to 30 mM. The concentration that increases mutation frequency 3-fold above spontaneous background (DC3C) was 12.7 mM for TGr and 2.3 for OUAr.
Executive summary:

The mutagenicity for mammalian cells of hexanal produced by lipid peroxidation was tested in V79 Chinese hamster lung cells either at the hypoxanthine-guanine phosphoribosyltransferase locus as resistance to 6-thioguanine or at the Na/K ATPase locus as resistance to ouabain in an assay simliar to OECD Guideline 476. The results show that hexanal induced a dose-dependent increase in the frequency over controls of both 6-thioguanine- and ouabain-resistant mutants at concentrations ranging from 3 to 30 mM. The concentration that increases mutation frequency 3-fold above spontaneous background (DC3C) was 12.7 mM for TGr and 2.3 for OUAr.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
not specified
GLP compliance:
no
Type of assay:
other: unscheduled DNA synthesis (UDS), in vitro DNA damagae and/or repair study
Species / strain / cell type:
hepatocytes: rat
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Rat hepatocytes were isolated from Sprague-Dawley male albino rats (200-250 g) by collagenase perfusion as described by Williams (1977). Cell suspensions less than 80 % viable, as evaluated by the trypan blue exclusion test, were discarded.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Isolated rat hepatocytes were suspended in Williams E medium supplemented with 10 % calf serum and gentamicin (50 µg/ml), and plated at a concentration of 1 × 10E6 in 35-mm Petri dishes coated with rat tail collagen
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
3, 10, 30, 100 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 3 h
- Exposure duration: 20 h
- Expression time (cells in growth medium): no expression time

STAIN (for cytogenetic assays): 10 µCi / mL [methyl-3H]thymidine

NUMBER OF REPLICATIONS: 2 independent assays

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Silver grains over the nucleus minus the grains over a randomly chosen equal-sized area in the cytoplasm were defined as net grains per nucleus. Cytoplasmic labeling was also considered in order to assess a possible effect on mitochondrial DNA.
Species / strain:
hepatocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentrations ranging from 3 to 100 mM produced a dose-dependent reduction in the fraction of viable trypan blue-excluding cells in human hepatocytes
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
NDMA (5 mM), used as a positive control, induced UDS in hepatocyte primary cultures from all human donors, thus demonstrating their metabolic competence
Additional information on results:
Any significant increase of net nuclear grains was absent in human hepatocytes and the number of cells in repair was always below 10 %.

Table 1: Percent survival of rat and human hepatocytes exposed for 20 h to hexanal as evaluated with the trypan blue exclusion test

Treatment

Dose(mM)

Percent survivalb

Control

 0

96.3 ± 2.18

Hexanal

3

95.7 ± 2.08

 

10

71.3 ± 18.01

 

30

49.3 ± 42.83

 

100

0

NDMA

5

93.0 ± 5.14

bData obtained from two human donors.

Table 2

UDS inhuman hepatocytes after 20-h exposure to hexanala

Treatment

conditions

NUC

(mean ± SD)

CYT

(mean ± SD)

NNG

(mean ± SD)

%Repair

Hexanal

3mM

10.87±4.31

9.13±2.80

1.74±4.03

 

10mM

12.47±5.33

11.67±4.54

0.8±5.85

 

30mM

13.89±6.05

10.81±5.12

3.08±5.31 *

NDMA

5mM

40.66±21.50

7.25±5.1

33.41±23.11*

aNUC, nuclear grain count; CYT, cytoplasmic grain count; NNG, net nuclear grains.Counts of 200 cells from four autoradiographs were carried out for each dose level. Grain counts include cells with no nuclear labeling encountered in the 50 cells counted for each slide. The percentage repair is the percentage of cells with net nuclear labeling 5 grains.

* Statistically significant (p < 0.001) compared to controlsas assessed by Student's t-test (two-tailed).

Conclusions:
After 20 h exposure, hexanal induced, even though to a modest extent a significant and dose-dependent amount of DNA repair synthesis in rat hepatocytes. As a matter of fact in rat, hepatocytes the value of 5 net nuclear grains, which is generally considered the minimum level for a frankly positive response, was never reached; nevertheless it has recently been agreed that no absolute threshold should be imposed for negative controls and for a positive response, and that a compound should be considered positive if a dose-dependent increase is observed in net nuclear grains for at least two points and both increased points are above the justified laboratory- specific threshold (as in our case), and/or statistical significance is demonstrated for both points (International Workshop on Standardization of Genotoxicity Test Procedures, Melbourne, 1993; G. Brambilla, personal communication).
Executive summary:

Hexanal was examined for cytotoxicity in an assay similar to OECD Guideline 482, as evaluated by the trypan blue exclusion test, and for genotoxicity, as evaluated by the induction of unscheduled DNA synthesis (UDS), in primary cultures of rat hepatocytes. After 20 h exposure, hexanal induced, even though to a modest extent a significant and dose-dependent amount of DNA repair synthesis in rat hepatocytes.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The umu-test which can detect the induction of DNA repair is applied in order to analyze the genotoxicity hexanal. The procedure was originally developed by Oda Y.and Nakamura S. et al.(1985), using Salmonella typhimurium TA1535 which involve a plasmid(pSK1002) carrying a fused gene umuC'-'lacZ. In this fused gene, the umu operon is induced by DNA-damaging agents. The intensity of DNA-repair after DNA damage is measured by b-galactosidase activity which is produced from the fused gene.
GLP compliance:
no
Type of assay:
SOS/umu assay
Specific details on test material used for the study:
no details given
Target gene:
fused gene umuC'-'lacZ. In this fused gene, the umu operon is induced by DNA-damaging agents.
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: involves a plasmid(pSK1002) carrying a fused gene umuC'-'lacZ.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from livers of male rats pretreated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
not specified
Vehicle / solvent:
not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-(2-FuryI)-3-(5-nitro-2-furyl)acrylamido
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Incubation in Luria broth(LB) at 37 deg.C. for 16 hours with shaking. Dilution of the culture 50 -fold into TGA medium. Incubation at 37 deg.C. for 3 hours (OD600 0.2-0.3) .
- Exposure duration: 2 h
- Expression time (cells in growth medium): none

SELECTION AGENT: 2-nitrophenyl-beta-D-galactopyranoside
Evaluation criteria:
The induction of beta-galactosidase against the solvent did not vary with the time course. The indicated value of blank in each time is set to be the base value (B). Net genotoxicity (beta-galactosidase) of the sample is calculated by subtracting this value (B) from the genotoxicity(A) of the sample in a response time. The judgment of genotoxicity in the umu test is shown as follows. The chemical whose value of the (A-B) /B gives over two is decided to have strongly positive genotoxicit.Hence, the following rank of genotoxic intensity is set:

(A-B)/B > 2.0: ++ strongly positive
2.0 > (A-B)/B > 1.0: + positive
1.0 > (A-B) /B > 0.5: +- weakly positive
0.5 > (A-B) /B: - negative
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: please refer to table 1

Table 1: Results of umu test for hexanal

Concentration [µg/mL/OD600]

(A-B)/B

S9-

(A-B)/B

S9 +

 680.3

 0.46 -

 0.65 +-

(A-B)/B > 2.0: ++ strongly positive

2.0 > (A-B)/B > 1.0: + positive

1.0 > (A-B) /B > 0.5: +- weakly positive

0.5 > (A-B) /B: - negative

Concentration indicates the minimum concentration which is defined to be positive.

S9 + With microsomal activation

S9 - Without microsomal activation

Conclusions:
Hexanal was weak positive in the umu assay with metabolic activation.
Executive summary:

Hexanal was tested in umu assay. The umu-test which can detect the induction of DNA repair is applied in order to analyze the genotoxicity hexanal. The procedure uses Salmonella typhimurium TA1535 which involve a plasmid(pSK1002) carrying a fused gene umuC'-'lacZ. In this fused gene, the umu operon is induced by DNA-damaging agents. The intensity of DNA-repair after DNA damage is measured by beta-galactosidase activity which is produced from the fused gene. Hexanal was weak positive in the umu assay with metabolic activation (S9 +).

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Alkaline elution was employed to study DNA damage in CHO-K1 cells treated with a series hexanal. DNA cross-linking was measured in terms of the reduction in the effect of methyl methanesulphonate on the kinetics of DNA elution.
GLP compliance:
no
Type of assay:
other: DNA cross-linking (DNA damage)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Hexanal was kindly supplied by Dr H. Esterbauer (Institute of Biochemistry, University of Graz, Austria).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: Cultures to be used for experiments grew exponentially with a doubling time of ca.14 h
- Number of passages: Cultures to be used for experiments were initiated weekly from stock cultures
- Methods for maintenance in cell culture: Stock cultures of CHO-K1 cells were propagated in Ham's F-12 medium (Flow Laboratories, Milan, Italy) supplemented with 10 % foetal calf serum, 100 unit/mL penicillin-G and 0.1 mg/mL streptomycin sulphate

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F-12 medium (Flow Laboratories, Milan, Italy) supplemented with 10% foetal calf serum, 100 unit/mL penicillin-G and 0.1 mg/mL streptomycin sulphate
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
0.5, 1.5, 4.5
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Bis(2-chloroethyl)methylarnine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Experimental cells were labelled for 24 h with 0.025 µCi/mL [2-14C]-thymidine (55 mCi/mmol; Amersham International, UK).
- Exposure duration: 90 min
- Expression time (cells in growth medium): none

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays): [2-14C]-thymidine (55 mCi/mmol; Amersham International, UK).

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Approximately
5 x 10E5 14C-labelled experimental cells were deposited on a Millipore mixed esters of cellulose filter (25 mm in diameter; 5 µm pore size). As an internal standard, an equal number of 3H-labelled cells exposed for 60 min to 1 mM MMS was included in each assay. The cells were washed on the filter with cold Merchant's solution and lysed with 5 ml 0.2% sodium lauroylsarcosinate, 2 M NaCl, 0.02 M Na2EDTA, pH 10, at room temperature (20-22°C). After washing with 0.02 M Na2EDTA, pH 10, single-stranded DNA was eluted from the filter in the dark by controlled flow of a solution of 0.02 M Na2EDTA, 0.06 M tetraethylammonium hydroxide, pH 12.3, at 0.15 ml/min. Eluted fractions were collected at 20 min intervals and assayed for radioactivity. Proteinase-K digestion was not performed, thus no distinction was made between DNA-DNA and DNA-protein cross-links.

NUMBER OF CELLS EVALUATED: 5 x 10E5

DETERMINATION OF CYTOTOXICITY
- Method: Trypan blue dye exclusion; at least 1000 cells/sample were examined
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Hexanal induced DNA single-strand breaks and/or alkali-labile sites. DNA-damage occurred at concentrations which did not reduce, or only minimally reduced, cell viability.
Conclusions:
Hexanal induced dose-dependent DNA damage in cultured mammalian cells at concentrations which did not reduce, or only minimally reduced, cell viability.
Executive summary:

Alkaline elution was employed to study DNA damage in CHO-K1 cells treated with hexanal. DNA cross-linking was measured in terms of the reduction in the effect of methyl methanesulphonate on the kinetics of DNA elution. Hexanal produced DNA single-strand breaks, or lesions which were converted to breaks in alkali.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
In the first experiment only 3 concentrations could be evaluated for mutagenicity, due to severe bacteriotoxicity. In consultation with the sponsor, no repetition of the first experiment was performed. It can be seen as uncritical, because the second experiment confirmed the lack of mutagenicity in five concentrations up to bacteriotoxic concentrations. The deviation was assessed and signed by the study director on 20. Mar. 2018.
Deviations:
yes
Remarks:
Please refer to 'Version/Remarks'
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz (8.04.2015)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), kept under inert gas.
Target gene:
Please refer to tables 1a and 1b
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
Test concentrations with justification for top dose:
0.05, 0.15, 0.5, 1.5, 5 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
methylmethanesulfonate
other: 2-amino-anthracene, 4-nitro-1,2-phenylene diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h
- Expression time (cells in growth medium): none
- Selection time (if incubation with a selection agent): 24 h

SELECTION AGENT (mutation assays): histidine, tryptophan, ampicillin, UV radiation, crystal violet solution

NUMBER OF REPLICATIONS: 3 replicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

DETERMINATION OF CYTOTOXICITY
The toxicity of the following concentration was tested: 5 µL/plate. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In all experiments, no precipitation of the test item Hexanal was observed at any of the tested concentrations up to 5 µL/plate .

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to table 3

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the highest concentration (5 µL/plate), bacterial growth and bacterial background lawn was not observed. In the next lower concentration (1.5 µL/plate), a decrease in the number of revertants was observed in all bacteria strains. The bacteria strain TA98 showed a decrease in number of revertants in the concentration 0.5 µL/plate, too.

Table 2      Mean Revertants First Experiment

Strain

TA97a

TA98

TA100

E.coli

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

73

84

36

33

92

108

n.d.

n.d.

21

22

sd

7.6

5.1

6.2

5.5

8.0

15.9

n.d

n.d

1.2

1.5

DMSO

Mean

74

110

38

36

89

91

60

62

26

18

sd

5.2

21.1

4.4

11.9

9.2

1.2

12.7

4.6

1.7

0.6

Positive
Controls*

Mean

479

463

268

176

379

1001

1001

1001

220

175

sd

67.2

132.3

28.0

24.3

62.3

0.0

0.0

0.0

10.6

31.1

f(I)

6.47

4.21

7.05

4.89

4.12

11.00

16.68

16.15

10.48

9.72

5 µL/plate

Mean

0

0

0

0

0

0

0

0

0

0

sd

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

f(I)

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

1.5 µL/plate

Mean

3

16

2

3

9

22

29

27

4

9

sd

0.6

2.9

1.2

0.6

0.6

4.6

8.4

5.5

0.6

1.2

f(I)

0.04

0.15

0.05

0.08

0.10

0.24

0.48

0.44

0.15

0.50

0.5 µL/plate

Mean

74

79

26

24

84

85

82

80

17

17

sd

8.0

16.0

4.0

1.2

3.2

13.3

24.8

14.9

2.5

2.5

f(I)

1.00

0.72

0.68

0.67

0.94

0.93

1.37

1.29

0.65

0.94

0.15 µL/plate

Mean

72

74

29

28

82

91

84

78

17

16

sd

10.5

12.1

2.5

7.0

4.9

11.4

1.5

7.0

2.1

3.5

f(I)

0.97

0.67

0.76

0.78

0.92

1.00

1.40

1.26

0.65

0.89

0.05 µL/plate

Mean

79

103

31

29

78

93

73

82

22

22

sd

3.2

25.5

1.5

1.5

5.3

10.3

10.6

7.0

3.0

2.5

f(I)

1.07

0.94

0.82

0.81

0.88

1.02

1.22

1.32

0.85

1.22

n.d. = not determined

f(I) = increase factor

* Different positive controls were used

Table 3      Historical Data of Spontaneous Revertants

Strain

 

TA97a

TA98

TA100

E.coli

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Demin. water

Mean

89

94

20

22

92

96

112

122

18

18

Min

60

63

6

8

51

64

85

67

6

7

Max

144

138

52

51

141

141

155

185

31

33

SD

18

16

11

10

15

14

23

41

6

6

Exp 1

73

84

36

33

92

108

n.d.

n.d.

21

22

Exp 2

79

76

37

37

85

93

n.d.

n.d.

10

12

DMSO

Mean

88

97

20

21

89

92

114

124

18

17

Min

58

67

7

8

44

62

79

80

8

6

Max

135

144

47

50

136

199

195

209

33

32

SD

17

17

11

10

16

17

38

44

6

6

Exp 1

74

110

38

36

89

91

60

62

26

18

Exp 2

79

95

37

42

75

84

97

87

11

12

Positive Controls*

Mean

537

520

407

113

490

764

1006

745

265

132

Min

264

237

100

39

220

273

1001

396

55

45

Max

1165

1181

1001

487

984

1912

1037

1001

515

712

SD

173

160

163

90

153

288

13

228

88

82

Exp 1

479

463

268

176

379

1001

1001

1001

220

175

Exp 2

577

433

621

264

699

717

1001

1001

249

113

* Different positive controls were used

1001 colonies per plate means the bacteria growth was too strong for counting.

n.d. = not determined

Exp 1: plate-incorporation method

Exp 2: pre-incubation method

Conclusions:
Based on the results of this study it is concluded that hexanal is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA1535 and one Escherichia coli (WP2) strain in the absence and presence of metabolic activation under the experimental conditions in this study.
Executive summary:

The test item hexanal showed no increase in the number of revertants in any bacteria strain with or without metabolic activation in an Ames test according to OECD Guideline 471 and in compliance with GLP criteria.

Nearly all negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that hexanal is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA1535 and one Escherichia coli (WP2) strain in the absence and presence of metabolic activation under the experimental conditions in this study.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz (8.04.2015)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), kept under inert gas.
Target gene:
Please refer to tables 1a and 1b
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
Test concentrations with justification for top dose:
0.01, 0.02, 0.05, 0.09, 0.19, 0.38, 0.75, 1.5 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
methylmethanesulfonate
other: 2-amino-anthracene, 4-nitro-1,2-phenylene diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Expression time (cells in growth medium): none
- Selection time (if incubation with a selection agent): 24 h
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): histidine, tryptophan, ampicillin, UV radiation, crystal violet solution

NUMBER OF REPLICATIONS: 3 replicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

DETERMINATION OF CYTOTOXICITY
The toxicity of the following concentration was tested: 5 µL/plate. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In all experiments, no precipitation of the test item hexanal was observed at any of the tested concentrations up to 5 µL/plate .

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to table 3

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item showed signs of toxicity in the following concentrations towards the bacteria strains:
• TA97a: 1.5 µL/plate
• TA98: 0.5 and 0.25 µL/plate
• TA100: 1.5 and 0.75 µL/plate
• Escherichia coli: 1.5 µL/plate
• TA1535: 1.5 µL/plate

The bacterial background lawn was observed at all concentrations

Table 2      Mean revertants in pre-incubation method

TA97a

TA100

E.coli

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

79

76

85

93

n.d.

n.d.

10

12

sd

3.2

11.3

9.5

12.5

n.d.

n.d.

2.1

3.5

DMSO

Mean

79

95

75

84

97

87

11

12

sd

15.9

5.8

18.9

7.6

5.3

9.0

2.5

4.0

Positive
Controls*

Mean

577

433

699

717

1001

1001

249

113

sd

182.5

32.3

25.7

37.0

0.0

0.0

72.0

29.5

f(I)

7.30

4.56

8.22

8.54

10.32

11.51

24.90

9.42

1.5 µL/plate

Mean

14

18

5

9

3

2

1

1

sd

3.8

5.3

2.0

4.6

0.6

1.0

0.0

0.6

f(I)

0.18

0.19

0.07

0.11

0.03

0.02

0.09

0.08

0.75 µL/plate

Mean

85

79

24

35

84

83

10

11

sd

4.6

15.6

7.5

4.4

2.5

12.0

3.2

1.5

f(I)

1.08

0.83

0.32

0.42

0.87

0.95

0.91

0.92

0.38 µL/plate

Mean

77

73

62

68

106

104

9

12

sd

6.1

8.0

18.8

5.5

19.1

18.1

0.0

5.1

f(I)

0.97

0.77

0.83

0.81

1.09

1.20

0.82

1.00

0.19 µL/plate

Mean

79

74

80

96

82

103

10

10

sd

8.7

8.5

14.0

13.5

15.3

6.2

2.5

1.2

f(I)

1.00

0.78

1.07

1.14

0.85

1.18

0.91

0.83

0.09 µL/plate

Mean

77

91

87

100

95

83

11

10

sd

13.8

13.7

10.3

13.1

14.5

18.0

2.5

1.7

f(I)

0.97

0.96

1.16

1.19

0.98

0.95

1.00

0.83

0.05 µL/plate

Mean

66

107

88

115

91

87

10

9

sd

17.1

17.0

12.0

10.1

15.6

26.0

1.5

1.2

f(I)

0.84

1.13

1.17

1.37

0.94

1.00

0.91

0.75

0.02 µL/plate

Mean

87

95

99

118

112

79

9

9

sd

13.5

18.1

8.1

12.5

5.3

12.8

2.3

1.2

f(I)

1.10

1.00

1.32

1.40

1.15

0.91

0.82

0.75

0.01 µL/plate

Mean

81

99

94

101

91

83

12

12

sd

9.8

20.1

28.0

21.0

9.2

18.0

1.2

4.0

f(I)

1.03

1.04

1.25

1.20

0.94

0.95

1.09

1.00

Strain

TA98

Induction

-S9

+S9

Demin.

water

Mean

37

37

sd

5.9

8.7

DMSO

Mean

37

42

sd

3.6

3.1

Positive
Controls*

Mean

621

264

sd

56.8

18.3

f(I)

16.78

6.29

0.5 µL/plate

Mean

13

17

sd

1.5

2.1

f(I)

0.35

0.40

0.25 µL/plate

Mean

16

15

sd

2.9

1.2

f(I)

0.43

0.36

0.13 µL/plate

Mean

43

42

sd

4.7

2.3

f(I)

1.16

1.00

0.06 µL/plate

Mean

40

39

sd

3.6

5.5

f(I)

1.08

0.93

0.03 µL/plate

Mean

40

45

sd

4.6

6.4

f(I)

1.08

1.07

0.02 µL/plate

Mean

40

39

sd

7.8

7.6

f(I)

1.08

0.93

0.008 µL/plate

Mean

33

38

sd

6.8

8.6

f(I)

0.89

0.90

0.004 µL/plate

Mean

30

42

sd

0.6

4.6

f(I)

0.81

1.00

n.d. = not determined

f(I) = increase factor

* Different positive controls were used

Table 3      Historical Data of Spontaneous Revertants

Strain

 

TA97a

TA98

TA100

E.coli

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Demin. water

Mean

89

94

20

22

92

96

112

122

18

18

Min

60

63

6

8

51

64

85

67

6

7

Max

144

138

52

51

141

141

155

185

31

33

SD

18

16

11

10

15

14

23

41

6

6

Exp 1

73

84

36

33

92

108

n.d.

n.d.

21

22

Exp 2

79

76

37

37

85

93

n.d.

n.d.

10

12

DMSO

Mean

88

97

20

21

89

92

114

124

18

17

Min

58

67

7

8

44

62

79

80

8

6

Max

135

144

47

50

136

199

195

209

33

32

SD

17

17

11

10

16

17

38

44

6

6

Exp 1

74

110

38

36

89

91

60

62

26

18

Exp 2

79

95

37

42

75

84

97

87

11

12

Positive Controls*

Mean

537

520

407

113

490

764

1006

745

265

132

Min

264

237

100

39

220

273

1001

396

55

45

Max

1165

1181

1001

487

984

1912

1037

1001

515

712

SD

173

160

163

90

153

288

13

228

88

82

Exp 1

479

463

268

176

379

1001

1001

1001

220

175

Exp 2

577

433

621

264

699

717

1001

1001

249

113

* Different positive controls were used

1001 colonies per plate means the bacteria growth was too strong for counting.

n.d. = not determined

Exp 1: plate-incorporation method

Exp 2: pre-incubation method

Conclusions:
Based on the results of this study it is concluded that hexanal is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA1535 and one Escherichia coli (WP2) strain in the absence and presence of metabolic activation under the experimental conditions in this study.
Executive summary:

The test item hexanal showed no increase in the number of revertants in any bacteria strain with or without metabolic activation in an Ames test according to OECD Guideline 471 and in compliance with GLP criteria. Nearly all negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that hexanal is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA1535 and one Escherichia coli (WP2) strain in the absence and presence of metabolic activation under the experimental conditions in this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-24 - 2018-06-18 (Experimental phase: 2018-05-15 - 2018-05-29)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 473, "In Vitro Mammalian Chromosome Aberration Test", adopted 29th July, 2016.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Council Regulation (EC) No 440/2008, B.10. Mutagenicity – IN VITRO MAMMALIAN CHROMOSOME ABRERRATION TEST, COMMISSION REGULATION (EU) 2017/735 adopted 14 February 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
n/a
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: purchased from ECACC (European Collection of Cells Cultures)
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Methods for maintenance in cell culture if applicable: The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2.
- Modal number of chromosomes: diploid, 2n=22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S9
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 62.5, 125, 250 and 500 μg/mL test item
with S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 125, 250, 500 and 750 μg/mL test item
Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at least for the highest test concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DME (Dulbecco’s Modified Eagle’s) medium
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database. The test item was dissolved in a concentration of 4 mg/mL DME (Dulbecco’s Modified Eagle’s) medium (stock solution) at the first step. A clear solution of Hexanal was obtained in DME medium up to a concentration of 4 mg/mL. There was no precipitation in the medium at any concentration tested.
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DME (Dulbecco’s Modified Eagle’s) medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: n/a
- Exposure duration: 3h (±S9) or 20h (-S9)
- Expression time (cells in growth medium): Cell counts were conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix)
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): 40h or 31h

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS:
Duplicate cultures were used at each test item concentration and the negative control cultures as well as the positive controls for treatment without and with S9 mix.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Preparation of Chromosomes: Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5% Giemsa for subsequent scoring of chromosome aberration frequencies.
Analysis of Metaphases Cells: All slides were independently coded before microscopic analysis and scored blind. 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

NUMBER OF CELLS EVALUATED: 300 well-spread metaphase cells (150 metaphases/slide) per incubation

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well-spread metaphase cells (150 metaphases/slide) per incubation

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)
Rationale for test conditions:
as indicated in the guideline
Evaluation criteria:
Evaluation of Result
Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance are considered together.
There is no requirement for verification of a clearly positive or negative response.
Statistics:
For statistical analysis the CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment dose using the adequate regression analysis by Microsoft Excel software.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Remarks:
solvent controls
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / Effects of osmolality: There were no relevant changes in pH or osmolality after treatment with the test item.
- Evaporation from medium: none known
- Water solubility / Precipitation: No precipitation of the test item was observed at any of the applied concentrations

RANGE-FINDING/SCREENING STUDIES:
Solubility and Dose Selection
A clear solution of Hexanal was obtained DME (Dulbecco’s Modified Eagle’s) medium up to a concentration of 4 mg/mL. There was no precipitation in the medium at any concentration tested.
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.
Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:
Experiment A with 3/20 h treatment/sampling time
without S9 mix: 62.5, 125, 250 and 500 μg/mL test item
with S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250 and 500 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 125, 250, 500 and 750 μg/mL test item
All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
3h/20h treatment/sampling time without S9-mix
incl. Gaps excl. Gaps
Mean 40.50 31.70
SD 3.51 3.88
Range 35-50 26-39
Lower CI 33.22 23.64
Upper CI 47.78 39.75
n 23 23

3h/20h treatment/sampling time with S9-mix
incl. Gaps excl. Gaps
Mean 46.07 39.43
SD 2.39 2.65
Range 39-51 34-46
Lower CI 41.11 33.95
Upper CI 51.02 44.92
n 23 23

- Negative (solvent/vehicle) historical control data: 3h/20h treatment/sampling time without S9-mix
incl. Gaps excl. Gaps
Mean 6.26 2.85
SD 0.75 0.65
Range 4-8 2-5
Lower CI 4.70 1.59
Upper CI 7.82 4.11
n 23 23

3h/20h treatment/sampling time with S9-mix
incl. Gaps excl. Gaps
Mean 6.39 3.02
SD 0.83 0.64
Range 5-9 2-5
Lower CI 4.66 1.69
Upper CI 8.12 4.35
n 23 23

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983
Conclusions:
The study was performed according to the OECD TG 473 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). Hexanal did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.
Executive summary:

The test item Hexanal, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix), in accordance with the current OECD Guideline 473, under GLP:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 62.5, 125, 250 and 500μg/mL test item

with S9 mix: 125, 250, 500 and 750μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 125, 250 and 500μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix:125, 250 and 500μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix:125, 250, 500 and 750μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at least for the highest test concentration.

No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation.

In the experiment A in the absence of metabolic activation one value at the dose of 500 μg/mL (5 aberrant cells excluding gaps/150 cells) was within the historical control range (2-5 aberrant cells without gaps/150cells) but slightly above the 95% control limits of the historical control data (upper limit approximately 4,11 aberrant cells excluding gaps/150 cells).

However, no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups. Therefore, the finding was considered as accidential and not being biologically relevant.

There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, Hexanal did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Thus, the test item is considered as being non-clastogenic in this system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal: OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet assay / OECD 489
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

In accordance with REACH Regulation (1907/2006) Annex VIII column 2 point 8.4 "appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII". Since hexanal was positive in mammalian gene mutation and cytogenicity assays (non GLP, Klimisch 2), an in vivo Comet assay conducted according to OECD 489 is considered appropriate to fulfil data requirements of Annex VIII of the REACH Regulation.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
Hexanal

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- Available GLP studies :
In-vitro Gene Mutation Study in Bacteria/OECD 471, in-vitro Chromosome Aberration/OECD 473.

- Available non-GLP studies :
Umu assay (Ono 1991), gene mutation in mammalian cells (Brambilla 1989), UDS in mammalian cells (Martelli 1994), DNA damage in mammalian cells (Marinari 1984), all Klimisch 2

- Historical human data :
no data

- (Q)SAR :
Profiling with OECD QSAR Toolbox v4.2: positive alerts for Ames and micronucleus mutagenicity due to aldehyde group (explanation: "All compounds carrying an aldehydic group can potentially undergo Schiff base formation with a primary amine. They are to be considered potentially genotoxic, as demonstrated in vivo ability to react with nucleobases, without metabolic activation, forming adducts, interbase cross-links (both intra- and inter-strand), and DNA-protein crosslinks. The length of carbon chain for aliphatic aldehydes, and in general molecular size, can strongly modulate the formation of every type of cross-link and even the accessibility of the DNA nucleobases. DNA-protein crosslinks have been reported as the primary DNA damage induced by the related substance formaldehyde (Speit et al. 2007)).

- In vitro methods :
in vitro assays conducted under GLP conditions: In-vitro gene mutation study in bacteria/OECD 471, in-Vitro Chromosome Aberration/OECD 473

- Weight of evidence :
A weight-of-evidence approach was performed using in vitro data from different publications (Ono 1991, Brambilla 1989, Martelli 1994, Marinari 1984) and the availble Ames test (Andres 2018) and Chromosomal aberration test (Beres 2018). The results were ambiguous. While hexanal was negative on bacterial strains in Ames test (GLP study) and did not induce chromosomal aberrations in V79 chinese hamster fibroblasts (GLP study), it was tested positive in umu assay (Ono 1991, Klimisch 2). In addition, results of in vitro assays indicate cytogenic and genotoxic potential of hexanal in rat hepatocytes and Chinese hamster cells (Martelli 1994b, Marinari 1984, Brambilla 1989, all Klimisch 2). Cytogenicity testing (UDS) in human hepatocytes, on the other hand, was negative (Martelli 1994a)

- Grouping and read-across :
Heptanal was negative in in vitro chromosomal aberration and gene mutation in mammalian cells. Pentanal, on the other hand, was tested positive in in vitro gene mutation assay and DNA repair studies in mammalian cells (ECHA registration dossiers). Due to these ambiguous results, a grouping approach was not considered appropriate to address this endpoint.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The following adaptations are possible according to REACH Regulation (1907/2006) Annex VIII column 2
8.4.2. The study does not usually need to be conducted
– if adequate data from an in vivo cytogenicity test are available or
– the substance is known to be carcinogenic category 1 or 2 or mutagenic category 1, 2 or 3.
8.4.3. The study does not usually need to be conducted if adequate data from a reliable in vivo mammalian gene mutation test are available.
The above mentioned adaptations are not applicable, hence a further in vivo Comet assay conducted according to OECD 489 is considered appropriate to fulfil data requirements of Annex VIII of the REACH Regulation.
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

gene mutation in bacterial cells

Hexanal showed no increase in the number of revertants in any bacteria strain with or without metabolic activation in two Ames tests (pre-incubation method and plate-incorporation method) (Andres 2018a and b). Nearly all negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that hexanal is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA1535 and one Escherichia coli(WP2) strain in the absence and presence of metabolic activation.

In addtion, hexanal was tested in umu assay. The umu-test which can detect the induction of DNA repair is applied in order to analyze the genotoxicity hexanal. The procedure uses Salmonella typhimurium TA1535 which involve a plasmid(pSK1002) carrying a fused gene umuC'-'lacZ. In this fused gene, the umu operon is induced by DNA-damaging agents. The intensity of DNA-repair after DNA damage is measured by beta-galactosidase activity which is produced from the fused gene. Hexanal was weak positive in the umu assay with metabolic activation (S9 +).

gene mutation in mammalian cells

The mutagenicity for mammalian cells of hexanal produced by lipid peroxidation was tested in V79 Chinese hamster lung cells either at the hypoxanthine-guanine phosphoribosyltransferase locus as resistance to 6-thioguanine or at the Na/K ATPase locus as resistance to ouabain. The results show that hexanal induced a dose-dependent increase in the frequency over controls of both 6-thioguanine- and ouabain-resistant mutants at concentrations ranging from 3 to 30 mM. The concentration that increases mutation frequency 3-fold above spontaneous background (DC3C) was 12.7 mM for TGr and 2.3 for OUAr.

Cytogenicity in mammalian cells

The test item Hexanal, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix), in accordance with the current OECD Guideline 473, under GLP:

Experiment Awith 3/20 h treatment/sampling time

without S9 mix:62.5, 125, 250 and 500μg/mL test item

with S9 mix:125, 250, 500 and 750μg/mL test item

Experiment Bwith 20/20 h treatment/sampling time

without S9 mix:125, 250 and 500μg/mL test item

Experiment Bwith 20/28 h treatment/sampling time

without S9 mix:125, 250 and 500μg/mL test item

Experiment Bwith 3/28 h treatment/sampling time

with S9 mix:125, 250, 500 and 750μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts at least for the highest test concentration.

No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation.

In the experiment A in the absence of metabolic activation one value at the dose of 500 μg/mL (5 aberrant cells excluding gaps/150 cells) was within the historical control range (2-5 aberrant cells without gaps/150cells) but slightly above the 95% control limits of the historical control data (upper limit approximately 4,11 aberrant cells excluding gaps/150 cells).

However, no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups. Therefore, the finding was considered as accidential and not being biologically relevant.

There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, Hexanal did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Thus, the test item is considered as being non-clastogenic in this system (Beres 2018).

Hexanal, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. Hexanal did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Alkaline elution was employed to study DNA damage in CHO-K1 cells treated with hexanal. DNA cross-linking was measured in terms of the reduction in the effect of methyl methanesulphonate on the kinetics of DNA elution. Hexanal produced DNA single-strand breaks, or lesions which were converted to breaks in alkali (Marinari 1984).

In addition, hexanal was evaluated for the induction of unscheduled DNA synthesis (UDS), in primary cultures of human hepatocytes. After 20 h exposure, human hepatocytes failed to induce UDS. However, in the same study, hexanal was tested positive in rat hepatocytes (Martelli 1994).

Conclusion

Hexanal was weakly positive in umu assay in bacterial cells with metabolic activation. Furthermore, the observed capability of eliciting UDS in rat hepatocytes and causing DNA-damage in CHO-K1 cells (Marinari 1984) agrees with the reported induction of an increased frequency of 6-thioguanine- and ouabain-resistant mutants in V79 Chinese hamster cells (Brambilla et al., 1989). This indicates intrinsic genotoxic activity of hexanal. On the other hand, hexanal was negative in a chromosomal aberration assay (OECD 473, GLP).

Justification for classification or non-classification

Based on the weight-of-evidence approach, there is some limited indication for genotoxicity of hexanal. However, in vivo cytogenicity data on the analogue isovaleraldehyde do not show clastogenicity of isovaleraldehyde, as well as the chromosome aberration test on hexanal did not induce chromosome aberrations. To draw a final conclusion concerning classification of hexanal, the results, if accepted, of the proposed in vivo comet assay are awaited.