Magnesium chloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 2 is applied because the original report is not available. But the study was perfomed according to the OECD 421 and this study was peer reviewed (SIDS, OECD HPV program). This study is available in the SIDS of magnesium chloride (SIDS Initial Assessment Report For SIAM 32, 18-21 April 2011, Paris, France)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2003

Reference 2

Reference Type:

publication

Title:

SIDS Dossier , OECD HPV Chemical Programme, SIDS Dossier approved at SIAM 32 (19-21 April 2011)

Author:

OECD HPV Chemical Programme

Year:

2011

Bibliographic source:

SIDS Dossier and SIDS Initial Assessment Report For SIAM 32

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium chloride

EC Number:

232-094-6

EC Name:

Magnesium chloride

Cas Number:

7786-30-3

Molecular formula:

Cl2Mg

IUPAC Name:

magnesium dichloride

Details on test material:

- Analytical purity: reagent grade

Specific details on test material used for the study:

Molecular formular: MgCl2
- Supplier:Sigma-Aldrich, Inc.
- Lot No.: 052K0893
- Purity: 98.0%
- Description: White powder
- Storage condition: Room temperature

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

[Test animals]
- Source: Samtako Bio Korea Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 33.9 to 37.5 grams
- Fasting period before study: Not reported
- Housing: Metallic cage
- Diet (e.g. ad libitum): ad libitum (Purina experimental diet for rats 5057 (Agri Brands Prina Korea, Inc.))
- Water (e.g. ad libitum): ad libitum (Filtered and sterilized drinking water)

[Environmental conditions]
- Temperature (°C): 20 to 25 °C
- Humidity (%): 50 +/-10 %
- Air changes (per hr): 10 to 15 per hours
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Chemical name: Distilled water
- CAS No.: Not reported
- Lot No.: Not reported
- Storage condition: Room temperature

Duration of treatment / exposure:

24 hours

Frequency of treatment:

two times per 24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6/male/dose

Control animals:

yes

Positive control(s):

- Chemical name: Mitomycin C (MMC)
- CAS No.: Not reported
- Supplier: Sigma-Aldrich Korea.
- Lot No.: 111K2511

Examinations

Tissues and cell types examined:

Eahc specimen was observed by microscope with a magnification of 1,000. To calculate the proportion of PCE in the total erythrocytes [PCE/(PCE+NCE)], sum of polychromatic eryhrocyte (PCE) and normochromatic erythrocyte (NCE) should be 1,000 and to calculate the proportion of MNPCE in the micronucleated polychromatic erythrocyte [MNPCE/(1,000 PCE)], polychromatic erythrocyte (PCE) were counted 2,000.

Details of tissue and slide preparation:

Bone marrow cells collection was carried out at the time decided in the induction of micronucleus, 24 hours. To collect the bone marrow cells, mice were euthanized by dislocation of cervical vertebrae, after that the 200 uL of fetal bovine serum (Hyclone, USA) was run through an extracted femur using a 1.0 mL disposable syringe with a needle. Finally, collected samples were centrifuged at 1,000 rpm, 4℃, for 5 miniutes. The supernatant was discarded and the cell pellet was mixed well and then smeared on the slide glass. Two specimens per animal were produced, in which they were air-dried and stained with a 5% Giemsa solution for 30 minutes and observed at 1,000 fold magnification under the microscope.

Evaluation criteria:

It was judged as a positive when MNPCE was increased significantly in dose dependent manner or there was reproducible increase in number of MNPCE at least one test level.

Statistics:

In accordance with the table of Kastenbaum and Bowman, statistical significance was tested for the induction frequency of micronucleus and the t-test was conducted for an appearance rate for polychromatic erytrocyte (PCE) in 5% significant levels in p-value statistically. We conducted the ANOVA test for a body weight change within 5% significant levels in p-value statistically.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

[Clinical signs and mortality]
- There were no treatment related mortality and clinical signs for all groups. No dead animals were founded in all groups.[Measurement of body weight (Table 2)]
- There was no significant body weight change for the all test groups during the the test period.[In vivo micronucleus test (Table 3)]
- The micronucleated polychromatic erythrocytes (MNPCE) were not increased all the time in the preliminary test to establish a bone marrow cells collection times. Therefore, collection time was decided at 24 hours after the treatment. The frequency of micronucleated polychromatic erythrocytes (MNPCE) induction per 1,000 polychromatic erythrocytes (PCE) was 0.8, 0.4, 0.8 and 1.1 for the negative control, 30, 60 and 120 mg/kg of treatment groups, respectively. Therefore, there was no significant difference in appearance rate of micronicleated polychromatic erythrocytes (MNPCE) in polychromatic erythrocytes (PCE) for all treatment groups in contrast with that of the negative control. In the positive control, the frequency of micronucleated MNPCE induction per 1,000 PCE was 79.0 and micronucleated PCE was significantly increased in contrast with that of the negative control. The PCE/(PCE+NCE) ratio, criterion of cytotoxicity, following dose levels 0 (negative control), 30, 60 and 120 mg/kg and positive control (MMC) were 0.499, 0.503, 0.499, 0.499 and 0.496, respectively. There was no significant difference between the treatment groups and control group.

Any other information on results incl. tables

Table 2. Change of body weight

Hours after treatment	Groups	G1	G2	G3	G4	G5
0	Dose (mg/kg)	0 (NC)	30	60	120	2.0 (PC)
	Mean	36	36.2	36	36	36
	SD	1.5	1.3	1.3	1.0	1.0
	N	6	6	6	6	6
24	Mean	35.2	34.9	35.1	34.3	35.2
	SD	1.3	1.7	1.2	1.8	1.6
	N	6	6	6	6	6

Table 3. Micronucleus test of magnesium chloride (CAS No.: 7786 -30 -3) in male ICR mice

Groups	Test substance	Dose (mg/kg)	N	Sampling time (Hr)	MNPCE/1,000 PCE (Mean+/-SD)	PCE/(PCE+NCE) (Mean +/- SD)
G1	Distilled water	0	6	24	0.8 +/- 0.5	0.499 +/- 0.005
G2	Magnesium chloride	30	6	24	0.4 +/- 0.5	0.503 +/- 0.005
G3		60	6	24	0.8 +/- 0.6	0.499 +/- 0.008
G4		120	6	24	1.1 +/- 0.4	0.499 +/- 0.008
G5	Mitomycin C	2	6	24	79.0^* +/-5.4	0.496 +/- 0.004

*: Indicate significan difference at p<0.01 level

Applicant's summary and conclusion

Conclusions:

In conclusion, the test substance, magnesium chloride did not influence on the micronucleus formation in the bone marrow cells in mice.

Executive summary:

This study was designed to determine the mutagenic potential of magnesium chloride in the micronucleus test using bone marrow cells in ICR mice. The serial doses of the test substance such as 30, 60 and 120 mg/kg were administrated by intraperitoneally (i.p).10 mL/kg of distilled water was also administrated by i.p to negative control group and 2.0 mg/kg of mitomycin C. In 24 hours after the treatment, all mice were sacrificed then bone marrow cells in thigh were collected. The specimens were stained with 5% Giemsa solution. The frequency of micronucleus induction was observed by a microscope. As a result, there was no significant difference in appearance rate (MNPCE/1,000 PCE) of micronucleated polychromatic erythrocytes (MNPCE) in polychromatic erythrocytes (PCE) for all treatment groups in contrast with that of the negative control. However, in the positive control group, an appearance rate of MNPCE was increased significantly in contrast with that of the negative control. The PCE/(PCE+NCE) ratio for all treatment groups was not different significantly in contrast with that of the negative control. In conclusion, the test substance, magnesium chloride did not influence on the micronucleus formation in the bone marrow cells in mice