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EC number: 208-293-9 | CAS number: 520-45-6
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Genetic toxicity in vitro
Description of key information
OECD Guideline 473 In Vitro Mammalian Chromosome Aberration Test: Dehydroacetic acid sodium salt; +/- metabolic activation, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0 mM test material tested. Not genotoxic.
In vitro cytogenicity / chromosome aberration study in mammalian cells test: Dehydroacetic acid sodium salt. Not genotoxic.
In vitro cytogenicity / chromosome aberration study in mammalian cells test: Chinese hamster cell line, maximum dose 0.25 mg/ml. Not genotoxic.
In vitro gene mutation study in bacteria test: S. typhimurium strains TA92, TA1535, TA100, TAI537, TA94 and TA98; +/- metabolic activation, maximum dose 10 mg/plate. Not genotoxic.
Chromosome aberrations and sister chromatid exchange test: Dehydroacetic acid sodium salt, doses 1x10-3, 1x10-4 and 1x10-5 M. Not genotoxic.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Circa 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline followed
- Version / remarks:
- No guideline stated, but tests conducted in a reputable Japanese facility.
- Principles of method if other than guideline:
- Please see details on test system below.
- GLP compliance:
- no
- Type of assay:
- other: chromosome aberration
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Remarks:
- Details on cell line given in details on test system section.
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- Max. dose 0.25 mg/ml
- Vehicle / solvent:
- Solvent was ethanol, no specific details of any dilutions thereafter.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- not specified
- Details on test system and experimental conditions:
- Chromosomal aberration tests in vitro using a Chinese hamster fibroblast cell line. The cell line was originally established from the lung of a new-born female at the Cancer Research Institute. Tokyo and was maintained by 4-day passages in Minimum Essential Medium (MEM) supplemented with 10% calf serum.
The cells were exposed to each sample at three different doses for 24 and 48 hr; no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated.
Chromosome preparations were made as follows Colcemid (final conc 0.2/µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution (0.075 M) for 13 min at room temperature. After centrifugation, the cells were fixed with acetic acid-methanol (1:3. v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which (he incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the DM was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/ml). TR values are relatively high for chemicals that show carcinogenic potential in animals. - Evaluation criteria:
- Please see details on test system.
- Statistics:
- Not specified.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
- Conclusions:
- At a maximum concentration of 0.25 mg/ml no genotoxicity was evident
- Executive summary:
In a chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line; cells were exposed to each sample of the test substance at three different doses for 24 and 48 hr; no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated. At a maximum concentration of 0.25 mg/ml dehydroacetic acid no genotoxicity was evident. The test substance was not clastogenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Circa 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline followed
- Version / remarks:
- No guideline stated, but tests conducted in a reputable Japanese facility.
- Principles of method if other than guideline:
- Please see details on test system below.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Not applicable.
- Species / strain / cell type:
- other: S. typhimurium strains TA92, TA1535, TA100, TAI537, TA94 and TA98.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S-9.
- Test concentrations with justification for top dose:
- Maximum 10 mg/plate.
- Vehicle / solvent:
- Solvent DMSO, no details on further dilutions.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- not specified
- Details on test system and experimental conditions:
- Ames Test: Reverse mutation assays using S. typhimurium strains TA92, TA1535, TA100, TAI537, TA94 and TA98 were carried out according to the method of Ames. For some samples, TA2637 was also used. All these test strains were provided by Dr B. N. Ames, University of California, Berkeley, USA. The liver microsome fraction (S-9) was prepared from the liver of Fischer rats pre-treated 5 days before with polychlorinated biphenyls (500mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 nM-NADPH, 4 mM-NADH, 33mM-KC1, 8 mM-MgCI2, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample. The number of revertant (his+) colonies was scored after incubation at 37°C for 2 days. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed.
- Evaluation criteria:
- See details on test system.
- Statistics:
- Not specified.
- Key result
- Species / strain:
- other: S. typhimurium strains TA92, TA1535, TA100, TAI537, TA94 and TA98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
- Conclusions:
- Dehydroacetic acid was not genotoxic in this Ames test.
- Executive summary:
In an Ames test, using S. typhimurium strains TA92, TA1535, TA100, TAI537, TA94 and TA98, Dehydroacetic acid, up to a maximum concentration of 10 mg/plate, was found not to be genotoxic.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- Chromosome aberrations and sister chromatid exchange
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- Circa 1977
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Please see test system described below.
- GLP compliance:
- no
- Type of assay:
- sister chromatid exchange assay in mammalian cells
- Specific details on test material used for the study:
- Sodium dehydroacetate - supplied by the National Institute of Hygienic Sciences, Japan.
- Target gene:
- Not applicable.
- Species / strain / cell type:
- other:
- Details on mammalian cell type (if applicable):
- A pseudo-diploid Chinese hamster cell line.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- not specified
- Test concentrations with justification for top dose:
- 1x10-3, 1x10-4 and 1x10-5 M. No details regarding dosage selection were iindicated in the publication.
- Vehicle / solvent:
- HBSS - Hanks Buffered Saline Solution.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- not specified
- Remarks:
- Known oncogens were tested in this study with positive results.
- Details on test system and experimental conditions:
- A pseudo-diploid Chinese hamster cell line (Don) was used. The cells were grown in Eagle's minimum essential medium supplemented with fetal calf serum (pH 7.2). Three hours after 1.0-1.2x106 cells per TD-40 culture bottle were seeded, BUdR (1 µg/ml) and test chemicals were added to the cultures under an yellow darkroom safety lamp. All chemicals were freshly dissolved or suspended in HBSS, ethanol, or DMSO to make final concentrations of l0-6, l0-5, l0-4, and l0-3 M. When necessary, higher or intermediate doses were also tested. The final doses of the solvents per ml medium did not exceed 0.1 ml for saline and 0.005 ml for ethanol and DMSO. For a given dose of each chemical, at least one culture was made; however, the experiments were repeated for some critical concentrations with most of the chemicals tested. One control culture containing BUDR and solvent was routinely prepared for each series of experiments. All cultures were kept in complete darkness at 37oC for 26 hours (this covered two rounds of cell cycle), and 0.25 µg colchicine/ml was added for the final 2 hours. Cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment (0.075 M KCl, 37°C, 20 min) and fixation in ice-cold methanol: acetic acid (3:1).
Sister chromatids were differentiated by the fluorescence or Giemsa staining techniques. An acridine orange technique was used for fluorescence, and a modified FPG technique was used for Giemsa staining. The chromosome slide was stained in aqueous solution of 33258 Hoechst (50µg/ml) for l0 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60W, at l2-cm distance) for I hour. The cover slip was removed by tap water, and the slide was incubated in I M NaH2PO4 (pH 8.0, 83-85'C) for 10 minutes, rinsed, and stained in 2.57% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. Chromosome aberrations were examined on 100 metaphase plates for each dose, and the frequency of aberrations, excluding gaps was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The number of SCE per cell was determined on the basis of 20-50 intact metaphases in which all chromosomes had a "harlequinized" appearance without gross chromosome aberrations. SCE in the centromeric region were not scored because they were indistinguishable from the twisting of the sister chromatids. - Evaluation criteria:
- Please see test system described above.
- Statistics:
- t-test used for sister chromatid exchange.
- Key result
- Species / strain:
- other: Chinese hamster cell line
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Remarks:
- See attached data tables.
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
- Additional information on results:
- Mitotic index was not appreciably decreased; no chromosome aberrations detected, sister chromatide exchange was negative.
- Conclusions:
- Sodium dehydroacetate (Dehydracetic acid sodium), at concentrations between 1x10-3 to 1x10-5 M was not genotoxic in this assay.
- Executive summary:
Sodium dehydroacetate (Dehydrocetic acid sodium), tested at concentrations between 1x10-3 to 1x10-5 M in an in vitro assay using Chinese hamster cells gave negative results for chromosome aberrations and sister chromatid exchange. It was not genotoxic.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 Jun - 12 Sep 2016
- 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)
- Version / remarks:
- adopted Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Ministry of Health, Italy
- Type of assay:
- other: chromosome aberration
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Sex, age and number of blood donors: 2 female donors, 30 and 33 years old
- Whether whole blood or separated lymphocytes were used: separated lymphocytes
- Methods for maintenance in cell culture: 2% (v/v) phytohaemagglutinin
MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 supplemented with 20% heat-inactivated FCS, 1.25% (v/v) L-glutamine (200 mM) and 0.25% (v/v) antibiotic solution - Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital - 5,6-benzoflavone
- Test concentrations with justification for top dose:
- 3h treatment with and without metabolic activation: 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0 mM
24h treatment without metabolic activation: 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0 mM - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (1%)
- Justification for choice of solvent/vehicle: DMSO was selected based on the survival of the cells and the S9 mix metabolic activity - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 24 h; 24 h treatment: 24 h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.2 ug/mL media
STAIN (for cytogenetic assays): Giemsa 3% (v/v) in tap water
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: A few drops of the treated lymphocyte culture suspension were dropped onto clean, wet, grease-free glass slides and air-dired. The coded slides were stained in 3% Giemsa in tap water and made permanent with Eukitt.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Rationale for test conditions:
- The mitotic index (MI) was determined for each of the treatment levels. This parameter is based on the number of metaphases observed per 1000 cells and is expressed as a percentage. The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial reduction in mitotic index compared with the solvent control. Ideally the reduction should be approximately to 45 ± 5% of the concurrent negative control.
In the absence of cytotoxic/cytostatic effects the highest treatment level was selected as the highest dose level for scoring.
Two lower dose levels were also selected for the scoring of chromosomal aberrations. Slides were independently coded before microscopic analysis for chromosomal aberrations. Metaphases that differed from the modal chromosomal complement by more than two centromeres were not scored. The number of chromosomes, the specific types and numbers of aberrations were recorded. Polyploid and endoreduplicated cells encountered were recorded, but not included in the count of eligible metaphases.
One hundred and fifty metaphases spreads were scored for chromosomal aberrations from each culture. - Evaluation criteria:
- The test item is considered as clearly positive if the following criteria are met:
- any dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps)
- the incidence of cells bearing aberrations is outside the normal distribution of historical control values
- the increase of cells bearing aberration is dose-related when evaluated with an appropriate trend test
The test item is considered as clearly negative if none of the above criteria is met. - Statistics:
- Fisher`s Exact Test was used to compare the number of cells bearing aberrations in control and treated cultures. Bonferroni`s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps. Cochran-Armitage trent test (one-sided) was performed to aaid determination of concentration response relationship.
The percentage of cells bearing aberrations excluding gaps was considered for the evaluation of the outcome of the study. - Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no remarkable variation of pH was observed
- Effects of osmolarity: no remarkable variation of osmolarity was observed
- Precipitation: no precipitation at the beginning or end of treatment was observed
RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the vehicle and the cell culture medium
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes - Conclusions:
- Dehydroacetic acid sodium salt did not induce chromosomal aberrations in human lymphocytes afterin vitro treatment, under the reported experimental conditions.
- Executive summary:
The test item Dehydroacetic acid sodium salt was assayed for the ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment both in the absence and presence of S9 metabolic activation. Dose levels of 10.0, 5.00,2.50,1.25, 0.625,0.313, 0.156 and 0.0781 mM (corresponding to 1900, 950,475,238,119, 59.4,29.7 and 14.8 µg/mL) were used for all treatment series. Appropriate negative and positive controls were included. Two replicate cell cultures were prepared at each test point.
Dehydroacetic acid sodium salt did not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source chemical is the sodium salt of the target. As such, both the source and the target are anticipated to behave similarly in biological systems.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Dehydroacetic acid, sodium salt [EC 224-580-1; CAS 4418-26-2]. Composition provided in TMI.
Target: Dehydroacetic acid [EC 208-293-9; CAS 520-45-6]. Purity/impurity profile as described in Section 1.2
3. ANALOGUE APPROACH JUSTIFICATION
The source chemical and the target are structurally identical apart from the sodium ion in the source replacing the hydrogen on the parent acid target. The sodium ion does not have any effect on genotoxic properties and therefore data on the sodium salt may be read across to the target. Both the source and the target have been shown to be non-genotoxic in bacterial reverse mutations assays with S. typhimurium. In addition, the target chemical was found to be non-clastogenic is a study performed in Chinese hamster fibroblasts (V79), without metabolic activation, which is in accordance with the results of the chromosome aberration study using the source (human lyphocytes; negative +/- metabolic activation; OECD 473).
4. DATA MATRIX
Refer to analogue justification provided in IUCLID section 13. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- Chromosome aberrations and sister chromatid exchange
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- other: chinese hamster cell line
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
Referenceopen allclose all
Table 1: Mitotic index results
Test item | Concentration | Mitotic Index |
in µg/mL | in % | |
Exposure period 3 h, fixation time 24 h, without S9 mix | ||
DMSO | 1.0% (v/v) | 100 |
Test substance | 0.0781 | 75 |
0.156 | 73 | |
0.313 | 74 | |
0.625 | 80 | |
1.25 | 87 | |
2.5 | 92 | |
5.00 | 87 | |
10.0 | 85 | |
Exposure period 3 h, fixation time 24 h, with S9 mix | ||
DMSO | 1.0% (v/v) | 100 |
Test substance | 0.0781 | 98 |
0.156 | 118 | |
0.313 | 108 | |
0.625 | 97 | |
1.25 | 109 | |
2.5 | 99 | |
5.00 | 102 | |
10.0 | 98 | |
CP | 18.0 ug/mL | 29 |
CP | 23.0 ug/mL | 26 |
Exposure period 24 h, fixation time 24 h, without S9 mix | ||
DMSO | 1.0% (v/v) | 100 |
Test substance | 0.0781 | 93 |
0.156 | 84 | |
0.313 | 86 | |
0.625 | 86 | |
1.25 | 46 | |
2.5 | 29 | |
5.00 | 25 | |
10.0 | 11 | |
MMC | 0.300 ug/mL | 60 |
MMC | 0.450 ug/mL | 69 |
MMC = Mitomycin C
CP = Cyclophosphamide
Table 2: Aberration results
Test item | Concentration | Mitotic Index | Aberrant cells in % | |
in µg/mL | in % | with gaps | without gaps | |
Exposure period 3 h, fixation time 24 h, without S9 mix | ||||
DMSO | 1.0% (v/v) | 100 | 0 | 0 |
Test substance | 2.5 | 92 | 5 | 2 |
5.0 | 87 | 0 | 0 | |
10.0 | 85 | 1 | 0 | |
Exposure period 3 h, fixation time 24 h, with S9 mix | ||||
DMSO | 1.0% (v/v) | 100 | 0 | 1 |
CP | 18 uL/mL | 29 | 0 | 0 |
Test substance | 2.5 | 99 | 0 | 1 |
5.0 | 102 | 0 | 1 | |
10.0 | 98 | 59 | 64 | |
Exposure period 24 h, fixation time 24 h, without S9 mix | ||||
DMSO | 1.0% (v/v) | 100 | 0 | 0 |
MMC | 0.300 g/L | 60 | 0 | 0 |
Test substance | 0.313 | 86 | 0 | 0 |
0.625 | 86 | 1 | 0 | |
1.25 | 46 | 137 | 110 |
MMC = Mitomycin C
CP = Cyclophosphamide
Table 3 Historical control data
Solvent/untreated controls | 24 h, without S9 | 24 h, with S9 | ||
+ gaps | - gaps | + gaps | - gaps | |
Mean | 0.7 | 0.3 | 0.7 | 0.2 |
SD | 0.9 | 0.4 | 0.9 | 0.4 |
n | 275 | 275 | 240 | 240 |
UCL | 2.5 | 1.5 | 2.5 | 1.0 |
LCL | 0.0 | 0.0 | 0.0 | 0.0 |
Positive controls | 24 h, without S9 (MMC) | 24 h, with S9 (CP) | ||
+ gaps | - gaps | + gaps | - gaps | |
Mean | 32.5 | 31.6 | 22.7 | 21.5 |
SD | 15.7 | 15.5 | 10.6 | 10.6 |
n | 70 | 70 | 157 | 157 |
UCL | 60.1 | 60.1 | 48.4 | 46.2 |
LCL | 14.5 | 13.7 | 9.5 | 8.4 |
UCL = upper confidence limit
LCL = lower confidence limit
MMC = Mitomycin C
CP = Cyclophosphamide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Dehydroacetic acid sodium salt DHA-Na, when dosed by oral gavage showed that there was no trend for genotoxicity in a micronucleus study. When dosed by the intraperitoneal route there was a positive trend for genotoxicity. However, as indicated in the OECD guideline 747, the appropriate route of exposure should be used, the appropriate route of potential exposure is most likely oral rather than intraperitoneal. The data therefore indicate that DHA-Na should be considered as not genotoxic.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Circa 1988
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Micronucleus test conducted on a Japanese government laboratories: National Institute of Hygiene Sciences and Public Health Laboratory.
- GLP compliance:
- no
- Specific details on test material used for the study:
- Dehydroacetic acid, sodium salt DHA-Na, CAS 4418-26-2
Supplied by Japan Food Additives Association
Purity: 99.6% - Species:
- mouse
- Strain:
- other: ddy - from a Japanese breeder
- Details on species / strain selection:
- Used by the laboratories.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- Eight-week-old male ddY mice (Shizuoka Agri-cultural Cooperative Association for Laboratory Animals, Shizuoka) were used at both laboratories, and were allowed food pellets CE-2 (Japan Clea, Tokyo) and water ad lib. throughout the experiments.
- Route of administration:
- other: Oral and intraperitoneal
- Vehicle:
- water or normal saline
- Details on exposure:
- See any other information on materials and methods below
- Duration of treatment / exposure:
- See any other information on materials and methods below
- Frequency of treatment:
- Multiple (four or five) injections with 24-hr intervals between the injections.
- Post exposure period:
- 24 hours
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- See results for route of exposure for dosages
- Dose / conc.:
- 37.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 250 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 2-6/group for the pilot and main test
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- Mitomycin C. Additionally a number of substances were tested in this study, some of which were suspected to be genotoxic.
- Tissues and cell types examined:
- See any other information on materials and methods below
- Details of tissue and slide preparation:
- See any other information on materials and methods below
- Evaluation criteria:
- See any other information on materials and methods below
- Statistics:
- A two-stage statistical procedure was used: the frequency of MNPCEs in each treatment group was compared with the binomial distribution specified by historical control data. In the second stage, the dose-response relationship was tested by the Cochran-Armitage trend test. A positive result was recorded only when one or more treatment group(s) showed a statistically significant difference (P <0.01) from the spontaneous level of MNPCEs and the trend test indicated a positive dose response (P < 0.05).
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: intraperitoneal injection route only positive
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: oral gavage
- Conclusions:
- Dehydroacetic acid, sodium salt DHA-Na, when dosed by oral gavage showed that there was no trend for genotoxicity. When dosed by the intraperitoneal route there was a positive trend for genotoxicity. However, as indicated in the OECD guideline 747, the appropriate route of exposure should be used, the appropriate route of potential exposure is most likely oral rather than intraperitoneal. The data therefore indicate that DHA-Na should be considered as not genotoxic.
- Executive summary:
Dehydroacetic acid, sodium salt DHA-Na, when dosed by oral gavage, at dose levels between 37.5 and 1250 mg/kg showed that there was no positive trend for genotoxicity. When dosed by the intraperitoneal
route there was a positive trend for genotoxicity. However, as indicated in the OECD guideline 747, the appropriate route of exposure should be used, the appropriate route of potential exposure is most
likely oral rather than intraperitoneal. The data therefore indicate that DHA-Na should be considered as not genotoxic.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source chemical is the sodium salt of the target. As such, both the source and the target are anticipated to behave similarly in biological systems.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Dehydroacetic acid, sodium salt [EC 224-580-1; CAS 4418-26-2]. Composition provided in TMI.
Target: Dehydroacetic acid [EC 208-293-9; CAS 520-45-6]. Purity/impurity profile as described in Section 1.2
3. ANALOGUE APPROACH JUSTIFICATION
The source chemical and the target are structurally identical apart from the sodium ion in the source replacing the hydrogen on the parent acid target. The sodium ion does not have any effect on genotoxic properties and therefore data on the sodium salt may be read across to the target. Both the source and the target have been shown to be non-genotoxic in bacterial reverse mutations assays with S. typhimurium. In addition, the target chemical was found to be non-clastogenic is a study performed in Chinese hamster fibroblasts (V79), without metabolic activation, which is in accordance with the results of the chromosome aberration study using the source chemical (human lyphocytes; negative +/- metabolic activation; OECD 473).
4. DATA MATRIX
Refer to analogue justification provided in IUCLID section 13. - Reason / purpose for cross-reference:
- read-across source
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: intraperitoneal injection
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: oral gavage
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Negative genotoxicity results in vitro and in vivo; classification is not warranted.
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