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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-02-4 to 1986-03-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: JAPAN: Guidelines for Toxicity Studies of Drugs
Version / remarks:
1984-02-15, Yakushin 118
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dihydroxyacetone
EC Number:
202-494-5
EC Name:
1,3-dihydroxyacetone
Cas Number:
96-26-4
Molecular formula:
C3H6O3
IUPAC Name:
1,3-dihydroxyacetone
Details on test material:
Name: Dihydroxyacetone

Test animals

Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals or test system and environmental conditions:
Seven to eight-week-old ddY male and female mice were purchased from SLC Inc. and used at 8 to 10 weeks of age. The mice were given commercial food pellets and tap water ad libitum throughout acclimation and experimental periods. For the main study, only male mice were used. Male and female mice were used for the acute toxicity test and the pilot study.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: physiol. saline (0.9% NaCl)
- Justification for choice of solvent/vehicle: good solubility and stability of test item
- Concentration of test material in vehicle: 125, 250 and 500 mg/mL (main study)
1, 10, 50, 100, and 500 mg/mL (pilot study)
100, 200, 300, 400, and 500 mg/mL (acute toxicity test)
- Volume of vehicle administered: 10 mL/kg bw
Details on exposure:
The test chemical solutions and physiological saline (negative control) were given as single doses intraperitoneally to mice in a volume of 10 mL/kg.
Duration of treatment / exposure:
Sampling time was 24 hours after administration of the test material based on the results of a small acute test and the pilot micronucleus test.
Frequency of treatment:
The animals were treated once.
Post exposure period:
24 h after administration the animals were sacrificed. The post exposure time was shorter than recommended by the guideline. However, the substance is very hydrophilic; DHA was administered intraperitoneally at doses far above the limit dose and the results showed a clear negative response. Based on these considerations, the shorter post exposure time does not have influenced the result of the study.
Doses / concentrationsopen allclose all
Dose / conc.:
1 250 mg/kg bw/day (nominal)
Dose / conc.:
2 500 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 male mice per dose in the main test
Control animals:
yes, concurrent vehicle
Positive control(s):
Main Study: Mitomycin C
- Justification for choice of positive control(s): Mitomycin C is recommended by Guideline
- Route of administration: i.p.
- Doses / concentrations: 2 mg/kg

Pilot Study:
Mitomycin C (2 mg/kg, i.p., sampling at 24h after treatment)
6-Mercaptopurine (50 mg/kg, i.p., sampling at 48h after treatment)
7,12-Dimethylbenz[a]anthracene (40 mg/kg, i.p., 72h after treatment)

Examinations

Tissues and cell types examined:
For microscopic investigation, at least two specimens from each animal were prepared and coded. The number of micronucleated polychromatic erythrocytes (MNPCEs) per 1000 polychromatic erythrocytes (PCEs) per animal was determined. The proportion of PCEs to total erythrocytes b(PCEs and normochromatic erythrocytes) based on 1000 erythrocytes per animal was also determined to evaluate the bone marrow toxicity of DHA. The number of evaluated cells is below the figures in the OECD guideline, however, based on the very high test dose exceeding the limit dose of the guideline (5000 mg/kg bw) and the clear negative result, this deviation is not considered to have an impact on the reliability of the study.
Details of tissue and slide preparation:
Mice were euthanized by cervical dislocation at each sampling time and the femoral marrow cells were flushed out with fetal bovine serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant discarded to get appropriate cell suspension concentrations. The suspensions were smeared onto clean glass slides and allowed to dry. The specimens were fixed by addition of methanol for 5 minutes, and then stained with 3% Giemsa solution containing 0.004% citric acid for 30 minutes. The slides were then rinsed and allowed to dry before micronucleus examination. At least two specimens were prepared per animal.
Evaluation criteria:
The result would be judged to be positive when the frequency of MNPCEs in treated animals was statistically significantly different from controls at the probability level of 5%.
Statistics:
Statistical analysis was only performed in the main study micronucleus test. The statistical significance of the difference between the incidence of MNPCEs in DHA treatment groups or the positive control group and the negative control group was evaluated by the methods of Kastenbaum and Bawman.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups compared to the negative control (0.17 % in control vs 0.17, 0.25, 0.2 % for the low mid and high dose group, respectively). The PCE ratio was not changed in the treatment groups. The number of micronucleated PCE in the positive control was 2.12 % and statistically significantly different to control.

For details please refer to attached table.

Applicant's summary and conclusion

Conclusions:
DHA was not mutagenic in this in vivo micronucleus assay in male mice under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

An in vivo mouse bone marrow micronucleus test was conducted with the test material to evaluate its potential for clastogenicity in male ddY mice. The maximal dose level of the test material was 5000 mg/kg on the basis of the maximal tolerated dose estimated by an acute toxicity test, with intermediate and low dose levels set at 2500 and 1250 mg/kg, respectively. The sampling time was 24 hr after administration, based upon the result of a pilot micronucleus test. The main study micronucleus test was performed under the optimal experimental conditions, which were based on the results from the small-scale acute toxicity test and the pilot micronucleus test. Dihydroxyacetone was intraperitoneally singly administered to mice, and the induction of micronuclei was evaluated in femoral bone marrow. The number of micronucleated PCE in animals treated with Dihydroxyacetone was in the control range and did not show a statistically significant increase compared to the negative control.

The results suggest that dihydroxyacetone is not clastogenic in vivo under these experimental conditions

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