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Registration Dossier
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EC number: 200-624-5 | CAS number: 66-25-1
Endpoint summary
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
- 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 for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- 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 %.
- 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
- 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
- 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 for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- 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 %.
- 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
- 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 for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- 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. QA statement)
- 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. - 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 for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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 - 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
- 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
- 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. QA statement)
- 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.
Referenceopen allclose all
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).
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.
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).
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
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 |
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
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 |
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 |
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
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
In vivo Mammalian Alkaline Comet Assay
Male HAN:WIST rats; DNA strand breaks in stomach, liver and kidney cells; via oral gavage; 2000, 1000 and 500 mg/kg body weight/day (OECD 489, GLP): no genotoxic activity
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Comet assay / OECD 489
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 JUN 2020 to 4 NOV 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Version / remarks:
- adopted 29 JUL 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian comet assay
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Rats are routinely tested in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at the lab.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop ZRT. H-1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 50 - 52 d
- Weight at study initiation: 222 - 246 g
- Assigned to test groups randomly: No. All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping will be controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
- Housing: 3 animals / cage; at the positive control group 2 animals / cages in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm - width x length x height) cages on certified laboratory wood bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 22.9
- Humidity (%): 41 - 66
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: sunflower oil (Helianthi annui oleum raffinatum)
- Justification for choice of solvent/vehicle: The applicability of test item formulations in sunflower oil was additionally checked in the frame of the present study in a non-GLP preliminary solubility test. The testing laboratory has validated an analytical method for the necessary dose formulation analysis and additionally an in-study partial validation was performed where recovery and stability of the 450 mg/mL test item solution was investigated. At the chosen target tissues, the suitability of sunflower oil vehicle in the in vivo Comet assay was evaluated in separate validation, reliability studies performed in the testing laboratory under the same conditions as the present study; furthermore it is confirmed with an available own laboratory’s historical control database: that is based on four experiments in the case of stomach and liver, and on two experiments in the case of kidney.
- Concentration of test material in vehicle: 400, 200 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw (test item), 10 mL/kg bw (positive control) - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared before each treatment. - Duration of treatment / exposure:
- 24h
- Frequency of treatment:
- twice, once on the day 0 and 24 hours thereafter
- Post exposure period:
- 3-4 hours after the second treatment (doses and vehicle control)
3-4 hours after the treatment (positive control) - Dose / conc.:
- 2 000 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- Dose / conc.:
- 500 mg/kg bw/day
- No. of animals per sex per dose:
- 6 / dose group and negative control group, 4 / positive control group
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulfonate (EMS)
- Justification for choice of positive control(s): Positive control group with known mutagen
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw - Tissues and cell types examined:
- - Target tissues for sample preparation: Stomach, liver, kidney and testis seminiferous tubules (gonadal cells)
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Based on the results of previous studies the maximum dose was set to 2000 mg/kg bw/day (the maximum limit dose). Furthermore this dose is the maximum dose recommended in the respective OECD guideline. Two additional doses, 1000 and 500 mg/kg bw/day were selected.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment: twice; once ontrypan day 0 and 24 hours thereafter
Sampling times: 3-4 hours after the second treatment
DETAILS OF SLIDE PREPARATION:
- After pre-treatment of the slides the cells were embedded. Before the use a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (~ 5 minutes). After the cell isolations each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter 85 or 165 μL (~1-9 x 104 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip*. The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentle slide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed.
METHOD OF ANALYSIS:
- Cytotoxicity was determined (as a first screening) on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling.
- DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis).
- For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment and per vehicle control; 450 per positive control). - Evaluation criteria:
- test chemical is clearly negative if:
- 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 historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.
test chemical is clearly positive if:
- 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 historical negative control data for given species, vehicle, route, tissue and number of administration; - Statistics:
- The statistical significance of % tail DNA values, tail length and OTM values; furthermore, the number of ghost cells was carried out using the appropriate statistical method, using SPSS software. The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test, the normal distribution of data was examined by Kolmogorov-Smirnov test. Following these analyses, a one-way analysis of variance following by Dunnett’s test was used to compare the vehicle control value and the corresponding values of test item doses and positive control.
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- no statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, liver or kidney cells
- Toxicity:
- no effects
- Remarks:
- No cytotoxicity (using Trypan blue dye exclusion method) was noticed in any test and control item treatments for any target tissue. Number of ghost cells in respective tissues did not differ from vehicle control or were within historical control data.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
Based on available information of previous studies the performance of a range finding study was not necessary. The highest dose was selected according to the criteria required by the OECD 489 Guideline.
The historical control data and summary tables of the results are found as attachments to this study entry. - Conclusions:
- The investigated test item Hexanal 0,05% Toco did not show genotoxic activity in the examined tissues in this Comet Assay.
- Executive summary:
The study was performed according to OECD TG 489, is fully reliable and in concordance with GLP.
In the study the test item Hexanal 0,05% Toco was investigated by the means of the in vivo comet assay on isolated stomach, liver and kidney cells under alkaline conditions in the male HAN:WIST rats. The test item was administered twice via oral gavage at the dose levels 2000, 1000 and 500 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. As negative (vehicle) control sunflower oil and as positive control ethyl methanesulfonate (EMS) was used.
Under the experimental conditions presented in this report, the test item Hexanal 0,05% Toco did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, liver or kidney cells. Concurrent controls confirmed the sensitivity and validity of the test.
The investigated test item Hexanal 0,05% Toco did not show genotoxic activity in the examined tissues in this Comet Assay.
Reference
Results
- All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met.
- At the highest concentration no mortality was observed.
- Expect eating of embedding of 2000 mg/kg bw/day, no toxic symptoms or any clinical signs were observed during the treatments in the remaining dose levels and controls.
- At the tissue isolation normal appearance and anatomy of liver, kidney and testis was noticed in all dose levels and controls.
- At the stomach openings test item smell and characteristic stomach content (bedding material or at 1000 mg/kg body weight/day in two cases conspicuously thin liquid) was noticed. Additionally, in several cases stomach tympanites were noticed. Smell intensity and bedding amount in the stomach showed a dose dependent tendency.
- No effect on body weights were stated.
- No cytotoxicity was noticed in any test and control item treatments for any target tissue (using Trypan blue dye exclusion method) .
- Number of ghost cells in the liver and kidney samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control.
- Despite slight dose related tendency and statistical difference from vehicle control (at highest dose level), number of ghosts cells in the stomach remained well within historical control data. The relatively higher number of ghost cells in stomach samples especially at the highest dose level seemed to be associated with toxic effects attributable to the test item, which was observed during macroscopic inspection of the tissue prior to cell isolation, whereas no cytotoxic effects (see above) were noted. Nevertheless, even at this local toxic effect concentration at the tissue of first contact no increased DNA migration was noted.
- Statistically significant increase of ghost cells was noticed at the EMS treatments in the stomach, liver and kidney samples (in line with historical control data).
- The mean median % tail DNA values of all dose groups did not differ statistically significantly from that of the vehicle control. All mean median % tail DNA values fell within corresponding historical control data.
- The tail length values of the stomach samples did not differ statistically significantly from that of the vehicle control in whole examined dose range. Statistically differences were observed in case of samples of the liver (at the two highest doses) and the kidney (at highest dose). However, these significances were considered as not relevant for mutagenicity assessment since the significances were observed for lower values (compared to the concurrent control) noted for the above dose levels. In addition, all values for test and control items were well within the corresponding historical control data ranges
- The Olive Tail Moment values in the stomach, liver and kidney of the test item treated groups did not differ statistically significant from that of the vehicle control and were within the established historical control data ranges.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
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).
In vivo Mammalian Alkaline Comet Assay
Hexanal 0,05% Toco was investigated by the means of the in vivo comet assay on isolated stomach, liver and kidney cells under alkaline conditions in the male HAN:WIST rats, according to OECD 489 and GLP. The test item was administered twice via oral gavage at the dose levels 2000, 1000 and 500 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. As negative (vehicle) control sunflower oil and as positive control ethyl methanesulfonate (EMS) was used. Cytotoxicity was determined (as a first screening) on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling and DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis).
Under the experimental conditions, the test item Hexanal 0,05% Toco did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, liver or kidney cells. Concurrent controls confirmed the sensitivity and validity of the test. Thus, the investigated test item Hexanal 0,05% Toco did not show genotoxic activity in the examined tissues in this Comet Assay.
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). Despite slight indications if genotoxic potential of Hexanal in in vitro studies, in an in vivo mammalian alkaline comet assay, Hexanal 0,05% Toco did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels (tested up to limit concentration of 2000 mg/kg bw) in stomach, liver or kidney cells. Thus, Hexanal 0,05% Toco is considered to have no genotoxic activity (OECD 489, GLP).
Justification for classification or non-classification
Based on the weight-of-evidence approach with in vitro data, there is some limited indication for genotoxicity of hexanal. However, the chromosome aberration test on hexanal did not induce chromosome aberrations. The results of an in vivo mammalian alkaline comet assay reveal that hexanal (tested as Hexanal 0,05% Toco) did not induce statistically significant increases in DNA strand breaks, up to limit concentration of 2000 mg/kg bw/day. Thus, the in vivo study confirms that hexanal does not posses genotoxic acitivity.
As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008.
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