Sodium dithionite

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

2008-06-24 to 2008-11-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attachment “Read-across concept – Human Health/Environment - Category approach for Inorganic sulfites/thiosulfates/dithionite" in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000-05-19

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2005-10-21

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphite

EC Number:

231-821-4

EC Name:

Sodium sulphite

Cas Number:

7757-83-7

Molecular formula:

NA2SO3

IUPAC Name:

disodium sulfite

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389)
- Test substance No.: 08/0250-1
- Date of production: 04 May 2008
- Physical state: solid, white
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed until 24 Apr 2010 as indicated by the sponsor, and the sponsor holds this responsibility.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (under N2; protected against humidity)

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The animals were selected according to the recommendations of the OECD and EU or on the basis of results published so far (Boller and Schmid (1970) and Heddle (1973))*. Moreover, there has been up to now most experience with or most data for NMRI mice.

*References:
- BOLLER, K.; SCHMID, W. Chemische Mutagenese beim Säuger. Das Knochenmark des chinesischen Hamsters als in-vivo-Testsystem. Hämatologische Befunde nach Behandlung mit Trenimon. Humangenetik, 11, 35 - 54 (1970)
- HEDDLE, J. A. A rapid in vivo test of chromosomal damage. Mut. Res., 18, 187 - 190 (1973)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks (information from the breeder)
- Assigned to test groups randomly: yes, under following basis: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.
- Weight at study initiation: mean 29.9 g
- Housing: individually in Makrolon cages, type M I, with Type Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was used as vehicle.
- Concentration of test material in vehicle: 25, 50, 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
- The stability of the test substance at room temperature in the vehicle purified water was determined analytically. For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations. These were kept at room temperature until the treatment of the last animal (approximately 1 hour) and then were kept deep-frozen. The determination of the concentrations in the vehicle was carried out by means of iodometric titration.

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 mice

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (20 mg/kg bw) and vincristine sulfate (0.15 mg/kg bw)

Examinations

Tissues and cell types examined:

polychromatic erythrocytes of bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute subcutan toxicity, deaths were observed down to 1500 mg/kg body weight. At 1000 mg/kg body weight all animals (male and female) survived showing clinical signs. The clinical sign only observed was making rough of the administration area. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 1000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
The two femora of the animals sacrificed by cervical dislocation was prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 uL fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10,000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value.
A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4). Slides were coded before microscopic analysis. Since the absolute values shown were rounded, but further calculation was based on
unrounded values, there may be deviations in the relative values given.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows: * p < 0.05, ** p < 0.01. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 mg/kg bw
- Clinical signs of toxicity in test animals: deaths were observed down to 1500 mg/kg body weight. At 1000 mg/kg body weight all animals (male and female) survived showing clinical signs.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.

Any other information on results incl. tables

Results:

Test group	Interval: 24 hours				Interval: 48 hours
	Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)		Polychromatic erythrocytes	Normocytes / total amount polychromatic erythrocytes	Cells with micronuclei (%)
Dose (mg/kg bw)			polychromatic	normochromatic			polychromatic	normochromatic
vehicle control	10000	4767	0.9	1.5	10000	4834	0.9	0.4
250	10000	5469	0.8	0.5
500	10000	5309	1.5	0.9
1000	10000	4211	1.4	1.4	10000	7289	1.2	0.5
20, cyclophosphamide	5000	3833	10.3	1.8
0.15, vincristine	5000	6097	48.4	1.3

Applicant's summary and conclusion

Conclusions:

The substance Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389) was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in purified water, was administered once subcutan to male animals at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case.
Purified water was used as the vehicle control, administered by the same route. Cyclophosphamide and vincristine sulfate were used as positive control substances.
The animals were sacrificed and the bone marrow of the 2 femora was prepared 24 and 48 hours after administration in the highest dose group of 1 000 mg/kg body weight and in the vehicle controls. In the test groups of 500 mg/kg and 250 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.
According to the results of the present study, the single subcutaneous administration of Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389) did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.
Under the experimental conditions chosen here, the test substance Natriumsulfit wasserfrei food grade (E 221) / (PRD 30042389) does not have any chromosome damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.