Magnesium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

magnesium chloride hexahydrate

IUPAC Name:

magnesium chloride hexahydrate

Constituent 2

Reference substance name:

7791-18-6

Cas Number:

7791-18-6

IUPAC Name:

7791-18-6

Details on test material:

water content (specification): 51-55% (53.4%)
Colour: colourless
Physical state: solid, crystals
Storage: at room temperature, in a tightly closed package
Solvent: water
Stability after opening: instable after repeated contact to air
pH: 5.5 - 7.0 (5% solution at 20° C)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Young healthy male and nulliparous, non pregnant, female rats [strain: Wistar Crl:WI] (Full-Barrier), were used in this study. The animals were derived from a controlled full barrier maintained breeding system (SPF) (Source: Charles River, 97633 Sulzfeld, Germany).
At the beginning of the study, the age of the animals was 8-9 weeks. The range of the body weight was:
Females: 144.32 to 216.48 g, (mean: 180.40 g, ± 20%= 36.08 g)
Males: 211.18 to 316.77 g, (mean: 263.98 g, ± 20%= 52.80 g).

ENVIRONMENTAL CONDITIONS
After an adequate acclimatisation period (at least five days), the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions: temperature: 22 ± 3 °C, relative humidity: 55 ± 10%, artificial light, sequence being 12 hours light, 12 hours dark, air change: 10 x / hour, free access to Altromin 1324 maintenance diet, free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

MgCl2, 6H2O was formulated in deionised water with administration volume of 10 mL/kg body weight. Control animals were handled identically as the treated groups and received deionised water in a similar volume as the treated groups.
MgCl2, 6H2O formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and female animals, and during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days. Dose volumes were adjusted weekly based on body weight measurement.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

There were 3 sorts of sample:
1/ Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
2/ Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.
3/ Samples for stability analysis were taken in the first week of the study. After preparation small portions (in triplicate) were frozen immediately at -20 °C (0 h) and small portions were kept for 6 hours at room temperature before they were frozen at -20 °C (6h) to determine the stability of the test item in the vehicle.

All formulation samples were stored frozen (approximately -20°C) till the shipment to analytic laboratory and analysis were performed.

In conclusion, the dose formulation analysis of samples collected during study week 1, 3, 5 and 7 from LD, MD and HD group showed very good recovery.

Duration of treatment / exposure:

28-29 days for males
maximum 54 days for females (14 days pre mating, 14 days mating, during gestation period and up to post natal day 3)

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Basis: actual ingested

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Basis: actual ingested

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

98 animals were included in the study. Control- 12/12 (Male/Female), LD- 10/10 (Male/Female), MD- 12/12 (Male/Female) and HD- 15/15 (Male/Female)

Control animals:

yes, plain diet

Details on study design:

The animals were randomly assigned (using Microsoft Excel template) to the dose/control groups, each animal was assigned a unique identification number and caged individually.
The test item was administered by gavage using a gavaging cannula. The maximum dose volume administered was 10 mL / kg body weight.
For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- General clinical observations were made twice a day except during weekend and holidays where observations were made only once, approximately at the same time each day and considering the peak period of anticipated effects after dosing
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Once before the first exposure, and once a week thereafter.
- Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalization, diarrhea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males weekly during the entire study period and at terminal sacrifice. Females were weighed weekly during pre mating period, on gestation day 0, 7, 14, 20 and on PND 1 (within 24 hours of parturition) and 4 along with pups.

FOOD CONSUMPTION: Yes
- Food consumption was measured on corresponding day of body weight (in males only during pre mating period) after beginning of the dose administration. Food consumption was not measured during mating period.

WATER CONSUMPTION No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of treatment
- Anaesthetic used for blood collection: Yes: Isoflourane anesthesia
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each group.
- Parameters checked in table were examined: haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count, platelet count, blood clotting time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 of treatment
- Animals fasted: Yes
- How many animals: five males and five females of each group
- Parameters checked in table were examined: cholesterol, urea, creatinine, total protein and albumin, two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase).

URINALYSIS: Yes
- Time schedule for collection of urine: day 28 of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- How many animals: seven randomly selected males of HD group, six randomly selected males of control and MD group and five randomly selected males of LD group at the terminal sacrifice.
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated).
- Dose groups that were examined: five randomly selected males and females from each group
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behaviour observations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Males were sacrificed after the completion of mating period (total dosing of 28-29 days) and females were sacrificed on respective post natal day 4 along with pups by using high dose of sodium pentobarbital. However, in the animals randomly selected for the blood collection, anaesthesia solution (Ketamin/Xylazin, 2:1) was used. At the time of sacrifice or death during the study, the adult animals were subjected to a full, detailed gross necropsy which includeed careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.

ORGAN WEIGHT: Yes
- Reproductive organs from all animals were weighed (testes, epididymides, prostate, seminal vesicle with coagulating glands as whole, ovaries, uterus with cervix as applicable). Apart from the reproductive organs from all animals, from five adult males and females randomly selected from each group, the wet weight of the liver, kidneys, adrenals, thymus, spleen, brain and heart were taken as soon as possible.
- Paired organs were weighed separately and no organ weights would be taken for animals found dead.
- The following tissues of same selected animals were preserved in 10% formalin and their intended subsequent histopathological examinations: all gross lesions, brain, spinal cord, liver, kidneys, adrenals, stomach, small and large intestines (including Peyer´s patches), thymus, thyroid, spleen, trachea and lungs, heart, urinary bladder, lymph nodes, peripheral nerve and section of bone marrow.

HISTOPATHOLOGY: Yes
- Full histopathology was carried out on the preserved organs and tissues of the randomly selected animals (Male and Female) in the control and high dose groups.
- Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals.
- For testis, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
- In decedents, lung, trachea, stomach (nonglandular and glandular), oesophagus, heart, adrenal gland and kidneys were evaluated. Macroscopic lesions, with the exception of observations made at mouth and nose, were evaluated in all study animals.

Other examinations:

No

Statistics:

Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and presented as percentage.

Dead animals were taken into the account in the analysis of parameters like body weight, food consumption, clinical signs and few reproductive parameters.

For statistical analysis, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. These statistics were performed with GraphPad Prism V.5 software (p<0.05 was considered as statistical significant).
In the evaluation of laboratory parameters, all values within a range of the mean value +/- the two fold standard deviation (x +/- 2s) were considered to be "normal“ values within a "normal“ population.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were premature mortalities recorded in MD and HD groups. In view of the clinical and macroscopic findings recorded, gavage error or regurgitation cannot be excluded as cause of death for these rats

Mortality:

no mortality observed

Description (incidence):

There were premature mortalities recorded in MD and HD groups. In view of the clinical and macroscopic findings recorded, gavage error or regurgitation cannot be excluded as cause of death for these rats

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Day of sacrifice
With the exception of some animals in MD and HD groups, all animals survived till the end of the study. Male animals were sacrificed on day 29 and 30. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactating females along with pups were sacrificed on respective post natal day 4.

Clinicals signs and mortality
No significant clinical findings were observed in the control and treatment groups of males and females, except for few incidental findings. They are not assumed to be related to systemic toxicity, but rather appear to be the sign of an acute local irritation caused by the test item.

Body weight
A statistically significant reduction in body weight development was observed in male HD group animals during the first week of pre mating period and the entire study duration. But for female animals, no such findings were reported in any of the treatment group as compared to the corresponding control group values. The statistically significant deviation observed in male HD group could be due to few low values in individual animals and may not be attributed to treatment. This finding was not correlated with the findings of food consumption.

Haematology
With some exception, no statistical significant deviations were recorded in any of the hematological parameters:
- The deviations from the biological range in the above parameters were slight and may not be attributed to treatment, but could be incidental.
- The statistically significant deviation observed for MCHC (Female MD) may not be attributed to toxicity due to lack of dose response effect.
- The statistically significant deviation observed in male HD group for the WBC mean value could be due to low values in two individual animals. No such deviation was statistically recorded for female animals. In keeping with the sensitivity of the female animals compared with males, this finding may not be directly attributed to treatment.

Clinical biochemistry
In most of the clinical biochemistry parameters evaluated, with exception, the data revealed no statistically significant deviation in both male and females. The exceptions are:
- The statistically significant deviation in GLU (female HD group) may not be attributed to treatment, but instead could be due to differential feeding of individual animals and were in the biological range.
- The deviation observed for ALBB value (male HD group) may not be toxicity related as macroscopically or microscopically no effect was observed in liver. This finding could be due to individual incidental values. The deviation observed for TP value (male HD group) is in correlation with the findings of ALBB value.

Gross pathology
At terminal sacrifice, the only macroscopic organ lesions seen were yellowish foci in the epididymis of single males of all four study groups. Histologically, theese were confirmed to be spermatic granulomas and were considered to be incidental findings.
Ten animals were found dead during the study. These were two females and one male of MD group (female Nos. 21, 26 and male No. 62) and four females and three males of HD group (female Nos. 32, 33, 35, 38 and male Nos. 73, 75, 80). Macroscopic findings in these decedents were seen at mouth and nose (foam or food in 9/10 decedents), in the trachea (foam in 6/10 decedents, bloody infiltration in 1/10 decedents), in the stomach (foam or fluid in 4/10 decedents, rest of test item in 1/10 decedents) and in the lung (bloody in 8/10 decedents, foam or fluid content in 2/10 decedents).

Histopathology
All other histopathological changes seen in this study, in reproductive and other organs, were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

other: All the parameters observed

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the condition of this study, for a period of 28-29 days for male and of 54 days (maximum) for female, the NOAEL of MgCl2, 6H2O by oral gavage to rat was 1000 mg/kg/day. No adverse effects were seen on general toxicity endpoints.

Executive summary:

In conclusion, the repeated dose administration of Magnesium chloride hexahydrate in deionised water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 250, 500 and 1000 mg/kg body weight revealed no major toxicological findings. The cause of death of animals during the conduct of the study could not be determined (a plausible cause may be gavaging error or regurgitation).

Based on the data generated from this combined repeated dose toxicity and reproduction/ developmental toxicity screening test with Magnesium chloride hexahydrate, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for Magnesium chloride hexahydrate in males and females in Wistar rats.