3-acetyl-6-methyl-2H-pyran-2,4(3H)-dione

Endpoint summary

Key value for chemical safety assessment

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

Reference

Reference 1

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.

Reference 2

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.

Reference 3

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.

Reference 4

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

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

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.

Reference 5

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

Reference 6

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

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

Reference

Reference 1

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.

Reference 2

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Negative genotoxicity results in vitro and in vivo; classification is not warranted.