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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in bacteria or mammalian cells, but induces chromosome aberrations in the absence of a metabolic activating system in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
20 µg - 5,000 µg/plate (both for standard plate test and preincubation test)
Vehicle / solvent:
Due to the good solubility of the test substance in water, water was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylendiamine (NOPD)
Remarks:
With S9-mix: 2-AA (All strains); Without S9-mix: MNNG (TA 1535, TA 100), NOPD (TA 98), 9-aminoacridine (TA 1537), 4-nitroquinoline-N-oxide (E. coli)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation and preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants; clearing or diminution of the background lawn; reduction in the titer
Evaluation criteria:
Acceptance criteria: Generally, the experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was >1E9/mL

Evaluation criteria: The test chemical is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system is observed. A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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:
E. coli WP2 uvr A
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:
A slight decrease in the number of revertants was occasionally observed.
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.
No precipitation of the test substance was found.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S-9 mix
Test concentrations with justification for top dose:
250, 500, 750 (experiment I); 600, 700, 800 (experiment II) µg/mL
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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
- Exposure duration: without S9 mix: 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphase plates per culture were scored for structural chromosome aberrations.

DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
- The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.

A test substance is generally considered nonclastogenic in this test system if:
- There was no significant increase in the number of chromosomally damaged cells at any dose, above concurrent control frequencies
- The aberration frequencies were within the historical control range
Statistics:
The proportion of metaphases with aberrations was calculated for each group. A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at app. 700µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line.
- Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations.
- On the basis from the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps without S-9 mix in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated.
- Osmolality and pH values were not influenced by test substance treatment
- According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under any of the experimental conditions

 Dose (µg/mL)  Metabolic activation  Cell Count *10^8/ml  Metaphases w/aberrations (excl. gaps)
 0  13.10  2.0%
 250  11.95 (91.2%) 5.5% 
 500  12.70 (97.0%)  3.0%
 750  11.05 (84.4%)  4.0%
 0  12.75  2.0%
 250 9.95 (78.0%)  4.0%
 500  -  11.25 (88.2%)  2.0%
 750  -  10.95 (85.9%)  8.0%*
 0  -  8.95  3.0%
 600  -  6.35 (71.0%)  9.0%*
 700  -  5.05 (56.4%)  15.5%*
 800  -  4.40 (49.2%)  19.0%*
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
1st experiment: without S9 mix: 0; 50; 100; 200; 400; 800 µg/mL; with S9 mix 0; 50; 100; 200; 400 ; 800 µg/mL
2nd experiment: without S9 mix: 0; 200; 400; 600; 800 µg/mL; with S9 mix (S9 fraction : cofactors = 1 : 9): 0; 100; 200; 400; 800 µg/mL; with S9 mix (S9 fraction : cofactors = 3 : 7): 0; 100; 200; 400; 800 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: methylcholanthrene
Remarks:
Without metabolic activation 300 µg ethyl methane sulfonate/mL culture medium added in a volume of 1.0 mL. With metabolic activation (S-9 mix) 10 µg methylcholanthrene/mL culture medium added in a volume of 1.0 mL.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
After incubation (5% CO2, 37°C and > 90% humidity) for 4 hours both without and with S-9 mix and the test substance, the serum-free medium was replaced by 10 mL Ham's F12 medium with 10% FCS after having been rinsed twice with Hanks' balanced salt solution (HBSS). Subsequently, the flasks were incubated for another 17 - 24 hours (4-hour treatment) ; two flasks were pooled in each case and then subcultured (1 st passage). After two further passages (duration of the expression period is about 1 week), cells were transferred into selection medium (TG medium) at the 4th passage.

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED:
For the selection of the mutants, 6 x 300,000 cells from each treatment group were seeded in six 75-cm2 flasks with selection medium (TG medium) at the end of the expression period and the flasks then returned to the incubator for about 1 week . At the end of the selection period, colonies were fixed with methanol, stained with Giemsa and counted.

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency 1 (survival): Parallel to the seeding of test cultures for the mutagenicity test, approx. 200 cells per dose group were taken in duplicate and seeded into Ham's F12 medium with 10% FCS (25 cm2 flasks): Approx. 24 hours after seeding and incubation, the cells,were treated with the test substance and then incubated for a further 4 hours, after which time the cells were washed twice with HBSS and the serum-free medium was replaced by 5 ml Ham's F12 medium with 10% FCS. The flasks were returned to the incubator for 1 week and afterwards colonies were fixed, stained and counted.
- Cloning efficiency 2 (viability): The cloning efficiencies were determined in parallel to the selection of mutants. For each treatment group approx. 200 cells of each dose (cells pooled from 2 flasks) were taken in duplicate, seeded into Ham's F12 medium with 10% FCS (25-cm2 flasks) and allowed to form colonies (incubator; 1 week). Afterwards, the colonies were fixed, stained and counted.

OTHER EXAMINATIONS:
pH values and osmolality were measured. The solubility of the test substance in the vehicle used and in the aqueous culture medium about 3 hours (both without and with S-9 mix) after treatment was checked to ensure proper culturing and to avoid extreme treatment conditions.

OTHER:
The experimental doses were determined from appropriate pretest with cultures exposed for the duration of 4 hours to a wide dose range of the test article, i.e. 1 µg/mL - 5,000 µg/mL culture medium both without S-9 mix and after adding a metabolizing system. Besides the "cloning efficiency" (range-finding cytotoxicity test), various parameters were checked for all or at least for some selected doses. Test substance precipitation in the culture medium was not observed. On the basis of the findings from the pretest, the concentrations for the main experiment were selected.
Evaluation criteria:
• Increases of the corrected mutation frequencies above the concurrent negative control values and above 15 mutants per 1E6 clonable cells and/or the evidence of a dose-response relationship in the increase in mutant frequencies .
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above 15 mutants per 1E6 clonable cells or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
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:
MAIN EXPERIMENT
The test substance did not cause any increase in the mutant frequencies either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative controls (untreated and vehicle controls) gave mutant frequencies within the range expected for the CHO cell line.
- Both of the positive control chemicals1, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without S-9 mix there was a slight decrease in the number of colonies at 800 µg/mL and a slightly reduced cell density.
With S-9 mix there was a decrease in the number of colonies from about 400 µg/mL onward but only in the 2nd experiment using a S-9 Mix preparation containing 1 volume of S-9 fraction and 9 volumes of cofactors .
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance was not clastogenic and did not cause gene mutations in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
A mouse bone marrow micronucleus test (in duplicate) was performed to evaluated the genetic toxicity of the test item in mice. Mice were injected intraperitoneally three times at 24-hour intervals with isobutyraldehyde dissolved in corn oil; the total dosing volume was 0.4 mL.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on exposure:
Available LD50 information was used to determine the doses to be tested. Male B6C3F1 mice were injected intraperitoneally three times at 24-hour intervals with isobutyraldehyde dissolved in corn oil; the total dosing volume was 0.4 mL. Solvent control animals were injected with 0.4 mL of corn oil only. The positive control animals received injections of cyclophosphamide.
Duration of treatment / exposure:
3 times at 24-hours interval
Frequency of treatment:
3 times
Post exposure period:
no data
Dose / conc.:
39 mg/kg bw/day
Dose / conc.:
78 mg/kg bw/day
Dose / conc.:
156 mg/kg bw/day
Dose / conc.:
312.5 mg/kg bw/day
Dose / conc.:
625 mg/kg bw/day
Dose / conc.:
1 250 mg/kg bw/day
No. of animals per sex per dose:
2 x 5
Control animals:
yes
Positive control(s):
Cyclophosphamide 25 mg/kg
Tissues and cell types examined:
Bone Marrow Polychromatic Erythrocytes.
Details of tissue and slide preparation:
The animals were killed 24 hours after the third injection, and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained; 2,000 polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in each of up to five animals per dose group.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposure group is less than or equal to 0.025 divided by the number of dose groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials. Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group (Margolin et al., 1990). In the presence of excess binominal variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Study 1       

   Dose (mg/kg)  Micronucleated PCEs/1000 PCEs  No. of animals
 Corn oil    1.3 +/- 0.3  5
 Cyclophosphamide  25  3.0 +/- 0.4  5
 Isobutyraldehyde  39.06  1.7 +/- 0.6  5
   78.13  0.7 +/- 0.2  5
   156.25  0.8 +/- 0.4  5
   312.5  1.4 +/- 0.4  5
   652  1.0 +/- 0.3  5
   1250  1.7 +/- 0.5  4
     p = 0.157  

Study 2

   Dose (mg/kg)  Micronucleated PCEs/1000 PCes  No. of animals
 Corn oil    1.0 +/- 0.4  5
 Cyclophosphamide  25  7.5 +/- 1.2  5
 Isobutyraldehyde  156.25  2.3 +/- 0.5  5
   312.5  1.8 +/- 0.6  5
   652  1.8 +/- 0.8  5
   1250  lethal  5
     p = 0.269  

P = Significance of micronucleated PCEs/1,000 PCEs tested by the one-tailed trend test; significant at P < 0.025 (Margolin et al., 1986)

Endpoint:
in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The test substance was assayed in the SLRL test by feeding for 3 days to adult Canton-S wild-type males no more than 24 hours old at the beginning of treatment. Because the response was negative, isobutyraldehyde was retested by injection into adult males.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Canton-S
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 24- to 72-hour old
Route of administration:
other: feed and injection
Vehicle:
An aqueous solution of isobutyraldehyde dissolved in saline was used.
Details on exposure:
Injection was performed either manually, by attaching a rubber bulb to the other end of the pipette and forcing through sufficient solution (0.2 to 0.3 μL) to slightly distend the abdomen of the fly, or by attaching the pipette to a micro injector that automatically delivered a calibrated volume. Flies were anesthetized with ether and immobilized on a strip of tape. Injection into the thorax, under the wing, was performed with the aid of a dissecting microscope.

Oral exposure was achieved by allowing Canton-S males to feed for 72 hours on a solution of isobutyraldehyde in 5% sucrose .
Duration of treatment / exposure:
single dose
Post exposure period:
24 hr
Remarks:
Doses / Concentrations:
50,000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
80,000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
6100 - 7700
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
Treated males were mated to three Basc females for 3 days and given fresh females at 2-day intervals to produce three matings of 3, 2, and 2 days (in each case, sample sperm from successive matings were treated at successively earlier post-meiotic stages). F1 heterozygous females were mated with their siblings and then placed in individual vials. F1 daughters from the same parental male were kept together to identify clusters. (A cluster occurs when a number of mutants from a given male result from a single spontaneous premeiotic mutation event, and is identified when the number of mutants from that male exceeds the number predicted by a Poisson distribution.) If a cluster was identified, all data from the male in question were discarded. Presumptive lethal mutations were identified as vials containing fewer than 5% of the expected number of wild-type males after 17 days; these were retested to confirm the response.
Evaluation criteria:
A test result was considered positive if the P value was less than or equal to 0.01 and the mutation frequency in the tested group was greater than 0.10 % or if the P value was less than or equal to 0.05 and the frequency in the treatment group was greater than 0.15%. A test was considered to be inconclusive if the P value was between 0.05 and 0.01 but the frequency in the treatment group was between 0.10 % and 0.15 % or the P value was between 0.10 and 0.05 but the frequency in the treatment group was greater than 0.10 %. A test was considered negative if P was less than or equal to 0.10 or if the frequency in the treatment group was less than 0.10 %.
Statistics:
SLRL data were analyzed by simultaneous comparison with the concurrent and historical controls, using a normal approximation to the binomial test.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
Injection: The percentage lethals was 0.13 (8/6165) in the treatment group compared to 0.08 (5/6145) in the control group.
Feed: The percentage lethals was 0.06 (4/6250) in the treatment group compared to 0.12 (9/7666) in the control group.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet Assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted as part of the JaCVAM validation of the comet assay
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Principles of method if other than guideline:
All test chemicals were provided by JaCVAM and were coded at the time of the studies. Chemical identities were revealed only after all data were submitted to the JaCVAM validation management team.
GLP compliance:
not specified
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 7 weeks
- Weight at study initiation: 189 - 264g
- Assigned to test groups randomly: yes, based on weight
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): app. 22°C
- Humidity (%): app. 50%
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Dosing amount: 10mL/kg
Frequency of treatment:
treatments at 0, 24, 45h
Post exposure period:
3h
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Positive control(s):
ethylmethanesulphonate
- Route of administration: oral, gavage
- treatment times: 24 and 45h after beginning of the study
- Doses / concentrations: 200mg/kg
- Justification for selection: based on effects observed in previous studies
Tissues and cell types examined:
Liver
Glandular stomach
Details of tissue and slide preparation:
Liver:
A50–100mg piece of liver was excised from the left lateral lobe within minutes of death of the animal (previous experiments in our lab with liver showed that delays greater than 10 min result in increased baseline % tail intensity) and was briefly rinsed in ice-cold mincing buffer (calcium- and magnesium-free Hank’s Balanced Salt Solution, HBSS [Gibco, Grand Island, NY], with 20mM disodium EDTA [Sigma–Aldrich, St. Louis, MO, USA], pH 7.5) containing 10% dimethylsulfoxide (DMSO; Sigma–Aldrich). The tissue was then finely minced in fresh cold buffer using small dissection scissors.

Glandular stomach:
The stomach was removed, cut open along the greater curvature and rinsed in ice-cold mincing buffer to remove food and debris. The forestomach was then removed and discarded, and the remaining tissue then gently stretched and pinned flat, with the mucosal surface up, to a cold rubber mat in a Petri dish on ice. The tissue was then submerged in cold mincing buffer and incubated on ice for approximately 20 min. The mucosal surface was then gently scraped with a scalpel to remove debris and surface layers of dead or sloughing cells. The mincing buffer was replaced and the surface then scraped more extensively to obtain cells and nuclei for the comet assay from deeper layers of the tissue. The resulting suspension was pipetted forcefully several times against the dish surface to further disrupt clumps.

Comet slides:
Each suspension was allowed to settle for about 30–60 s and 30µL of supernatant then mixed with 300µL 0.5% molten agarose (approximately 37°C; NuSieve GTG low gel temperature agarose). An additional sample of each suspension was quickly frozen on dry ice and stored at approximately−70 ◦C for additional comet assays, if needed. Thewarmcell/agarose mixture was spread on pre-coated microscope slides (coated with 1% normal gel temperature agarose), cover slips were added, and slides were then placed on a metal tray on ice to solidify for 3–10 min. Cover slips were then removed and the slides immediately placed in ice-cold lysing solution (100mM disodium EDTA, 2.5M sodium chloride, 10mM Tris hydroxymethyl aminomethane, pH10.0 with 1% Triton-X 100, by volume, and 10% DMSO, by volume, added on the day of use) and held at 4–10 ◦C overnight, protected from light.
Lysed slides were rinsed briefly in water before submerging in alkaline unwinding solution (300mM sodium hydroxide) on the platform of an electrophoresis box connected to a circulating refrigerated water bath. Slides were distributed across the platform and across electrophoresis runs in a balanced fashion with respect to dose group and animal. After 20 min for unwinding, slides were electrophoresed for 20 mins at 26V (approximately 0.7 V/cm, 300 mA). Temperature was maintained at 6–8 ◦C for unwinding and electrophoresis. Slides were then neutralized, dehydrated in ethanol, air-dried and coded for scoring.
Fifty comets from different areas of each of two slides per tissue sample were scored. Comets that were mis-shaped, overlapped, or debris-covered were not scored. Comets exhibiting very high levels of DNA damage (“hedgehogs”, small or non-existent heads and large diffuse tails) were not scored but the percentage of hedgehogs among scorable comets was determined.
Evaluation criteria:
Positive result:
Statistically significant increase in the % DNA in tail for at least one dose level in comparison with the concurrent negative control (p-value <= 0.03, adjusted based on in-house validation results for this assay)
Effects should be dose related and should exceed historical vehicla control group values for that tissue.
Effects should be observed at a dose not associated with evidence of acute toxic effects as assessed by examiniation of the percentage of cells with very low molecular weight DNA (hedgehogs) histomorphology of the target tissue, serum liver enzyme levels, and / or other measured toxicity endpoints.

Acceptance criteria:
Vehicle controls should not exceed the typical historical control range
(liver: 0.337 +/- 0.449, 24 studies with a low of 0.009 and a high of 1.86)
(stomach: 6.3 +/- 2.08, 11 studies with a low of 4.07 and a high of 11.3)
Positive EMS control in the range of 13.2 +/- 2.3 for liever and 29.0 +/- 3.9 for stomach (4 studies)
Statistics:
one-way ANOVA
Threshold for a positive response was set at p<= 0.03
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
decreased activity 2h post dosing, slight, but significant decrease in body weight change (high dose)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Histopathology of liver and stomach mucosa was unremarkable.
Formation of micronuclei in the bone marrow was also evaluated in the same animals. There was no increase after treatment with isobutyraldehyde.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Many studies exist that investigate the genetic toxicity of isobutyraldehyde. However, several of these lack essential documentation (Val. 4), follow unsuitable protocols or use severely flawed methodologies (Val. 3). Below a summary of the reliable (klimisch 1 and 2) studies is given.


 


Genetic toxicity in vitro


Ames


In an Ames test performed by BASF in 1999 according to OECD 471 the mutagenic potential of the test substance was evaluated in the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation (rat liver S-9). Concentrations from 20 ug to 5,000 ug/plate were tested in triplicate in both, the standard plate test and the preincubation test. A slight decrease in the number of revertants was occasionally observed. An increase in the number of his+ or trp+ revertants did not occur in any experiment. No precipitation of the test substance was found. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.


 


NTP (1999) describes an Ames preincubation test with strains Salmonella typhimurium TA97, TA98, TA100, TA102, TA1535, TA1537, and TA104 with and without metabolic activation (Aroclor induced rats and hamster S-9 mix (5, 10 or 30%)). Bacteria were exposed to levels between 0 and 10,000 µg/plate. Vehicle and positive controls were considered reliable. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions chosen here.


 


Aeschbacher et al., (1988) performed an Ames preincubation test with strains TA98, TA100, TA102 with and without addition of Aroclor-induced rat liver S-9 as metabolic activation system. Solvent and positive controls were tested in parallel. Exposure to 0.08 - 7920 ug/plate (1.1 nmol - 0.11 mmol per plate) test substance with and without metabolic activation did not result in a mutation factor of at least 1.5. Under these conditions, isobutyraldehyde does not cause gene mutations in bacteria.


 


Dillon et al. (1998) tested mutagenicity using S. typhimurium strains TA100, TA102, and TA104 in a preincubation experiment. The exact concentrations were determined in a preliminary dose range finding test in order to perform the main tests up to cytotoxic concentrations. The strains were exposed to 50 - 5000 µg/plate with metabolic activation (Aroclor induced mouse and rat liver S9 mix) and without metabolic activation. Positive controls and vehicle controls were performed. Under the test conditions, isobutyraldehyde was negative with and without metabolic activation.


 


HPRT


An in vitro mammalian cell gene mutation assay in Chinese Hamster Ovary cells (HPRT locus) was performed according to OECD 476 and in compliance with GLP (BASF 1999). In the first experiment the cells were exposed to 0; 50; 100; 200; 400; 800 µg/mL with or without S-9 mix (S-9 fraction : cofactors = 3 : 7). In the second experiment the test concentration were 0; 200; 400; 600; 800 µg/mL without S-9 mix, 0; 100; 200; 400; 800 µg/mL with S-9 mix (S-9 fraction : cofactors = 1 : 9) and 0; 100; 200; 400; 800 µg/mL with S-9 mix (S-9 fraction : cofactors = 3 : 7). Solvent, negative and positive controls were performed in parallel. The test substance did not cause any increase in the mutant frequencies either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. Under these conditions, the test substance was not mutagenic in mammalian cells.


 


Chromosome aberration


An in vitro chromosome aberration assay in V79 cells (BASF 1999) was performed according to OECD guideline 473. The cells were exposed to 250, 500, 750µg/mL (experiment I) and 600, 700, 800µg/mL (experiment II) with and without metabolic activation (rat liver S-9 mix). The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. The test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps without S-9 mix in two experiments performed independently of each other. Clear and dose dependent cytotoxicity was observed (cell count 49 – 71% compared to the control) at all concentrations tested in the second experiment (600 – 800µg/mL). At the one positive concentration of 750µg/mL in the first experiment, cytotoxicity was less pronounced, even though the dose was in the same range. No increase in the frequency of cells containing numerical aberrations was demonstrated. Also, no positive response was obtained after addition of S9-mix. Thus, under the experimental conditions of this assay, the test substance was clastogenic in the absence of a metabolic activation system.


 


NTP (1999) describes a chromosomal aberration test performed with and without metabolic activation. Chinese hamster ovary cells were exposed to 16, 50, 160, 500, 1600, 3000, 4000 µg/mL test substance (-S9 mix and +S9 mix). In the second experiment concentrations of 500, 1000, 1500, 2000 µg/mL (-S9 mix) and 100, 250, 500, 750, 1000, 1500, 2000 µg/mL (+S9 mix) were used. Solvent and positive controls were tested in parallel. Results were positive only in the absence of S9-mix, while a negative result was obtained after the addition of S9-mix. No data on cytotoxicity has been provided.


 


Allemang et al. (2020) performed an in vitro mammalian cell micronucleus test in TK6 cells without metabolic activation. The cells were exposed to 0, 625, 1250, 2500, 5000, and 10000 µM test substance in triplicate. There was a significant dose-dependent increase in micronclei starting from 2500 µM. The top dose resulted in a relative survial of 59%. Hence, under the conditions of the test, the substance was genotoxic without metabolic activation.


 


Thougaard et al. (2014) performed an in vitro mammalian cell micronucleus test in TK6 cells with and without metabolic activation. The cells were exposed to 0, 391, 444, 587, 667, 880, 1000 µM test substance in duplicate. Solvent and positive controls were performed in parallel. Under the condition of the test, the substance was not genotoxic.


 


Westerink et al., 2011 performed an in vitro micronucleus assay with the rodent cell line CHO-k1 and human hepatoma cell line HepG2. The aim of the study was the development of a method to discriminate aneugens from clastogens based on size-classification of the micronuclei. The cells were exposed to a serial dilution with a maximum concentration of 1mM with and without metabolic activation. Solvent and positive controls were performed. The test was performed in duplicate on two different 96-well plates. All experiments were repeated at least twice, independently. Under the conditions of the test the substance was not genotoxic.


 


 


Mouse Lymphoma


NTP (1999) describes a mouse lymphoma (L5178Y) mutagenicity test performed without metabolic activation. In the first experiment 62.5, 125, 250, 500, 1000, and 1500 µg/mL test substance was tested. Based on the cytotoxicity observed in the first experiment 62.5, 125, 250, 500, and 750 µg/mL were used in the second experiment. All experiments were performed in triplicate, including solvent and positive controls. In the first experiment: 125, 250, 500 and 1000 µg/mL resulted in significant positive responses (P0.05) and in the second experiment: 125, 250, and 500 µg/mL resulted in significant positive responses (P0.05). 750, 1000 (one out of 3 plates) and 1500 µg/mL were completely lethal to the cells. Reductions in relative total growth (41 – 73%) and cloning efficiency (42 – 77%) were already observed at 125µg/mL and decreased further dose-dependently. There is no information on colony size, so no conclusion can be drawn, if this increase is due to gene mutations or chromosomal aberrations. Considering the results from the other assays (positive results in vitro were only observed in assays for chromosomal aberrations, but not in tests for gene mutations) it seems likely that this response was caused by a clastogenic mechanism.


 


Other information


NTP (1999) describes a Sister Chromatid Exchange test performed with and without metabolic activation. Chinese hamster ovary cells were exposed to 5, 16, 50, 160, 500 µg/mL (-S9 mix) and 16, 50, 160, 500, 1600 µg/mL (+S9 mix) of the test substance in the first experiment. In the second experiment concentration of 10, 25, 50, 160, 250, 500 µg/mL (-S9 mix) and 500, 750, 1000, 1250 µg/mL (+S9 mix) were used. Solvent and positive controls were tested in parallel. The test substance induced a strong, dose-related increase in SCEs, with and without S9. In the absence of S9, positive responses were noted with test substance concentrations of 5 to 500μg/mL; cell cycle delay occurred at the 250 and 500μg/mL without S9, and culture times were extended accordingly. With S9, doses of 160 to 1,250μg/mL produced significant increases in SCEs; no cell cycle delay was noted at any of the doses tested in the presence of S9. No information on cytotoxicity is available.


 


Kerckaert et al. (1996) performed a Syrian Hamster Embryo Cell Transformation Assay. At least two trials were performed. Cells were exposed to 0, 200, 300, 400, 575, and 750 µg/mL (exposure duration 24 hours) and 0, 200, 375, 550, 725, and 900 µg/mL (exposure duration 7 days). Solvent controls and positive controls were performed in parallel. No significant differences in morphological transformation frequencies were observed after exposure for 24 hours and 7 days compared to the control.


 


Matthews et al. (1993) performed an in vitro mammalian cell transformation assay in mouse Balb/c-3T3 cells. The cells were exposed to 0.964, 1.93, 2.89, 3.85 mM for 48 hours in two trials. The doses covered a range of cytotoxic responses of approximately 10-100 % relative cloning efficiency. Positive and solvent controls were performed in parallel. In both trials which had relatively high sensitivities to detect chemical-induced transformation, the test substance had a limited activity was evaluated as inactive in this assay.


 


Duerkensen-Hughes et al. (1999) used a mammalian in vitro assay for genotoxicity based on the ability of cells to increase their level of the tumor suppressor protein p53 in response to DNA damage. An NCTC 929 mouse fibroblast cell line was exposed for 6 and 17 hours to several doses of the test substance between 1 and 100 µg/L. Vehicle and positive controls were tested in parallel. The cells were lysed for ELISA analysis and each point was measured in triplicate. No significant increase in p53 protein levels was observed after 6 and 17 hours exposure.


 


Van der Linden et al., 2014 validated two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. The human U2OS cell line was exposed to a serial dilutions with and without metabolic activation. 1.0E-3 M was the maximum test concentration. After the addition of the compounds, the plates were incubated for 24 h. In the case of exposure in combination with S9, plates were incubated for three hours. After removal of the test substance and S9 mix the cells were incubated for another 16 h. The induction of the p53 CALUX and Nrf2 CALUX assays were under the cut-off value of 1.5 and 2.0-fold induction, respectively. Under the condition of the test, the substance was negative for genotoxicity and oxidative stress.


 


Hughes et al. (2012) developed and validated a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens in TK6 cells. The results were compared with the results of GADD45a linked to GFP expression in TK6 cells. Both assays were exposed to a maximum concentration of 721 µg/mL (10mM) with and without metabolic activation. Positive, solvent and background controls were performed in parallel. Under the conditions of this test with and without metabolic activation the test substance did not induce the expression of the GADD45a gene above the threshold of 1.8 (-S9 mix) or 1.5 (+S9-mix) in the TK6 cells.


 


Khoury et al. (2013) used HepG2 cells for the quantification of the phosphorylation of the histone H2AX (gamma-H2AX). The phosphorylation of the histone H2AX (named gamma-H2AX) occurs after a DNA double strand break in a cell and reflects a global genotoxic insult that may originate from different types of DNA damage: DNA adducts, DNA single-strand breaks, DNA replication or transcription blocking lesions. The highest test concentration was 1 mM (app. 7.2µg/ml) and tests were performed in duplicate. Positive and solvent controls were tested. No phosphorylation of the histone H2AX was observed after exposure to a relatively low concentration of isobutyraldehyde.


 


Becker et al., 1998 exposed isolated supercoiled DNA from the phage PM2 to the isomers n- and isobutyraldehyde alone or in combination with Cu(II) to determine single and double strand breaks and 8 -OHdG formation. For the determination of strand breaks cells were exposed to 25 or 0.5 mM butanal or n-butanal with or without CuCl2 (0.1mM). For the 8 -OHdG formation assay, the test concentration were 0.6, 6, 30 mM iso-butanal or n-butanal with CuCl2 (1mM) and 30mM without CuCl2. The supercoiled DNA was exposed for 10 min, 1 hour, and 3 hours and the formation of (8-OHdG) was determined by HPLC-ECD. Both isomers iso-butanal and n-butanal induced DNA single and double strand breaks in combination with CuCl. Deoxyguanosine was hydroxylated by iso-BuA/CuCl2 in a concentration and time dependent manner, whereas n-BuA/CuCl2 produced only traces of 8-OHdG under the same conditions. Based on these results it is likely that the oxidation of iso-BuA mainly results in damage by OH-radicals.


 


Genetic toxicity in vivo


A mouse bone marrow micronucleus test (in duplicate) was performed to evaluated the genetic toxicity of the test item in male B6C3F1 mice (NTP 1999). Mice were injected intraperitoneally three times at 24-hour intervals with the test substance dissolved in corn oil at doses of 39, 78, 156, 312.5, 625, 1250 mg/kg/day; the total dosing volume was 0.4 mL. 2000 polychromatic erythrocytes (PCEs) were scored for the frequency of micro-nucleated cells in each of up to five animals per dose group. There was no significant difference between control and treated animals. The same bone marrow micronucleus test was performed with male Fischer 344 rats using the same methods as above (NTP 1999). Again, there was no significant difference found in occurrence in micro-nucleated PCEs. In conclusion, isobutyraldehyde was non-genotoxic in mice or rats in these assays.


 


The test substance was also evaluated for potential genotoxicity in a bone marrow chromosomal aberration test (performed in duplicate) (NTP 1999). Male B6C3F mice (10 animals per exposure group) were injected intraperitoneally with the test substance dissolved in corn oil (500, 1000, 1500, 2000 mg/kg (experiment 1); 1000, 1200, 1500, 1750 mg/kg (experiment 2)). Solvent control mice received equivalent injections of corn oil only. The animals were killed 17 hours later. The mice were subcutaneously implanted with a BrdU tablet 18 hours before the scheduled harvest. Responses were evaluated as the percentage of aberrant metaphase cells, excluding gaps. Significant increases in the frequency of aberrant cells were seen only at doses that produced notable clinical signs of toxicity, i.e., 1500 and 1750mg/kg. No details on clinical signs or mortality rates were provided, but the MTD was likely exceeded in these dose groups. 2000mg/kg was lethal in this study, 3 injections of 1250mg/kg were lethal in a simultaneously performed study by NTP. In conclusion, there was no increase in the number of aberrant cells in the absence of significant systemic toxicity. The genotoxic potential is evaluated as negative.


 


In the context of a validation exercise for the comet assay initiated by JaCVAM, Kraynak et al. treated 5 male rats per group with 500, 1000, and 2000mg/kg b.w. Animals were gavaged 0, 24, and 45h after the beginning of the study, and euthanized 3h after the last treatment. Slight toxicity was observed in high dose animals in the form of hypoactivity 2h post-dosing and slightly, but significantly reduced body weight change. Histopathology of the examined tissues (liver and glandular stomach) was unremarkable. There was no increase in the percentage of DNA in the tail in both tissues compared to vehicle control (corn oil treated) animals. Treatment with EMS yielded the expected increase in %DNA in tail. In the same study, the authors also examined formation of micronuclei in erythrocytes from the bone marrow. All results from the treatment groups were within historical control values.


 


In addition, the test substance was assayed in the drosophila SLRL test by feeding the test substance for 3 days to adult Canton-S wild-type males (NTP 1999). Drosophila were either exposed to 80,000 ppm test substance via feed or to 50,000 ppm via injection. When the test substance was administered via feed, the percentage of lethals was 0.06 (4/6250) in the treatment group compared to 0.12 (9/7666) in the control group. After injection, the percentage lethals was determined as 0.13 (8/6165) in the treated group compared to 0.08 (5/6145) in the control group. Therefore, the test substance was considered non-genotoxic under the conditions chosen in this study.

Justification for classification or non-classification

The substance is not mutagenic in bacteria or mammalian cells, but induces chromosome aberrations in the absence of a metabolic activating system in vitro. In vivo, three bone marrow micronucleus tests, one comet assay, and a drosophila SLRL assay for gene mutations produced negative results. An additional bone marrow chromosome aberration test was only positive at significantly toxic concentrations. Overall, the test substance does not cause gene mutations in vitro and in vivo and is not clastogenic in vivo. This conclusion is also supported by two negative carcinogenicity studies in rats and mice.

Consequently, no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.