Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dehydroacetic acid, sodium salt DHA-Na assessed in an Ames test at concentrations up to 1820 μg/plate (with or without metabolic activation) was not mutagenic.

Dehydroacetic acid sodium salt (with or without metabolic activation) did not induce chromosomal aberrations in human lymphocytes after in vitro treatment.

Dehydroacetic acid sodium salt, at concentrations between 1x10^-3to 1x10^-5M was not genotoxic in a chromosome aberration and sister chromatid exchange study.

Dehydroacetic acid sodium salt is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

Remarks:

Chromosome aberrations and sister chromatid exchange

Type of information:

experimental study

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

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 chromatid 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 (Dehydracetic 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 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Strain with AT base pair missing; not tested up to the maximum test concentration for soluble non-cytotoxic substances

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

yes

Remarks:

Strain with AT base pair missing; not tested up to maximum test concentration for soluble non-cytotoxic substances

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats and hamsters treated with Aroclor 1254.

Test concentrations with justification for top dose:

33, 100, 333, 1000 and 1820 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive control substance:

other: -S9: sodium azide for TA100 and TA1535; 9-aminoacridine for TA1537; 4-nitro-o-phenylendiamine for TA98; +S9: 2 aminoanthracene (2.5 or 5 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A chemical was judged to be mutagenic or weakly mutagenic if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable if a reproducible increase of his+ revertants did not meet the criteria for either mutagenic or weakly mutagenic, or if only single doses produced an increase in his+ revertants in repeat trials.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

other: TA 100, TAA 1535 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: ≥ 1000 µg/plate; + S9: ≥1820 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test substance was tested inititally in a toxicity assay to determine the appropriate dose range (data not shown).

Table 1 Test Results

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	0	122 ± 24.2	5 ± 1.7	14 ± 2.5	4 ± 2.0
–	33	107 ± 2.9	6 ± 0.9	12 ± 0.7	5 ± 1.2
–	100	115 ± 14.1	6 ± 1.3	11 ± 0.3	5 ± 2.2
–	333	104 ± 5.5	3 ± 0.9	12 ± 0.6	5 ± 0.6
–	1000	100 ± 5.8	4 ± 1.2	8 ± 1.9	3 ± 0.9
–	1820	85 ± 9.5	4 ± 1.0	8 ± 0.9	4 ± 1.8
Positive controls, –S9	Name	SA	SA	4-NOP	9-AA
	Mean No. of colonies/plate (average of 3 ± SD)	571 ± 9.9	168 ± 5.9	407 ± 11.3	1088 ± 11.7
+	0	158 ± 5.0	6 ± 2.3	26 ± 4.5	6 ± 1.8
+	33	148 ± 12.2	4 ± 1.2	17 ± 4.2	6 ± 1.2
+	100	161 ± 14.7	5 ± 0.6	26 ± 1.0	5 ± 1.2
+	333	146 ± 12.7	7 ± 2.0	17 ± 1.5	4 ± 1.2
+	1000	134 ± 28.7	5 ± 0.3	17 ± 2.6	7 ± 1.7
+	1820	138 ± 23.6	6 ± 1.3	11 ± 2.0	5 ± 1.7
Positive controls, +S9 (rat)	Name	2AA	2AA	2AA	2AA
	Mean No. of colonies/plate (average of 3 ± SD)	3149 ± 19.0	80 ± 4.7	1621 ± 71.4	129 ± 9.2

Conclusions:

Dehydroacetic acid, sodium salt DHA-Na (with or without metabolic activation) was not mutagenic in this Ames test.

Executive summary:

Dehydroacetic acid, sodium salt DHA-Na assessed in an Ames test at concentrations up to 1820µg/plate (with or without metabolic activation ) was shown to be not mutagenic.

Reference 3

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:

29 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 (with or without metabolic activation) did not induce chromosomal aberrations in human lymphocytes
after in 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 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29th August - 2nd October 2017

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)

Version / remarks:

July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Sprague Dawley rat liver induced with phenobarbital/ β-naphthoflavone

Test concentrations with justification for top dose:

0.5, 1, 2, 4, 6, 8 and 10 mM
Based on pre-experiment for toxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI cell culture medium (RPMI + 5% HS).
- Justification for choice of solvent/vehicle: Based on results of a solubility test

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1E+07

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable


SELECTION AGENT (mutation assays): Trifluorothymidine

SPINDLE INHIBITOR (cytogenetic assays): Not applicable

STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup]) analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: none
- Evaporation from medium: Not applicable
- Water solubility: Soluble
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Results of preliminary test shown in Tables 2 & 3 below.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
EMS (300µg/mL): Mean 726.5; range 318.7 - 2919.0; SD 203.5; RSD 28.6%; n=211
MMS (10 µg/mL): Mean 763.5; range 376.4 - 2416.1; SD 421.6; RSD 55.2%; n=254
B[a]P (2.5 µg/mL): Mean 635.9; range 303.6 - 1267.2; SD 167.8; RSD 26.4%; n=255

- Negative (solvent/vehicle) historical control data:
-S9: Mean 87.9; range 50.1 - 170.3; SD 25.5; RSD 29.0%; n=447
+S9: Mean 85.1; range 50.1-165.9; SD 24.3; RSD 28.6%; n=653

Results of Preliminary test:

Table2:  Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concen-tration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	362000	839000	1460000	12.2	100.5
C2		314000	860000	1410000	12.1	99.5
1	0.2	330000	904000	1380000	12.5	102.4
2	0.5	349000	926000	1420000	13.1	107.9
3	2.5	306000	828000	1520000	12.6	103.3
4	5	329000	738000	1550000	11.4	93.9
5	7.5	301000	664000	1340000	8.9	73.0
6	10	298000	668000	1180000	7.9	64.7

 

C:         Negative control

a:         Suspension Growth, SG = [((value 24h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))];

             * : for value 24 h > 3x10⁵then value 24 h = 3x10⁵

b:          Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

 

Table3:  Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concen-tration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	263000	712000	1260000	9.0	96.1
C2		265000	764000	1270000	9.7	103.9
1	0.2	227000	625000	1270000	7.9	85.0
2	0.5	251000	712000	1260000	9.0	96.1
3	2.5	214000	640000	1270000	8.1	87.1
4	5	230000	634000	1240000	7.9	84.2
5	7.5	218000	588000	1240000	7.3	78.1
6	10	216000	491000	1050000	5.2	55.2

 

C:         Negative control

a:         Suspension Growth, SG = [((value 24h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))];

             * : for value 24 h > 3x10⁵then value 24 h = 3x10⁵

b:          Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Conclusions:

In the described mutagenicity test under the experimental conditions reported, the test item Dehydroacetic acid sodium salt is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item Dehydroacetic acid sodium salt was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations (without and with metabolic activation): 0.5, 1, 2, 4, 6, 8 and 10 mM.

No precipitation of the test item was noted in the experiment. No growth inhibition was observed without and with metabolic activation. No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Dehydroacetic acid sodium salt is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

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

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:

not specified

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

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: oral gavage

Additional information on results:

See attached table

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 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.