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EC number: 202-860-4 | CAS number: 100-52-7
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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Benzaldehyde was tested in contemporary OECD TG 487 (2016) and TG 490 (2016) compliant studies. An OECD 487 (2016) in vitro micronucleus study in human lymphocytes was conducted up to toxic concentrations for 3 hours in the absence and presence of rat liver metabolic activation system (S9) and for 24 hours in the absence of S9. Under the experimental conditions, it was concluded that benzaldehyde did not induce biologically relevant increases in the frequency of micronuclei.
The OECD 490 (2016) in vitro mammalian cell gene mutation test was conducted up to precipitating concentrations. Under the experimental conditions, benzaldehyde did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence or presence of a rat liver metabolic activation system (S9).
A number of historical ames studies published in the scientific literature all resulted in a negative response both with and without metabolic activation.
With the completion of contemporary OECD TG 487 and TG 490 studies, the overall Weight of Evidence supports the classification of benzaldehyde as "not genotoxic". The whole of the literature points to benzaldehyde as not genotoxic, with only a small number of studies concluding "positive" results. In particular the following citations however these studies are not compliant with current OECD test guidelines with methodological deviations identified.
Citation |
Test System |
Conclusion |
Kasamaki et al (1982) |
Chinese hamster cell line CH-B-241 |
Equivocal |
Sofuni et al (1985) |
Chinese hamster cells |
Positive in the absence of S9 |
Jansson et al (1988) |
Primary lymphocytes obtained from healthy non-smoking donors |
Positive |
Heck et al (1989) |
Tk +/- 3.7.2C heterozygote of L5178Y mouse lymphoma cell line |
Equivocal |
McGregor et al (1991) |
Tk +/- 3.7.2C heterozygote of L5178Y mouse lymphoma cell line |
Positive without S9, increases in mutant fraction were "very close to highly toxic doses" |
Several in vitro and in vivo genotoxicity tests with the test substance are available. The NTP report on the test substance (NTP, 1990) concluded that benzaldehyde was not mutagenic in six strains of S. typhimurium and did not induce chromosomal aberrations in CHO cells, with or without exogenous metabolic activation. Benzaldehyde induced increases in trifluorothymidine-resistant mouse lymphoma cells in the absence of exogenous metabolic activation (McGregor et al, 1991) and increased sister chromatid exchanges in CHO cells in both the presence and absence of metabolic activation. Sex-linked recessive lethal mutations were not induced in the germ cells of adult male D. melanogaster administered benzaldehyde by feeding or by injection. However taking into consideration the key studies from the contemporary OECD 487 and 490 compliant GLP studies with the historical studies (with some methodological short comings), it is concluded that benzaldehyde is considered not to be genotoxic.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 October 2018 - 17 December 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical BV (The Netherlands); Lot No. 1803-1
- Expiration date of the lot/batch: 2020-01-15
- Purity test date: 2018-09-18
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C, protected from light under nitrogen
- Stability under test conditions: no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparations of test article were employed
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock solutions were prepared by formulating Kalama® Benzaldehyde FCC grade under subdued lighting in DMSO, with the aid of vortex mixing, to give
the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4 hours of initial formulation.
FORM AS APPLIED IN THE TEST (if different from that of starting material) : colourless liquid - Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Blood from two healthy, non-smoking Female volunteers from a panel of donors at Covance was used for each experiment.
- Suitability of cells: The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype (Evans & O’Riordan, 1975).
- Cell cycle length, doubling time or proliferation index: Cell cycle length: 13 ± 2 hour
- Sex, age and number of blood donors if applicable: Blood from two healthy, non-smoking Female volunteers (Range-Finder: 34, 30 years; Micronucleus Experiment: 23, 34 Years)
- Whether whole blood or separated lymphocytes were used if applicable: human lymphocyte cultures prepared from the pooled blood of two female donors - Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254.
- Test concentrations with justification for top dose:
- Cytotoxicity Range-Finder Experiment:
3+21 Hour Treatment (-S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
3+21 Hour Treatment (+S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
24+24 Hour Treatments (-S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
A maximum concentration of 1062 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a maximum concentration equivalent to 10 mM (a suitable maximum concentration for in vitro genetic toxicology assays of this type), based on the test article molecular weight of 106.121. No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1062 µg/mL), compared to the concurrent vehicle controls. The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Micronucleus Experiment.
Micronucleus Experiment:
3+21 Hour Treatments (-S9): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 950, 1062 µg/mL
3+21 Hour Treatments (+S9): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 950, 1062 µg/mL
24+24 Hour Treatments (-S9): 50, 100, 150, 200, 250, 300, 350, 400, 450, 600 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Vinblastine (VIN): Treatment Regime: -S9: 24+24 (concentration: 0.04 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: approximately 48 hours
- Exposure duration: 3 (±S9) or 24 hours (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): -S9 (3 hour treatment): 72 hours; -S9 (24 hour treatment): 96 hours; + S9 (3 hour treatment): 72 hours
SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (Cyto-B (formulated in DMSO) added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.
STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline (PBS), pH 6.8
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.
NUMBER OF CELLS EVALUATED: A minimum ofone thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. For the 24 hour treatment in the absence of S9, an additional 1000 binucleate cells from each culture (therefore 4000 per concentration) were analysed from the test article concentrations selected for analysis.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Relative index (RI)
- Any supplementary information relevant to cytotoxicity: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:
RI = (number binucleate cells + 2 (number multinucleate cells) / total number of cells in treated cultures)
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures / RI of vehicle controls) x 100
Cytotoxicity (%) is expressed as (100 – Relative RI)
A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred. - Rationale for test conditions:
- Please see 'Any other information on materials and methods incl. tables' for information on Rationale for Test Conditions.
- Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations (Thybaud et al., 2007). - Statistics:
- After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p ≤0.05 were accepted as significant. - Key result
- Species / strain:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1062 µg/mL), compared to the concurrent vehicle controls (individual data not reported).
The proportion of micronucleated binucleate (MNBN) cells in negative (vehicle) control cultures fell within (or very close to) the 95th percentile of the current observed historical vehicle control (normal) ranges. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.
All acceptance criteria were considered met and the study was accepted as valid.
Validity of Study
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the 3+21 hour and 24+24 hour treatments in the absence of S9. Statistically significant heterogeneity (p≤0.01) was observed for the 3+21 hour treatment in the presence of S9, primarily due to large differences in MNBN cell frequencies between replicates for the vehicle control and the lowest test article concentration analysed (400 µg/mL). However, none of the MNBN cell frequency values exceeded the normal range under this treatment condition, therefore the observed heterogeneity did not affect the interpretation of the data
2. The frequency of MNBN cells in vehicle controls fell within the normal ranges with the exception of two of the four vehicle control cultures analysed for the 24+24 hour treatment in the absence of S9, one of which fell within the observed range. However, the mean vehicle control MNBN cell frequency was within the normal range and the data were considered acceptable.
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range.
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.
5. The maximum concentration analysed under each treatment condition met the criteria specified. - Conclusions:
- The test material (Kalama® Benzaldehyde FCC grade) did not induce biologically relevant increases in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described.
- Executive summary:
In a key OECD Guideline 487 study, the test material (Kalama® Benzaldehyde FCC grade) was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254-induced rats.
The test material was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest concentrations analysed in the Micronucleus Experiment were limited by cytotoxicity under each treatment condition and were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test material concentrations for micronucleus analysis were selected by evaluating the effect of Kalama® Benzaldehyde FCC grade on the replication index (RI). Micronuclei were analysed at three concentrations.
Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within (or very close to) the 95thpercentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.
All acceptance criteria were considered met and the study was accepted as valid.
Treatment of cells with the test material for 3+21 hours in the absence and presence of S9and for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were generally similar to and not significantly different (at the p≤0.05 level), compared to those observed in the concurrent vehicle controls, at any concentration analysed under each treatment condition. The MNBN cell frequencies fell within the normal ranges at all concentrations analysed with the exception of one culture at the highest concentration analysed following the 24+24 hour treatment in the absence of S9 (1% at 200 µg/mL, which gave 55% mean cytotoxicity). However, there were no statistically significant increases in MNBN cell frequency at any concentration analysed following the 24+24 hour treatment in the absence of S9 and no statistically significant linear trend, therefore this isolated observation was considered not biologically relevant. A statistically significant linear trend (p≤0.05) was observed following the 3+21 hour treatment in the presence of S9 but as there were no statistically significant increases in MNBN cell frequency at any concentration analysed, this observation was also considered not biologically relevant.
It was concluded that the test material (Kalama® Benzaldehyde FCC grade) did not induce biologically relevant increases in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-10-25 to 2019-12-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical BV (The Netherlands); Lot #: 1803-1
- Expiration date of the lot/batch: 2020-01-15
- Purity test date: 2018-09-18
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C, protected from light under nitrogen
- Stability under test conditions: no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparations of test article were employed
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock solutions were prepared by formulating Kalama® Benzaldehyde FCC grade under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3.5 hours of initial formulation.
FORM AS APPLIED IN THE TEST (if different from that of starting material): colourless liquid - Target gene:
- tk (thymidine kinase) locus in mouse lymphoma L5178Y cells
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance
- Suitability of cells: The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells is capable of detecting both gene mutations and chromosome aberrations.
- Methods for maintenance in cell culture if applicable: The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1°C. When the cells were growing well, subcultures were established in an appropriate number of flasks.
MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free - Metabolic activation:
- with and without
- Metabolic activation system:
- Obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- Cytotoxicity Range-Finder Experiment (±S9): 0, 33.19, 66.38, 132.8, 265.5, 531, and 1062 µg/mL
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 33.19 to 1062 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide greater than 10% relative suspension growth (RSG) was 531 µg/mL, which gave 41% and 32% RSG in the absence and presence of S9, respectively.
In the Mutation Experiment ten concentrations, ranging from 100 to 1062 µg/mL, were tested in the absence and presence of S9. Two days after treatment the highest concentration analysed to determine viability and TFT resistance was 800 µg/mL, which gave 16% and 17% relative total growth (RTG) in the absence and presence of S9, respectively.
Mutation Experiment (±S9): 0, 100, 200, 400, 500, 600, 700, and 800 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): At least 10^7 cells in a volume of 17.8 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A to give a final concentration of 5% serum) were used.
DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days:
NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments)
NUMBER OF CELLS EVALUATED:
Plating for Viability – Mutation Experiment:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^4 cells/mL in readiness for plating for TFT resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well).
Plating for TFT Resistance - Mutation Experiment:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL) was diluted approximately 100 fold into these suspensions to give a final concentration of 3 µg/mL. Using an eight-channel pipette, 0.2 mL of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x 10^3 cells/well).
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth
- Any supplementary information relevant to cytotoxicity: Relative Suspension Growth (RSG) is a measure of the growth in suspension during treatment and the expression period relative to the mean control. - Rationale for test conditions:
- Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL. The solubility limit in culture medium was in excess of 1195 µg/mL, as indicated by the absence of precipitate at this concentration 24 hours after test article addition, with warming at 37°C. A maximum concentration of 1062 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a maximum concentration equivalent to 10 mM (a suitable maximum concentration for in vitro genetic toxicology assays of this type), based on the test article molecular weight of 106.121. Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
- Evaluation criteria:
- For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was statistically significant.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis. - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on statistics
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Cytotoxicity Range-Finder Experiment:
Six concentrations were tested in the absence and presence of S9, ranging from 33.19 to 1062 µg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test material to the cultures, precipitate was observed at the highest two concentrations tested in the absence and presence of S9 (531 and 1062 µg/mL) but no post-treatment precipitate was observed under either treatment condition. The highest concentration to provide greater than 10% RSG was 531 µg/mL, which gave 41% and 32% RSG in the absence and presence of S9, respectively (Table 1).
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1062 µg/mL), compared to the concurrent vehicle controls.
Mutation Experiment:
Ten concentrations, ranging from 100 to 1062 µg/mL, were tested in the absence and presence of S9. Upon addition of the test article to the cultures, precipitate was observed at the highest five concentrations tested in the absence and presence of S9 (700 to 1062 µg/mL) but no post-treatment precipitate was observed under either treatment condition. Two days after treatment the highest three concentrations tested in the absence of S9 (900 to 1062 µg/mL) and the highest concentration tested in the presence of S9 (1062 µg/mL) were considered too toxic for selection to determine viability and TFT resistance. All other concentrations in the absence and presence of S9 were selected. However, the highest two concentrations selected in the presence of S9 (900 and 1000 µg/mL) were later rejected from analysis due to extreme toxicity (<10% RTG). The highest concentration analysed was 800 µg/mL, which gave 16% and 17% RTG in the absence and presence of S9, respectively (Table 2).
The acceptance criteria were met, with one exception. The mean vehicle control MF value in the presence of S9 was 46.53 mutants per 106 viable cells, which fell slightly below the acceptable range of 50 to 170 mutants per 106 viable cells. However, there were clearly no notable increases in MF (above the GEF) in any treated culture in the presence of S9 and the positive control chemical B[a]P showed clear induction of mutation, therefore the data were considered acceptable and valid.
When tested up to toxic concentrations in the Mutation Experiment, the MF values of the concentrations plated were all less than the sum of the mean control MF plus the GEF, indicating a negative result. A statistically significant linear trend was observed in the presence of S9, but in the absence of any increases in MF which exceeded the GEF in any treated culture under this treatment condition, this observation was considered not to be biologically relevant.
In addition, for the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the vehicle controls, the proportion of small colony mutants in the absence and presence of S9 ranged from 38% to 42%. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P. - Conclusions:
- The test material (Kalama® Benzaldehyde FCC grade) did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S9) under the experimental conditions described.
- Executive summary:
In a key OECD Guideline 490 in vitro gene mutation study, the test material (Kalama® Benzaldehyde FCC grade)was assayed for the ability to induce mutation at the tk locus (5‑trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9). The test material was formulated inanhydrous analytical grade dimethyl sulphoxide(DMSO).
A 3 hour treatment incubation period was used for each experiment.
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 33.19 to 1062 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide greater than 10% relative suspension growth (RSG) was 531 µg/mL, which gave 41% and 32% RSG in the absence and presence of S9, respectively.
In the Mutation Experiment ten concentrations, ranging from 100 to 1062 µg/mL, were tested in the absence and presence of S9. Two days after treatment the highest concentrationanalysed to determine viability and TFT resistance was 800 µg/mL, which gave 16% and 17% relative total growth (RTG) in the absence and presence of S9, respectively.
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S9. Mutant frequencies (MF) in vehicle control cultures fell within (or very close to) acceptable ranges. Clear increases in mutation were induced by the positive control chemicals, Methyl methane sulphonate (without S9) and Benzo[a]pyrene (with S9). Therefore the study was accepted as valid.
When tested up to toxic concentrations in the Mutation Experiment, the MF values of the concentrations plated were all less than the sum of the mean control MF plus the Global Evaluation Factor (GEF, 126 mutants per 106viable cells), indicating a negative result.A statistically significant linear trend was observed in the presence of S9, but in the absence of any increases in MF which exceeded the GEF in any treated culture under this treatment condition, this observation was considered not biologically relevant.
Based on the results observed, the study authors concluded that the test material (Kalama® Benzaldehyde FCC grade) did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S9) under the experimental conditions described.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1978 - 1998
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Regarding the requirements under Annex X, Section 8.4.1 "In vitro gene mutation study in bacteria", while there is not a single study performed exactly to guideline, there are numerous studies which collectively cover all of the strains and conditions set forth in the current OECD 471.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Dillon et al. (1998)
- Deviations:
- yes
- Remarks:
- Did not evaluate tester strains TA1535 or TA98.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Florin et al. (1980)
- Deviations:
- yes
- Remarks:
- Did not evaluate E. coli WP2 uvrA/E. coli WP2 uvrA (pKM101) or S. Typhimurium TA102
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Fujita et al. (1992)
- Deviations:
- yes
- Remarks:
- This study only evaluted TA97 and TA102.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Haworth et al. (1983)
- Deviations:
- yes
- Remarks:
- This study did not evaluate E. coli WP2 uvrA/E. coli WP2 uvrA (pKM101)/TA 102
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Heck et al. (1989)
- Deviations:
- yes
- Remarks:
- This study did not evaluate E. coli WP2 uvrA/E. coli WP2 uvrA (pKM101)/TA 102. In addition, there is no mention of positive controls.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Kasamaki et al. (1982)
- Deviations:
- yes
- Remarks:
- This study only evaluates TA 98 and TA100.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Nohmi et al. (1985)
- Deviations:
- yes
- Remarks:
- Evaluated only two common strains of S. typhumurium (TA98 and TA100), and one non-standard (TA2637).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Sasaki et al. (1978)
- Deviations:
- yes
- Remarks:
- Only strains TA98 and TA100 were evaluated. Only abstract available, with no mention of positive controls.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Yamada and Honma (2018) summarizes previous studies.
- Principles of method if other than guideline:
- - Principle of test:
- Short description of test conditions:
- Parameters analysed / observed: - GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
Dillon et al. (1998) – Obtained from Radian Corporation (Austin, TX), but original supplier listed as LaPine Scientific. Stated as “72 – 91% purity”. No expiration date mentioned.
Florin et al. (1980) – Source/purity/Lot No./Expiration Date not specified.
Fujita et al. (1992) – Wako Pure Chemicals, purity/Lot No./expiration date not specified.
Haworth et al. (1983) – Obtained from Radian Corporation (Austin, TX), but original supplier listed as LaPine Scientific. Stated as “72 – 91% purity”. Lot No. 45,519, expiration date not specified.
Heck et al. (1989) – Source/purity/Lot No./Expiration Date not specified.
Kasamaki et al. (1982) – Obtained from Nakarai Pharmaceutical Company (Japan). Purity/Lot No./Expiration Date not specified.
Nohmi et al. (1985) – Source/purity/Lot No./Expiration Date not specified.
Sasaki and Endo (1978) – Source/purity/Lot No./Expiration Date not specified. - Species / strain / cell type:
- S. typhimurium, other: TA 100, TA 102 and TA 104
- Remarks:
- Dillon et al. (1998)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Remarks:
- Florin et al. (1980)
- Species / strain / cell type:
- S. typhimurium, other: TA97, TA102
- Remarks:
- Fujita et al. (1992)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Remarks:
- Haworth et al. (1983)
- Species / strain / cell type:
- S. typhimurium, other: TA1535, TA1537, TA1538 and TA98
- Remarks:
- Heck et al. (1989)
- Species / strain / cell type:
- S. typhimurium, other: TA98 and TA100
- Remarks:
- Kasamaki et al. (1982)
- Species / strain / cell type:
- S. typhimurium, other: TA100, TA2637, TA98
- Remarks:
- Nohmi et al. (1985)
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA100
- Remarks:
- Sasaki and Endo (1978)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 was prepared from Aroclor 1254-induced male Fischer F344 rats and male B6C3F1 mice
- Test concentrations with justification for top dose:
- Dillon et al. (1998): 33 – 3333 microg/plate, top dose was limited by toxicity as determined by thinning of background lawn or a reduction in the number of colonies per plate (or both).
Florin et al. (1980) – A spot test at 3 micromol/plate was performed initially. If toxicity was observed, the definitive study was done at lower concentrations. Where the spot test did not demonstrate toxicity, the definitive test was run at 0.03, 0.3, 3.0 and 33.0 micromol/plate.
Fujita et al. (1992) – 0, 0.01, 0.05, 0.1, 0.5 and 1.0 mg/plate. No justification for dose range is given, and toxicity is not mentioned.
Haworth et al. (1983) – 0, 10.0, 33.0, 100.0, 333.0 and 1000.0 microg/plate, top dose limited by toxicity as determined in a preliminary toxicity screen.
Heck et al. (1989) – 37,500 nl/plate (corresponds to 3,900 microg/plate), toxicity was not pre-screened, but toxicity during main study was taken into consideration.
Kasamaki et al. (1982) – 0.05 to 5000.0 microg/plate, rationale for top dose is not specified.
Nohmi et al. (1985) – 0, 0.1, 0.2, 0.5, 1.0 and 2.0 mg/plate, top dose was limited by toxicity.
Sasaki and Endo (1978) – Information is in abstract form only, no details on concentrations available. - Vehicle / solvent:
- Dillon et al. (1998) – vehicle is not mentioned.
Florin et al. (1980) – Ethanol
Fujita et al. (1992) – DMSO
Haworth et al. (1983) – Vehicle controls were run, but no specific mention of ID of vehicle for benzaldehyde.
Heck et al. (1989) – vehicle is not mentioned.
Kasamaki et al. (1982) – DMSO
Nohmi et al. (1985) – DMSO
Sasaki and Endo (1978) – vehicle is not mentioned. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- methylmethanesulfonate
- mitomycin C
- other: 2-Aminoanthracene used as + control for all strains w/ S9; without S9, formaldehyde or crotonaldehyde was used as + control for TA104.
- Remarks:
- Dillon et al. (1998)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-aminoanthracene
- Remarks:
- Florin et al. (1980)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- mitomycin C
- other: 2-aminoanthracene
- Remarks:
- Fujita et al. (1992)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 2-aminoanthracene and 4-nitro-o-phenylenediamine
- Remarks:
- Haworth et al. (1983)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- not specified
- Remarks:
- Heck et al. (1989)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- benzo(a)pyrene
- other: aflatoxin B1
- Remarks:
- Kasamaki et al. (1982)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- other: 2-aminoanthracene; 2-(2-furyl)-3-(5-nitrol-2-furyl)-acrylamide
- Remarks:
- Nohmi et al. (1985)
- Negative solvent / vehicle controls:
- not specified
- Positive controls:
- not specified
- Remarks:
- Sasaki and Endo (1978)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION (Cell density at seeding, if applicable):
Dillon et al. (1998) – Preincubation, cell density not specified.
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – Preincubation, cell density not specified.
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."
DURATION
- Preincubation period:
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – per Yahagi et al. (1975)
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."
- Exposure duration:
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – 20 minutes with test material
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."
- Expression time (cells in growth medium):
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – colonies counted 48 hours post pre-incubation.
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."
- Selection time (if incubation with a selection agent):
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – per Yahagi et al. (1975)
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."
SELECTION AGENT (mutation assays):
Dillon et al. (1998) – per Maron and Ames (1983)
Florin et al. (1980) – per Ames et al. (1975)
Fujita et al. (1992) – per Maron and Ames (1983)
Haworth et al. (1983) – per Yahagi et al. (1975)
Heck et al. (1989) – per Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – per Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – per Ames et al. (1975)
Sasaki and Endo (1978) – Abstract only, references "procedure recommended by Ames et al."
NUMBER OF REPLICATIONS:
Dillon et al. (1998) – Three plates per dose
Florin et al. (1980) – not specified, assumed to follow Ames et al. (1975)
Fujita et al. (1992) – mean of three plates
Haworth et al. (1983) – mean of three plates
Heck et al. (1989) – not specified, assumed to follow Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – not specified, assumed to follow Ames et al. (1975) and McCann et al. (1975)
Nohmi et al. (1985) – not specified, assumed to follow Ames et al. (1975)
Sasaki and Endo (1978) – not specified - Evaluation criteria:
- Dillon et al. (1998) – From section titled "Data Analyses": "The significance of mean revertant counts at individual dose levels was assessed using Dunnett's (-test and dose-response effects were analysed by two methods, Wahrendorf ranking and linear regression (Mahon et al, 1989). The latter was used to obtain slopes of the responses and both methods were only applied in the absence of a decline in revertant count at high dose levels. These analyses were used for guidance, but did not dictate the summary responses. Responses were judged mutagenic (+) when there was a reproducible, dose-related response and weakly mutagenic (+w) when reproducible, low level increases were obtained or when both positive and equivocal responses were seen in repeat trials. Responses were judged equivocal (?) when the increases in revertants were not reproducible or were seen only at a single dose.
Florin et al. (1980) – not detailed, assumed to follow Ames et al. (1975)
Fujita et al. (1992) – Statistical significance determined by Kruskal Wallis analysis.
Haworth et al. (1983) – from the section titled "Data Evaluation": "The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not."
Heck et al. (1989) – not detailed, assumed to follow Ames et al. (1973) and McCann et al. (1975)
Kasamaki et al. (1982) – from the section titled "Materials and Methods": "For mutagenic potency, a positive result was defined as a reproducible, dose-related increase in the number of revertant colonies per plate, and a greater than 2-fold increase in spontaneous mutation rate was obtained, according to the protocol of Ames (McCann et al., 1975)."
Nohmi et al. (1985) – at least 2-fold, dose dependent increase in mutant frequency.
Sasaki and Endo (1978) – no details. - Statistics:
- See "Evaluation Criteria" section.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Dillon et al. (1998)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Remarks:
- Dillon et al. (1998)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA 104
- Remarks:
- Dillon et al. (1998)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Florin et al. (1980)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Florin et al. (1980)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Florin et al. (1980)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Florin et al. (1980)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 97
- Remarks:
- Fujita et al. (1992)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Remarks:
- Fujita et al. (1992)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Haworth et al. (1983)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Haworth et al. (1983)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Haworth et al. (1983)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Haworth et al. (1983)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Heck et al. (1989)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Heck et al. (1989)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Heck et al. (1989)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Heck et al. (1989)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Kasamaki et al. (1982)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Kasamaki et al. (1982)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA2637
- Remarks:
- Nohmi et al. (1985)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Nohmi et al. (1985)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Nohmi et al. (1985)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 97
- Remarks:
- Sasaki and Endo (1978)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Sasaki and Endo (1978)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Sasaki and Endo (1978)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- Please see attached PDF tabular summary of all tests.
- Conclusions:
- While there is not a single study on in vitro bacterial mutagenicity which fully meets the criteria of the current OECD TG 471 (1997 version), there are several studies in the published literature performed in a manner similar to the guideline. These studies all either follow the original Ames design, or some minor variation. Most notably, there is not a single study which evaluates all recommended strains in the current OECD TG471, but the combined studies do cover the 5 combinations:
1. S. typhimurium TA1535, and
2. S. typhimurium TA1537 or TA97 or TA97a, and
3. S. typhimurium TA98, and
4. S. typhimurium TA100, and
5. E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.
In addition, while only one study reached the recomended maximum exposure concentration of 5 mg/plate, one other was relatively close (3.3 mg), and the remaining studies' top doses were limited by toxicity.
The most notable thing is that not one of the 8 studies summarized in this endpoint study record gave a positive result for benzaldehyde. Taken together, these 8 studies can be considered to comprise a Weight of Evidence approach to demonstrating that benzaldehyde is not considered mutagenic when evaluated in an in vitro bacterial mutagenicity assay. - Executive summary:
Despite there not being one study that meets all of the criteria of the current OECD TG 471, the results of 8 studies performed in a manner similar to the guideline support that benzaldehyde is not mutagenic in an in vitro bacterial mutagenicity assay.
Referenceopen allclose all
Table 4. Range-Finder: -S9: Results of the 3+21 Hour Treatments |
|||||||
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
64 |
125 |
11 |
200 |
0.74 |
|
B |
52 |
133 |
15 |
200 |
0.82 |
|
|
Total |
116 |
258 |
26 |
400 |
0.78 |
- |
|
3.853 |
A |
NSc |
- |
- |
- |
- |
- |
6.422 |
A |
NSc |
- |
- |
- |
- |
- |
10.70 |
A |
NSc |
- |
- |
- |
- |
- |
17.84 |
A |
NSc |
- |
- |
- |
- |
- |
29.73 |
A |
42 |
148 |
10 |
200 |
0.84 |
0 |
49.55 |
A |
58 |
139 |
3 |
200 |
0.73 |
6 |
82.58 |
A |
49 |
140 |
11 |
200 |
0.81 |
0 |
137.6 |
A |
54 |
135 |
11 |
200 |
0.79 |
0 |
229.4 |
A |
52 |
138 |
10 |
200 |
0.79 |
0 |
382.3 |
A |
75 |
123 |
2 |
200 |
0.64 |
18 |
637.2 |
A |
96 |
104 |
0 |
200 |
0.52 |
33P |
1062 |
A |
183 |
17 |
0 |
200 |
0.09 |
89P |
P = Precipitation observed at treatment
NSc = Not scored
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
Table 5. Range-Finder: +S9: Results of the 3+21 Hour Treatments |
|||||||
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
50 |
139 |
11 |
200 |
0.81 |
|
B |
56 |
127 |
17 |
200 |
0.81 |
|
|
Total |
106 |
266 |
28 |
400 |
0.81 |
- |
|
3.853 |
A |
NSc |
- |
- |
- |
- |
- |
6.422 |
A |
NSc |
- |
- |
- |
- |
- |
10.70 |
A |
NSc |
- |
- |
- |
- |
- |
17.84 |
A |
64 |
122 |
14 |
200 |
0.75 |
7 |
29.73 |
A |
55 |
133 |
12 |
200 |
0.79 |
2 |
49.55 |
A |
58 |
137 |
5 |
200 |
0.74 |
9 |
82.58 |
A |
45 |
138 |
17 |
200 |
0.86 |
0 |
137.6 |
A |
59 |
132 |
9 |
200 |
0.75 |
7 |
229.4 |
A |
57 |
138 |
5 |
200 |
0.74 |
8 |
382.3 |
A |
78 |
118 |
4 |
200 |
0.63 |
22 |
637.2 |
A |
96 |
102 |
2 |
200 |
0.53 |
34P |
1062 |
A |
177 |
23 |
0 |
200 |
0.12 |
86P |
P = Precipitation observed at treatment
NSc = Not scored
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
Table 6. Range-Finder: +S9: Results of the 24+24 Hour Treatments |
|||||||
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
28 |
146 |
26 |
200 |
0.99 |
|
B |
39 |
142 |
19 |
200 |
0.90 |
|
|
Total |
67 |
288 |
45 |
400 |
0.95 |
- |
|
3.853 |
A |
NSc |
- |
- |
- |
- |
- |
6.422 |
A |
NSc |
- |
- |
- |
- |
- |
10.70 |
A |
NSc |
- |
- |
- |
- |
- |
17.84 |
A |
31 |
148 |
21 |
200 |
0.95 |
0 |
29.73 |
A |
24 |
152 |
24 |
200 |
1.00 |
0 |
49.55 |
A |
23 |
155 |
22 |
200 |
1.00 |
0 |
82.58 |
A |
48 |
144 |
8 |
200 |
0.80 |
15 |
137.6 |
A |
80 |
118 |
2 |
200 |
0.61 |
35 |
229.4 |
A |
124 |
74 |
2 |
200 |
0.39 |
59 |
382.3 |
A |
119 |
74 |
7 |
200 |
0.55 |
53 |
637.2 |
A |
194 |
6 |
0 |
200 |
0.03 |
97P |
1062 |
A |
199 |
1 |
0 |
200 |
0.01 |
99P |
P = Precipitation observed at treatment
NSc = Not scored
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
Table 7. Micronucleus Experiment: -S9: Results of the 3+21 Hour Treatments |
|||||||
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
112 |
360 |
28 |
500 |
0.83 |
|
B |
127 |
347 |
26 |
500 |
0.80 |
|
|
C |
85 |
380 |
35 |
500 |
0.90 |
|
|
D |
88 |
378 |
34 |
500 |
0.89 |
|
|
Total |
412 |
1465 |
123 |
2000 |
0.86 |
- |
|
100.0 |
A |
134 |
344 |
22 |
500 |
0.78 |
|
B |
114 |
365 |
21 |
500 |
0.81 |
|
|
Total |
248 |
709 |
43 |
1000 |
0.80 |
7 |
|
200.0 |
A |
143 |
328 |
29 |
500 |
0.77 |
|
B |
160 |
315 |
25 |
500 |
0.73 |
|
|
Total |
303 |
643 |
54 |
1000 |
0.75 |
12 |
|
400.0 |
A |
140 |
348 |
12 |
500 |
0.74 |
|
B |
152 |
337 |
11 |
500 |
0.72 |
|
|
Total |
292 |
685 |
23 |
1000 |
0.73 |
15 # |
|
500.0 |
A |
183 |
311 |
6 |
500 |
0.65 |
|
B |
170 |
325 |
5 |
500 |
0.67 |
|
|
Total |
353 |
636 |
11 |
1000 |
0.66 |
23 |
|
600.0 |
A |
213 |
286 |
1 |
500 |
0.58 |
|
B |
244 |
255 |
1 |
500 |
0.51 |
|
|
Total |
457 |
541 |
2 |
1000 |
0.55 |
36 # |
|
650.0 |
A |
246 |
249 |
5 |
500 |
0.52 |
|
B |
237 |
261 |
2 |
500 |
0.53 |
|
|
Total |
483 |
510 |
7 |
1000 |
0.52 |
39 |
|
700.0 |
A |
225 |
271 |
4 |
500 |
0.56 |
|
B |
279 |
219 |
2 |
500 |
0.45 |
|
|
Total |
504 |
490 |
6 |
1000 |
0.50 |
41 |
|
750.0 |
A |
265 |
234 |
1 |
500 |
0.47 |
|
B |
285 |
213 |
2 |
500 |
0.43 |
|
|
Total |
550 |
447 |
3 |
1000 |
0.45 |
47 |
|
800.0 |
A |
305 |
193 |
2 |
500 |
0.39 |
|
B |
294 |
206 |
0 |
500 |
0.41 |
|
|
Total |
599 |
399 |
2 |
1000 |
0.40 |
53 # |
|
850.0 |
A |
308 |
191 |
1 |
500 |
0.39 |
|
B |
303 |
194 |
3 |
500 |
0.40 |
|
|
Total |
611 |
385 |
4 |
1000 |
0.39 |
54 |
|
950.0 |
A |
348 |
151 |
1 |
500 |
0.31 |
|
B |
381 |
117 |
2 |
500 |
0.24 |
|
|
Total |
729 |
268 |
3 |
1000 |
0.27 |
68 P |
|
1062 |
A |
473 |
27 |
0 |
500 |
0.05 |
|
B |
456 |
44 |
0 |
500 |
0.09 |
|
|
Total |
929 |
71 |
0 |
1000 |
0.07 |
92 P |
|
MMC, 0.30 |
A |
250 |
249 |
1 |
500 |
0.50 |
|
B |
244 |
255 |
1 |
500 |
0.51 |
|
|
Total |
494 |
504 |
2 |
1000 |
0.51 |
41 # |
P = Precipitation observed at treatment
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
# Highlighted concentrations selected for analysis
Table 8. Micronucleus Experiment: +S9: Results of the 3+21 Hour Treatments |
|||||||
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
132 |
341 |
27 |
500 |
0.79 |
|
B |
117 |
342 |
41 |
500 |
0.85 |
|
|
C |
115 |
369 |
16 |
500 |
0.80 |
|
|
D |
110 |
366 |
24 |
500 |
0.83 |
|
|
Total |
474 |
1418 |
108 |
2000 |
0.82 |
- |
|
100.0 |
A |
144 |
343 |
13 |
500 |
0.74 |
|
B |
106 |
366 |
28 |
500 |
0.84 |
|
|
Total |
250 |
709 |
41 |
1000 |
0.79 |
3 |
|
200.0 |
A |
124 |
364 |
12 |
500 |
0.78 |
|
B |
103 |
379 |
18 |
500 |
0.83 |
|
|
Total |
227 |
743 |
30 |
1000 |
0.80 |
2 |
|
400.0 |
A |
148 |
343 |
9 |
500 |
0.72 |
|
B |
130 |
363 |
7 |
500 |
0.75 |
|
|
Total |
278 |
706 |
16 |
1000 |
0.74 |
10 # |
|
500.0 |
A |
204 |
292 |
4 |
500 |
0.60 |
|
B |
187 |
310 |
3 |
500 |
0.63 |
|
|
Total |
391 |
602 |
7 |
1000 |
0.62 |
25 |
|
600.0 |
A |
250 |
247 |
3 |
500 |
0.51 |
|
B |
239 |
259 |
2 |
500 |
0.53 |
|
|
Total |
489 |
506 |
5 |
1000 |
0.52 |
37 # |
|
650.0 |
A |
258 |
237 |
5 |
500 |
0.49 |
|
B |
255 |
244 |
1 |
500 |
0.49 |
|
|
Total |
513 |
481 |
6 |
1000 |
0.49 |
40 |
|
700.0 |
A |
230 |
269 |
1 |
500 |
0.54 |
|
B |
274 |
226 |
0 |
500 |
0.45 |
|
|
Total |
504 |
495 |
1 |
1000 |
0.50 |
39 |
|
750.0 |
A |
270 |
227 |
3 |
500 |
0.47 |
|
B |
270 |
230 |
0 |
500 |
0.46 |
|
|
Total |
540 |
457 |
3 |
1000 |
0.46 |
43 |
|
800.0 |
A |
321 |
177 |
2 |
500 |
0.36 |
|
B |
303 |
197 |
0 |
500 |
0.39 |
|
|
Total |
624 |
374 |
2 |
1000 |
0.38 |
54 # |
|
850.0 |
A |
353 |
145 |
2 |
500 |
0.30 |
|
B |
317 |
181 |
2 |
500 |
0.37 |
|
|
Total |
670 |
326 |
4 |
1000 |
0.33 |
59 |
|
950.0 |
A |
370 |
128 |
2 |
500 |
0.26 |
|
B |
366 |
134 |
0 |
500 |
0.27 |
|
|
Total |
736 |
262 |
2 |
1000 |
0.27 |
67 P |
|
1062 |
A |
415 |
84 |
1 |
500 |
0.17 |
|
B |
403 |
96 |
1 |
500 |
0.20 |
|
|
Total |
818 |
180 |
2 |
1000 |
0.18 |
77 P |
|
CPA, 3.00 |
A |
181 |
312 |
7 |
500 |
0.65 |
|
B |
151 |
342 |
7 |
500 |
0.71 |
|
|
Total |
332 |
654 |
14 |
1000 |
0.68 |
17 |
|
CPA, 5.00 |
A |
218 |
280 |
2 |
500 |
0.57 |
|
B |
235 |
264 |
1 |
500 |
0.53 |
|
|
Total |
453 |
544 |
3 |
1000 |
0.55 |
|
|
CPA, 7.00 |
A |
244 |
255 |
1 |
500 |
0.51 |
|
B |
243 |
257 |
0 |
500 |
0.51 |
|
|
Total |
487 |
512 |
1 |
1000 |
0.51 |
37 # |
P = Precipitation observed at treatment
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
# Highlighted concentrations selected for analysis
Table 9. Micronucleus Experiment: -S9: Results of the 24+24 Hour Treatments |
|||||||
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
33 |
347 |
120 |
500 |
1.17 |
|
B |
41 |
326 |
133 |
500 |
1.18 |
|
|
C |
38 |
328 |
134 |
500 |
1.19 |
|
|
D |
32 |
366 |
102 |
500 |
1.14 |
|
|
Total |
144 |
1367 |
489 |
2000 |
1.17 |
|
|
50.0 |
A |
46 |
353 |
101 |
500 |
1.11 |
|
B |
53 |
342 |
105 |
500 |
1.10 |
|
|
Total |
99 |
695 |
206 |
1000 |
1.11 |
6 # |
|
100.0 |
A |
113 |
363 |
24 |
500 |
0.82 |
|
B |
124 |
346 |
30 |
500 |
0.81 |
|
|
Total |
237 |
709 |
54 |
1000 |
0.82 |
30 # |
|
150.0 |
A |
151 |
331 |
18 |
500 |
0.73 |
|
B |
174 |
320 |
6 |
500 |
0.66 |
|
|
Total |
325 |
651 |
24 |
1000 |
0.70 |
40 |
|
200.0 |
A |
264 |
230 |
6 |
500 |
0.48 |
|
B |
222 |
269 |
9 |
500 |
0.57 |
|
|
Total |
486 |
499 |
15 |
1000 |
0.53 |
55 # |
|
250.0 |
A |
337 |
158 |
5 |
500 |
0.34 |
|
B |
266 |
222 |
12 |
500 |
0.49 |
|
|
Total |
603 |
380 |
17 |
1000 |
0.41 |
65 |
|
300.0 |
A |
285 |
188 |
27 |
500 |
0.65 |
|
B |
312 |
175 |
13 |
500 |
0.40 |
|
|
Total |
697 |
363 |
40 |
1000 |
0.44 |
62 |
|
350.0 |
A |
212 |
250 |
38 |
500 |
0.65 |
|
B |
262 |
202 |
36 |
500 |
0.55 |
|
|
Total |
474 |
452 |
74 |
1000 |
0.60 |
49 |
|
400.0 |
A |
275 |
211 |
14 |
500 |
0.48 |
|
B |
291 |
199 |
10 |
500 |
0.44 |
|
|
Total |
566 |
410 |
24 |
1000 |
0.46 |
61 |
|
450.0 |
A |
348 |
147 |
5 |
500 |
0.31 |
|
B |
363 |
134 |
3 |
500 |
0.28 |
|
|
Total |
711 |
281 |
8 |
1000 |
0.30 |
75 |
|
600.0 |
A |
478 |
21 |
1 |
500 |
0.05 |
|
B |
478 |
22 |
0 |
500 |
0.04 |
|
|
Total |
956 |
43 |
1 |
1000 |
0.05 |
96 |
|
VIN, 0.04 |
A |
195 |
225 |
80 |
500 |
0.77 |
|
B |
227 |
199 |
74 |
500 |
0.69 |
|
|
Total |
422 |
424 |
154 |
1000 |
0.73 |
38 # |
Mono = Mononucleate
Bi = Binucleate
Multi = Multinucleate
RI = Replication index
# Highlighted concentrations selected for analysis
Table 10. Summary of Results of the Micronucleus Test |
|||||
Treatment |
Concentration (µg/mL) |
Cytotoxicity (%)$ |
Mean MNBN Cell Frequency (%) |
Historical Control Range (%)# |
Statistical Significance |
3+21 hour -S9 |
Vehiclea |
- |
0.60 |
0.00 to 1.01 |
- |
400.0 |
15 |
0.60 |
NS |
||
600.0 |
36 |
0.45 |
NS |
||
800.0 |
53 |
0.50 |
NS |
||
*MMC, 0.30 |
41 |
7.00 |
p≤0.001 |
||
|
|||||
3+21 hour +S9 |
Vehiclea |
- |
0.50 |
0.10 to 1.20 |
- |
400.0 |
10 |
0.35 |
NS |
||
600.0 |
37 |
0.90 |
NS |
||
800.0 |
54 |
0.80 |
NS |
||
*CPA, 7.00 |
37 |
3.10 |
p≤0.001 |
||
|
|||||
24+24 hour -S9 |
Vehiclea |
- |
0.75 |
0.10 to 0.80 |
- |
50.0 |
6 |
0.60 |
NS |
||
100.0 |
30 |
0.40 |
NS |
||
200.0 |
55 |
0.88 |
NS |
||
*VIN 0.04 |
38 |
2.95 |
p≤0.001 |
a Vehicle control was DMSO
* Positive control
# 95thpercentile of the observed range
$ Based on replication index
NS Not significant
Table 1: RSG Values - Range-Finder Experiment |
||
Concentration (µg/mL) |
3 Hour Treatment (-S9) % RSG |
3 Hour Treatment (+S9) % RSG |
0 |
100 |
100 |
33.19 |
98 |
65 |
66.38 |
95 |
100 |
132.8 |
91 |
77 |
265.5 |
87 |
65 |
531 P |
41 |
32 |
1062 P |
2 |
3 |
% RSG: Percent relative suspension growth
P: Precipitation observed at the time of treatment
Table 2: Summary of Mutation Data |
|||||
3 Hour Treatment -S9 |
3 Hour Treatment +S9 |
||||
Concentration µg/mL |
%RTG |
MF § |
Concentration µg/mL |
%RTG |
MF § |
0 |
100 |
52.67 |
0 |
100 |
46.53 |
100 |
73 |
52.90 |
100 |
69 |
66.75 |
200 |
52 |
85.52 |
200 |
63 |
74.33 |
400 |
44 |
68.00 |
400 |
48 |
78.03 |
500 |
29 |
88.62 |
500 |
38 |
72.73 |
600 |
28 |
75.80 |
600 |
33 |
82.08 |
700 P |
16 |
50.53 |
700 P |
21 |
111.11 |
800 P |
16 |
58.64 |
800 P |
17 |
99.83 |
MMS 15 |
48 |
316.78 |
B[a]P 2 |
35 |
575.56 |
MMS 20 |
42 |
370.08 |
B[a]P 3 |
30 |
640.12 |
Linear trend test on mutant frequency in the absence of S9: p-value = 0.4554, not significant
Linear trend test on mutant frequency in the presence of S9: p-value = 0.001 (** P < 0.01)
§: 5-TFT-resistant mutants/106 viable cells 2 days after treatment
% RTG: Percent Relative Total Growth
P: Precipitation noted at time of treatment
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vitro:
The test substance was not mutagenic in Salmonella gene mutation assays (Florin et al., 1980; Kasamaki et al., 1982; Haworth et al., 1983).
It exhibited genotoxic activity in the mouse lymphoma assay (McGregor et al., 1989) and in assays for sister chromatid exchanges in both Chinese hamster ovary (CHO) cells (Galloway etal., 1986). Induction of chromosomal aberrations by the test substance was also reported in Chinese hamster lung cells at a dose stated to be 50 nM (5.3 ng/ml) (Kasamaki et al., 1982); however, the National Toxicology Program (NTP), using concentrations of the test substance which were approximately 10,000 times higher, found no increase in aberrations in CHO cells (Galloway et al., 1986). This basic pattern of no mutagenic activity in bacterial systems but possible weak clastogenic effects in some mammalian cell assays is also reflected in test results from metabolites of the test substance, i.e., benzoic acid.
In in-vivo sex-linked recessive lethal mutation assays with Drosophila melanogaster the test substance was tested negative after oral and ip administration.
No adequate in vivo data are available that confirm the weakly positive results reported in in vitro tests.
Justification for selection of genetic toxicity endpoint
The study in mammalian cells was chosen over that in bacterial
cells, as the higher level study.
Justification for classification or non-classification
The available data do not lead to classification for genotoxicity according to DSD and CLP.
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