Magnesium hydroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

A 90-day repeated dose oral toxicity study of magnesium chloride in F344 rats

Author:

Takizawa T. et al.

Year:

2000

Bibliographic source:

Bulletin of National Institute of Health Sciences 2000; 118: 63-70

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

10 animals at each dose level instead of 20 animals at each dose level

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium chloride hexahydrate

EC Number:

616-575-1

Cas Number:

7791-18-6

IUPAC Name:

Magnesium chloride hexahydrate

Specific details on test material used for the study:

- Purity: 98%
- Appearance: Colourless to white deliquescent crystalline powder

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: The rats were subjected to testing at 6 weeks old.
- Housing: 3-4 rats per cage in plastic cages laid on a soft chip floor cloth in a barrier system room.
- Diet: Gave 2.5%, 0.5% and 0.1% magnesium chloride mixed feed to treatment groups and basic feed to the control group.
- Water: tap water, ad libitum
- Acclimation period: 8 days acclimatising

ENVIRONMENTAL CONDITIONS
- Temperature (°C):23 ±2°C
- Humidity (%):55±5%
- Air changes (per hr): Ventilation of 18 cycles/hr (all fresh air)
- Photoperiod (hrs dark / hrs light): 12 hour light cycle (light from 07.00 to 19.00)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with: Magnesium chloride hexahydrate was mixed with basic feed and solid mixed feed shaped as pellets was used.
- Storage of food: After refrigerating and storing shielded from light in a sealed package for 3 months, and verifying its safety after storing indoors in an open container for 2 weeks at room temperature in scattered light, it was stored in a refrigerated feed storehouse until use, replacing the feed givent o rats accordingly within the period during which its safety was verified.

VEHICLE
- Justification for use and choice of vehicle : Feed-The magnesium chloride hexahydrate concentration was determined in mixed feed based on a preliminary 2-week study conducted beforehand. In order to confrim mixed feed avoidance and preliminary toxicity in rats, powdered feed mixed with 5% magnesium chloride hexahydrate was administered, as well as a half dose of 2.5%.

See table 1 for details on food consumption and the amount of magnesium chloride consumed

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The stability of this compound diet was proven in a storage test comprising 3 months closed storing under cooling at a dark place followed by 2 weeks open storing under diffuse light at ambient temperature.

Duration of treatment / exposure:

Rats received dietary supplementation of magnesium chloride at 2.5, 0.5, 0.1 and 0 % for 90 days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.5 other: %

Remarks:

nominal in diet

Doses / concentrations 2

Dose / conc.:

0.5 other: %

Remarks:

nominal in diet

Doses / concentrations 3

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

The test substance concentrations in the compound diet were determined from the results of a 2-week pretest conducted before the present study. In this pretest in which rats were subjected to 2-week administration of a pulverised compound diet with a test substance concentration of 5% and 2.5%, no decrease in diet consumption was observed in both groups. However, diarrhea, increase of water consumption and a tendency of inhibited body weight increase occurred in the two groups. Furthermore, autopsy revealed softening of caecum and colon content. From these results, the maximum concentration applied in this final study was set at the concentration (i.e. 2.5%) at which the administration of the test substance is likely to show effects.
The animals of the test substance groups were fed arbitrarily with compound diet with magnesium chloride concentrations of 0.1%, 0.5%, or 2.5% respectively.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Within the test period, the general condition was verified daily and whether they were dead or alive, and the amount of food and water consumed was measured once a week.

BODY WEIGHT: Yes
Body weight was recorded weekly

FOOD AND WATER CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption and water consumption were recorded weekly

HAEMATOLOGY: Yes
Red blood cell count, hemoglobin, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count and platelet count were measured using a multi-automatic blood cell counter.
In addition, wright-stained smear samples were prepared and, using an automatic blood cell analyzer, bandneutrophilocytes (Band), segmented neutrophilocytes, eosinocytes, basocytes, lymphocytes, monocytes and nucleated erythrocytes were measured.

CLINICAL CHEMISTRY: Yes
As for the serum, total protein, albumin, albumin/globulin, total cholesterol, γ-glutamyl transpeptidase, cholinesterase, alanine transaminase, aspartic acid transaminase, lactic acid dehydrogenase, alkaline phosphatase, blood urea nitrogen, creatinine, calcium, inorganic phosphorus, sodium, potassium and chloride were measured.

Sacrifice and pathology:

After blood-taking the animals were killed by exsanguination and subjected to autopsy. Brains, thymus glands, lungs, spleens, livers, adrenal glands, suprarenal glands, kidneys, and testis were weighed. Furthermore, for histopathological examination of the animals of the control group and the maximum dose group, the lymph nodes (neck section and mesenteries), thymus glands, bones and bone marrow (sternal bones and thighbones), thymus glands, tracheas, lungs, hearts, thyroids and parathyroids, tongues, gullets, proventriculuses, glandular stomachs, duodenums, small intestines (jejunum and ileum), colons (cecum, intestinum colon, rectum), livers, pancreases, urinary bladders, kidneys, suprarenal glands, vesicular glands, prostate gland, epididymis, ovaries and oviducts, uteruses, vaginas, brains, hypophyses, sciatic nerves, skeletal muscles, spinal cords, eyeballs, and adnexal organs were fixed with 10% neutral-buffered formalin, or, as for the testis (from 5 animals of the control group and 5 animals from the maximum dose group), Bouin's fixature, and, then, cut into thin slices according to conventional methods; successively the slices were stained with hematoxylin-eosin stain.

Statistics:

The results of body weight, hematological analysis, serum biochemistry analysis and organ weight were compared during the treatment period for each group using one 0-way analysis of variance. For items shown to be significant at a 5% level of significance, the Dunnett two-tailed test at a 5% and 1% level of significance was used to compare results with the control group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In treatment week 2 surviving male and female rats in the 2.5% group were observed with stained fur likely attributable to soft stool. This disappeared during the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male rat in the control group had to be slaughtered because in the treatment week 5 he was observed with a 3 -4 cm diameter tumour on his left gluteus, discolouration (anaemia) of the ears and eyes, and decreased body weight. Autopsy revealed a swollen spleen and discolouration in the liver.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males in the 2.5% group were observed with decreased body weight starting in treatment week 5, which was significant with body weight during the final week of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No clear difference was observed between the control group and treatment groups with regard to the amount of food consumed. The average daily amount of food consumed in treated rats in the 0.1% group was 62 mg/kg for males and 59 mg/kg for females, 308 mg/kg for males and 299 mg/kg for females in the 0.5% group, and 1,600 mg/kg for males and 1,531 mg/kg for females in the 2.5% group, which increased relative to magnesium chloride concentration.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The amount of water consumed by male and female rats in the 2.5% group was observed to increase continually from treatment week 1 throughout the treatment period.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In hematological tests, although among males the eosinophil ratio in red blood cell count, hemoglobin, hematocrits and while blood cells was lower in the 2.5% group versus the control group, while red blood cell count, hemoglobin and hematocrits were lower in the 0.5% group as well, these levels were lower than those in the 2.5% group and changes were not correlative to dosage levels. Among female, hemoglobin and MCH were higher in the 2.5% group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In serum biochemical tests, males in the 2.5% group were observed with decreased cholesterol, cholinesterase, alkaline phosphatase and inorganic phosphorus, while the same changes were observed for cholinesterase, alkaline phosphatase and inorganic phosphorus. Females in the 2.5% group were observed with decreased lactate dehydrogenase. Though a number of other items with statistical significance were observed in serum biochemical tests, they were all poorly correlative with dosage levels.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Among males, rats in the 2.5% group experienced a mild decrease in body weight at the time of autopsy, while the actual weight of the spleen decreased along with the actual weight and relative weight of the liver. Although the relative weight of the liver decreased in the 0.5% and 0.1% groups, changes in the 0.1% group were the largest and did not correlate with dosage levels. Among females, rats in the 2.5% group showed a decrease in actual and relative weight of the liver.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

While basophilia of the renal tubule and eosinophilic bodies in the tubular epithelium were seen in both the treatment groups and control group, no difference in changes were seen between the treatment groups and control group.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathologically, the tumor on the left gluteus of the slaughtered rat in the control group was a malignant epithelial tumor due to extensive necrosis.

Details on results:

No treatment-related death was observed during the study. Transient soft stool and increase in water consumption in males and females, and slight reduction in body weight gain in males, and changes in liver weight in females were noted at the higher dose level of 2.5%. There were no toxic changes in food consumption, organ weights, hematology and biochemistry, and histopathological examinations in any treated-groups.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

308 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

water consumption and compound intake

2

Dose descriptor:

NOAEL

Effect level:

299 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, the NOAEL was estimated to be 308 mg/kg bw/day in males and 299 mg/kg bw/day in females.

Executive summary:

In a 90-day repeated dose study, conducted similar to OECD guideline 408, 10 male and female Wistar rats were treated with magnesium chloride hexahydrate at doses of 0.1, 0.5, and 2.5% for 90 days. In the 2.5% group, soft stool, decreased body weight gain in males and increased water intake was observed. Among females, rats in the 2.5% group showed a decrease in actual and relative weight of the liver. The soft stool disappeared during the treatment period. No treatment-related death was observed during the study.

Based on the results of this study, a NOAEL of 299 mg/kg bw/day for females (0.5% dose group) and 308 mg/kg bw/day for males (0.5% dose group) was determined.