Sodium glyoxylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 04 July 2008 and 17 September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: in vivo micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium glyoxylate

EC Number:

220-298-8

EC Name:

Sodium glyoxylate

Cas Number:

2706-75-4

Molecular formula:

C2H2O3.Na

IUPAC Name:

sodium oxoacetate

Specific details on test material used for the study:

Name: Sodium glyoxylate (GOA-Na)
Lot No. C080501
Purity: 100%
Appearance: White powder
Storage conditions: Stored at room temperature (actual temperatures from the day of receipt to the final day of use: 16.6°C to 22.7°C, acceptable range: 10° to 30°C) shielded from light in an airtight container filled with nitrogen

Test animals

Species:

rat

Strain:

other: Crl: CD(SD)

Details on species / strain selection:

Rats are commonly used in micronucleus tests and are appropriate for evaluation of micronucleus induction. Rats are suitable for evaluation of micronucleus induction regardless of strain with minimal differences in sensitivity among strains. In addition, there is abundant historical data at the test facility and a large number of animals are available.

Sex:

male

Details on test animals or test system and environmental conditions:

Supplier: Tsukuba Breeding Center, Charles River Laboratories Japan Inc.
Age at arrival: 7 weeks old
Quarantine and acclimation: Animals were weighed on the day of their arrival (July 9, 2008) and at the end of the quarantine period (July 14, 2008) and observed for their general health condition once a day for 5 days (quarantine period). All animals were confirmed to be healthy with normal increases in their body weights. The animals were acclimated during the quarantine period and thereafter until the initiation of administration. During this period, the animals were observed once a day for their general health condition. Animals without abnormal clinical signs were used in the experiment.
Animal allocation to groups: The animals were randomly assigned to treatment groups based on their body weights measured on the day of animal allocation (July 14, 2008) by stratification based on their body weights to give homogeneous distribution of body weights among the groups.
Age at dosing: 8 weeks old
Rationale for age selection: It was reported that animals of this age are susceptible enough for evaluation of the micronucleus induction, and young adult rodents are recommended in the applied guidelines. In addition, a large number of animals are available from the breeder.
Body weight at initiation of administration: At the initiation of administration, the body weights of the animals ranged from 307 to 335 g, which were within the mean body weight± 20% (256.0 to 384.0 g)
Identification: Upon arrival and before allocation, animals were identified by marking the animal number (quarantine number, 19001 to 19035) on their tails with oil-based ink. During the quarantine period (before allocation), the cages were labeled with the study number, cage number, quarantine number, species, strain and sex. After the allocation, the cages were labeled with the study number, group name, dose, animal number, species, strain and sex.

Animal husbandry
Room temperature: Acceptable range: 19. 0 ° to 25.0°C (actual range: 21.0 ° to 23.3°C)
Relative humidity: Acceptable range: 35.0% to 75.0% (actual range: 47.9% to 65.5%)
Ventilation: 10 to 3 0 times/hour with filtered fresh air
Lighting period: 12 hours/day (7:00 to 19:00)

Housing instruments
Cages: Polycarbonate cages, TR-PC-200 (265Wx426Dx200H mm, Tokiwa Kagaku Kikai Co., Ltd. ) sterilized by autoclave were used and exchanged at animal allocation to groups.
Feeders: Stainless steel containers for pellet diet (Tokiwa Kagaku Kikai Co., Ltd. ) sterilized by autoclave were used and exchanged at animal allocation to groups.
Water bottles: Polycarbonate water bottles (700 mL, Tokiwa Kagaku Kikai Co., Ltd.) sterilized by autoclave were used and exchanged at animal allocation to groups.
Rack: A stainless steel rack (Tokiwa Kagaku Kikai Co., Ltd. ) disinfected with benzalkonium detergent (Micro Q uat®, Ecolab K.K. ) was used and exchanged at animal allocation to groups.

Bedding
Type: Autoclaved bedding for experimental animals (Beta-Chip®, Charles River Laboratories Japan, Inc.) and autoclaved stainless steel draining boards (Tokiwa Kagaku Kikai Co., Ltd.) placed on the bedding were used and exchanged at the same time as the cages.
Contaminant analysis: The results of the periodical test conducted by Eurofins Scientific Analytics were obtained from Charles River Laboratories Japan, Inc. and contaminants such as pesticide residue were confirmed to meet the criteria described in the standard operating procedure (SOP) of the test facility.

Diet
Pellet diet for experimental animals (MF, Oriental Yeast Co. , Ltd., lot No. 0 80 522)
The feed was given ad libitum. It was supplemented once 2 days after animal receipt, and was exchanged at group allocation.
Contaminant analysis: The results of analytical test conducted by Eurofins Scientific Analytics were obtained from Oriental Yeast Co., Ltd., and contaminants such as pesticide residue of the used lot were confirmed to meet the criteria described in SOP of the test facility.

Water supply
Description
Tap water was provided after filtration through a filter (pore size: 5 μm) and irradiation with UV light.
Watering
The tap water was given ad libitum. The tap water was exchanged 2 and 5 (day of group allocation) days after animal receipt.
Analysis
Tap water was analyzed periodically (twice a year) by Mitsubishi Chemical Analytech Co., Ltd. , and the analytical data of samples (collected on August 28, 2007 and February 19, 2008) were confirmed that the quality of the water met the specifications of SOP of the test facility.

Animal housing: Three or less animals were housed in a cage throughout the experimental period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Carboxymethyl cellulose sodium salt (CMC-Na) - 0. 5 w/v%
- Lot/batch no. (if required): PER2830

Details on exposure:

Preparation of test substance formulations
The test substance formulations were prepared just before each administration. The test substance was ground with a pestle, and a few drops of the vehicle (0.5 w/v% CMC-Na) were added into the mortar, and the test substance was mixed sufficiently to obtain a homogenous mixture. The paste was transferred into a measuring glass, and the mortar and pestle were rinsed with the vehicle and the resulting rinse solution was transferred into the measuring glass. The vehicle was added to the measuring glass to adjust the final volume. The test substance formulations were stored in an airtight container filled with nitrogen. This condition was maintained until just before administration.

Duration of treatment / exposure:

Bone marrow samples were taken 24 hrs after final dosing, therefore total duration was 48 hrs.

Frequency of treatment:

The dosing frequency was twice at a 24-hour interval.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 other: mg/kg (nominal)

Doses / concentrations 2

Dose / conc.:

500 other: mg/kg (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 other: mg/kg (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 other: mg/kg (nominal)

No. of animals per sex per dose:

5 males per test concentration, negative and positive control

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): This substance is commonly used as a positive control in micronucleus tests and is recommended in the applied guideline.
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg, dosing frequency the same as test susbtance

Examinations

Tissues and cell types examined:

Bone marrow: polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In acute dose oral toxicity study of GOA-Na using female Rats (Study No. B080605) compliant with GLP regulations, the test substance was administered to rats at a single dose of 2000 mg/kg. As a result, no mortality was observed; however, decreased body weight and hunchback position were noted in one of six animals, as well as diarrhea in one of six animals. A slight decreased in locomotor activity was noted i n all six animals and continuously thereafter following the day after dosing in two of six animals.
Therefore, the highest dose for this study was set at 2000 mg/kg, and the lower doses of 250, 500 and I 000 mg/kg were set with a common ratio of 2 in consideration the dosing frequency (twice). Furthermore, the negative and positive control groups treated with the vehicle (0.5 w/v% CMC-Na) and 20 mg/kg of CP, respectively, were also set. The dose volume was set at I 0 mL/kg of body weight. The dosing volume for each animal was calculated based on the body weight measured just before each administration.

TREATMENT:
A 5 mL disposable syringe with a gastric tube was used for administration of the dosing formulations. The test substance dosing formulations were stirred with a magnetic stirrer, and then the homogeneous formulation was administered.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were collected 24 hours after the final dosing.
Preparation of specimens
The specimens were prepared from the animals for evaluation:
1) The animals were anesthetized with an intraperitoneal injection of sodium thiopental (RAVONAL ®, Mitsubishi Tanabe Pharma Corporation, manufacturing No. 77005, concentration: 50 mg/mL) at 0.4 mL/body. The abdominal cavity was opened, and
the animals were euthanized by exsanguination from the abdominal aorta.
2) Bone marrow cells were collected by washing the cavity with 10% neutral buffered formalin (Muto Pure Chemicals Co., Ltd. , lot No. 080219).
3) The cell suspension was mixed further with 0. 5 mL of 10% neutral buffered formalin and then left to stand for 5 minutes to obtain the supernatant.
4) The supernatant was mixed with 1 mL of 10% neutral buffered formalin and centrifuged at about 170 x g (1000 rpm) for 5 minutes. The supernatant was discarded.
5) The precipitate was resuspended in a small amount of 10% neutral buffered formalin, and the resulting cell suspension was stored in a serum tube at room temperature.
6) The cell suspension was mixed and stained with the same volume of 500 μg/mL aqueous solution of acridine orange (Wako Pure Chemical Industries Ltd., lot No. WKR2239) just before the microscopic observation, and was spread on a slide.

METHOD OF ANALYSIS:
Evaluated animals:
In the negative and positive control groups, all survival animals were used for the evaluation. Three high dose groups of the test substance were used for the evaluation since no animals died in any test substance group.

Microscopic observation:
Bone marrow cell specimens were observed under a fluorescence microscope with a B excitation filter in a blind manner. One thousand erythrocytes [including both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE)] per animal were examined to determine the ratio of PCEs among the total erythrocytes. A total of 2000 PCEs (in two areas of view under a microscope, total of 2000 PCEs) was examined for the number of micronucleated PCEs (MNPCEs). Micronuclei in the cytoplasm and the types of erythrocytes were identified according to the methods of Hayashi et al. Small bodies with yellowish green fluorescence in the cytoplasm were recognized as micronuclei. Erythrocytes with red fluorescence in the cytoplasm were recognized as PCEs, and erythrocytes without any fluorescence in the cytoplasm were recognized as NCEs.

Evaluation criteria:

The ability of the test substance to induce MNPCEs was judged positive when the test substance significantly increased the number of MNPCEs as compared to the negative control with a dose-responsive.

Statistics:

The number of MNPCEs, percentage of PCEs among total erythrocytes and body weight of animals were statistically analyzed. Among the tests listed below requiring the significance levels of both 5% and I%, the significance level of 5% was applied at first. When there was no significant difference at the 5% level, the significance level of 1% was not applied.

Number of micronucleated polychromatic erythrocytes (MNPCEs)
(1) The statistical method of Kastenbaum and B owman was applied to compare the number of MNPCEs in each treatment group with that in the negative control group at the one-tailed significance level of 5% and 1%.
(2) Since no significant differences were noted in any of the test substance groups, Cochran-Armitage test for examining a dose-relationship was not applied.

Percentage of polychromatic erythrocytes (PCEs):
(1) The percentage of PCEs among total erythrocytes was statistically analyzed with a Toxicological Data Processing System (MiTOX, Mitsui Zosen System Research Inc.).
(2) All the data except for the positive control data were tested by Bartlett's test for homogeneity of variance among the groups (significance level: 5%).
(3) Williams' test was applied to determine the statistical difference between the test substance groups and the negative control group (two-tailed significance level: 5%), since the variance was homogeneous.
(4) Since there were no significant differences by Williams' test, Dunnett's test was performed to compare the mean values between each test substance group and that of the negative control group (two-tailed significance levels: 5% and 2%).
(5) Data of the positive control group was compared with that of the negative control group by the F test for homogeneity of variance between two groups (significance level: 5%).
(6) As the variance was homogeneous, the Student's t-test was applied to compare the mean values of the two groups (two-tailed significance levels: 5% and 1%).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Body weight
There were no significant differences between the test substance groups and the negative control group. However, the mean body weight decreased from the previous measurement just before the second dosing (after the first dosing), and decreased body weight was noted before preparation of specimens (after the second dosing) in one of five animals in the 2000 mg/kg test substance group.

Clinical signs
Loose stool was observed 3 hours after the first dosing in one of five animals in the 2000 mg/kg test substance group.

Micronucleus test
The total numbers of micronucleated polychromatic erythrocytes (MNPCEs) per 10000 (2000 cells x 5 animals/group) polychromatic erythrocytes (PCEs) were 10, 11, 11 and 10 at 0 (negative control), 500, 1000 and 2000 mg/kg, respectively. No statistically
significant increases were observed in the incidence of MNPCEs in the test substance groups as compared with the negative control group. No statistically significant differences were detected in the percentage of PCEs between the test substance groups
and the negative control group. In contrast, the number of MNPCEs per 10000 PCEs in the positive control group was 612, which was significantly higher than the negative control value (p ≤ 0.01). The percentage of PCEs in the positive control group was
significantly lower than that of the negative control group, indicating a sign of bone marrow toxicity (p ≤ 0.01).
The individual data of MNPCE incidence in the negative control group were 0.10 ± 0.06, which were within the historical control data of the test facility (0.13 ± 0.24% [mean ± 3SD, number of animals = 307]), whereas statistically significant increase
was observed in the positive control group. These results prove the validity of this study.

Applicant's summary and conclusion

Conclusions:

It was concluded that GOA-Na was negative for micronucleus induction in rat bone marrow, and that GOA-Na does not have genotoxic potential to induce chromosome aberration in vivo, under the conditions employed in this study.

Executive summary:

The test substance GOA-Na was tested in an in vivo micronucleus test in male Crl:CD(SD) rats (8 weeks old at dosing) to examine its ability to induce micronucleated cells in the bone marrow.

The animals (5 animals per group) received oral gavage doses administration of the negative control substance (0.5 w/v% CMC-Na), test substance (250, 500 , 1000 and 2000 mg/kg) and positive control substance (cyclophosphamide monohydrate, 20 mg/kg) twice at a 24-hour interval. Bone marrow cells were collected 24 hours after the final administration. Bone marrow cell specimens were prepared to determine the incidence of micronucleated polychromatic erythrocytes (MNPCEs) and percentage of polychromatic erythrocytes (PCEs).

No statistically significant difference was detected in the number of MNPCEs per 10000 PCEs (2000 cells x 5 animals/group) between the test substance groups and the negative control group. No significant decrease was detected in the percentage of PCEs in any of the test substance groups as compared to the negative control group, indicating no bone marrow toxicity. In contrast, the number of MNPCEs significantly increased, and the percentage of PCEs significantly decreased in the positive control group as compared to the negative control group.

The incidence of MNPCEs in the negative control group was within the range of the historical control data of the test facility. Significant increases were detected in the incidence of MNPCEs in the positive control group. These results prove the validity of this study.

It was concluded that GOA-Na does not induce micronucleated erythrocytes in rat bone marrow cells under the conditions employed in this study.