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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11 November 2009 - 01 February 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study As this study is being used for read-across, the reliability has been amended to reflect this. Read-across from magnesium chloride hexahydrate to magnesium carbonate is justified on the following basis. Due to the presence of acid, mainly in the form of hydrochloric acid, in the stomach, magnesium carbonate will be converted into magnesium chloride when orally ingested. Furthermore, magnesium chloride is significantly more soluble than the carbonate salt and therefore represents the worst case in terms of its bioavailability for systemic absorption. In addition, both salts have been shown to have no acute toxicity and do not exhibit evidence of systemic toxicity when tested at a concentration of 2000 mg/kg bw in acute oral studies. Magnesium carbonate and magnesium chloride also occur in the natural environment and humans are widely exposed to naturally occurring magnesium carbonate and chloride, e.g. via drinking water and food on a day to day basis. Ingested magnesium, carbonate and chloride ions are actively regulated by the body. Any systemic toxicity is likely to be caused by absorption of the magnesium ion rather than either the carbonate or chloride counterions and hence studies on magnesium salts can be read across from one to the other. This study is therefore deemed reliable for read across to magnesium carbonate as it represents the worst case for potential repeated dose toxicity via the oral route.

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
magnesium chloride hexahydrate (see endpoint summary for justification of read-across)
IUPAC Name:
magnesium chloride hexahydrate (see endpoint summary for justification of read-across)
Constituent 2
Reference substance name:
7791-18-6
EC Number:
616-575-1
Cas Number:
7791-18-6
IUPAC Name:
7791-18-6
Details on test material:
- Name of test material (as cited in study report): Magnesium chloride hexahydrate
- Molecular formula (if other than submission substance): MgCl2.6H2O
- Physical state: solid, crystals
- Analytical purity: 100.8 (as hexahydrate)
- Batch No.: M 1197
- Expiration date of the batch: 28 July 2012
- Storage condition of test material: at room temperature, in a tightly closed package
- 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
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Females: 144.32 - 2116.48 g (mean: 180.40 g); Males: 211.18 - 316.77 g (mean: 263.98 g)
- Housing: Animals were housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulfur acidified to a pH of approximately 2.8
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 times per hour
- Photoperiod (hrs dark / hrs light): Artificial light; 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was suspended in deionised water and administered by gavage using a gavaging cannula. The maximum dose volume administered was 10 mL/kg bw.

VEHICLE
- Justification for use and choice of vehicle: Water was chosen due to its non-toxic characteristics.
- Batch no.: 17.11.2009, 26.11.2009, 10.12.2009, 23.12.2009
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analysed for nominal concentration. samples were taken in study week 1 (first week of pre-mating period), 3 (first week of mating ), 5 (gestation) and 7 (gestation/ lactation).
Duration of treatment / exposure:
The test substance was administered daily during 14 days pre-mating and 14 days mating in both male and females, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.
Frequency of treatment:
Daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Control: 12 animals/sex
250 mg/kg bw/day (Low dose, LD): 10 animals/sex
500 mg/kg bw/day (Mid dose, MD): 12 animals/sex
1000 mg/kg bw/day (High dose, HD): 15 animals/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and a NOAEL.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily except during weekend and holidays where observations were made only once.
- Cage side observations: Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and once a week thereafter.
- Observations: Detailed clinical observations were made in all animals outside the home cage in a standard arena. They included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation and 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 also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed 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 and 4 along with pups.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: measured on corresponding days to the body weight measurements (in males only during pre-mating period). Food consumption was not measured during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on sacrifice
- How many animals: 5 males and 5 females randomly selected from each group
- Anaesthetic used for blood collection: Yes (Ketamin/ xylazin, 2:1)
- Parameters examined: haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count, platelet count, blood clotting time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on sacrifice
- How many animals: 5 males and 5 females randomly selected from each group
- Anaesthetic used for blood collection: Yes (Ketamin/ xylazin, 2:1)
- Parameters examined: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, 2 enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase)

URINALYSIS: Yes
- Time schedule for collection of urine: on sacrifice
- How many animals: 7 randomly selected males of the HD group, 6 randomly selected males of the control and MD group and 5 randomly selected males of the LD group

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Observation occurred during the last week of treatment in males and on day 3 of lactation in females.
- Dose groups that were examined: 5 randomly selected males and 5 lactating females from each group
- Battery of functions tested: sensory activity to different modalities, grip strength, motor activity and other behaviour observations
Sacrifice and pathology:
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.

GROSS PATHOLOGY: Yes: 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 reproductive organs. The number of implantation sites and corpora lutea were recorded for each parental female. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating glands as a whole) and all organs showing macroscopic lesions of all adult animals were examined.

HISTOPATHOLOGY: Yes: The following tissues were examined: all gross lesions, brain, spinal cord, liver, kidneys, adrenals, stomach, small and large intestines, thymus, thyroid, spleen, trachea and lungs, heart, urinary bladder, lymph nodes, peripheral nerve, section of bone marrow.
Full histopathology was originally carried out on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. However, these examinations were extended to animals of all other dosage groups as no treatment related changes were observed in the high dose group.

ORGAN WEIGHT: Reproductive organs from all animals were weighed. The wet weight of the liver, kidneys, adrenals, thymus, spleen, brain and heart were taken from 5 adult males and 5 adult females randomly selected from each group.
Statistics:
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No significant findings were observed in the control and treatment groups of males and females, except for a few incidental findings in individual male animals of control, LD and MD groups. There were premature mortalities recorded in MD and HD group. The following clinical findings were observed in these dead animals:
MD: male - moving the bedding post application
HD: male - salivation, moving the bedding post application
female - nibbling on fur
The cause of premature death could not be determined nevertheless a gavaging error or regurgitation could not be excluded. The deaths were therefore not thought to be treatment releated.
No abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalisation.
No convulsions, tremors, stereotypy or bizarre behaviour were observed for the animals of any group.
For supported and unassisted rears no abnormalities were detected. Responses to reflex testing were normal in all groups.
On measures such as counts of urination and defaecation, differences could not be detected.

BODY WEIGHT AND WEIGHT GAIN
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 groups as compared to the corresponding control group values. The statistically significant deviation observed in male HD group could be due to low values in individual animals and may not be due to treatment.

FOOD CONSUMPTION
The mean food consumption showed slightly lower values in male/female MD and HD groups during the first week of pre-mating and mating/post mating period. The statistical evaluation of food consumption did not show any statistically significant effect in any of the treatment groups as compared to corresponding control values.

HAEMATOLOGY
With the exception of WBC (male HD group), MCHC (female MD group) no statistical significant deviation was recorded in any of the haematological parameters between the treatment group and the corresponding control values. However, some individual or mean values of WBC, RBC, HGB, MCV, MCH, MCHC, PTT, aPTT were below or above the biological range. These differences were slight and may not be attributed to treatment but could be incidental.

CLINICAL CHEMISTRY
In most of the clinical biochemistry parameters evaluated, with exception of GLU (female HD group), TP (male HD group) and ALBB (male MD group), the data revealed no statistically significant deviation in both males and females.
However, some individual or mean values of GLU, CHOL, TP and ALBB were below or above the biological range. These differences may not have been caused by treatment.

URINALYSIS
The urinalysis performed in male animals revealed no test item related effect in any of the treatment groups compared to corresponding control.

NEUROBEHAVIOUR
No relevant differences were observed concerning functional and behavioural examination in males and females.

ORGAN WEIGHTS
In males, except for absolute kidney weight (LD group), absolute thymus weight (MD group) and absolute testes weight (HD group), statistically no significant differences in the absolute and relative organ weights of the treatment groups was observed when compared to the controls. The deviation observed in absolute kidney, thymus and testes weight cannot be attributed to toxicity as there was no such deviation for relative weights in these organs.
In females, a statistically significant difference was observed for relative spleen weight (MD group) when compared with the controls. This finding cannot be attributed to toxicity due to lack of dose response effect.

GROSS PATHOLOGY
Ten animals were found dead during the study. Macroscopic findings in these decedents were seen at the mouth and nose, in the trachea, in the stomach and in the lung.
At terminal sacrifice, the only macroscopic organ lesions seen were yellowish foci in the epididymis of single males of all 4 study groups. Histologically, these were confirmed to be spermatic granulomas and were considered to be incidental findings and not test material related.
In pups, no gross external abnormalities related to treatment were observed at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the female and male reproductive organs, no histopathological lesions considered to be test item related were noted.
All other histopathological changes seen in the 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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
MgCl2.6H2O
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Table 1:Mortality

Group

Treatment period

Male

Female

C (0 mg/kg)

0/12

0/12

LD (250 mg/kg)

0/10

0/10

MD (500 mg/kg)

1/12

2/12

HD (1000 mg/kg)

3/15

4/15

Animals affected/ total no. of animals

 

Table 2: Clinical observations – Main group: Male

Clinical findings

C (0 mg/kg)

LD (250 mg/kg)

MD (500 mg/kg)

HD (1000 mg/kg)

Total no. of animals examined

12

10

12

15

Injury

1

0

0

0

Nibbling fur

1

0

0

0

Dyspnoea

0

1

0

0

Found dead

0

0

1

3

 

Table 3: Clinical observations – Main group: Female

Clinical findings

C (0 mg/kg)

LD (250 mg/kg)

MD (500 mg/kg)

HD (1000 mg/kg)

Total no. of animals examined

12

10

12

15

Regurgitation

0

0

1

0

Found dead

0

0

2

4

Nibbling fur

0

0

0

1

 

Table 4: Body weight (g) – Males

Day of treatment

Group

-

C (0 mg/kg)

LD (250 mg/kg)

MD (500 mg/kg)

HD (1000 mg/kg)

Pre-mating

1

Mean

292.00

292.20

288.58

294.07

SD

19.54

13.18

13.11

9.47

N

12

10

12

15

7

Mean

310.17

310.00

301.75

304.33

SD

23.98

17.28

14.21

10.67

N

12

10

12

15

14

Mean

320.83

320.40

308.83

313.64

SD

30.32

19.59

17.33

11.85

N

12

10

12

14

Mating/ post mating

7

Mean

328.33

336.50

325.73

326.50

SD

31.59

22.11

20.34

17.49

N

12

10

11

12

14

Mean

340.08

336.50

325.73

326.50

SD

34.28

22.11

20.34

17.49

N

12

10

11

12

T.S

Mean

342.42

338.40

329.46

327.33

SD

34.41

22.57

19.08

17.91

N

12

10

11

12

T.S = terminal sacrifice

 

Table 5: Body weight (g) – Females – Pre-mating

Day of treatment

Group

-

C (0 mg/kg)

LD (250 mg/kg)

MD (500 mg/kg)

HD (1000 mg/kg)

Pre-mating

1

Mean

186.90

191.13

188.82

188.58

SD

12.08

7.55

6.52

4.48

N

10

8

11

12

8

Mean

193.10

197.75

193.45

193.00

SD

9.26

9.24

6.20

6.98

N

10

8

11

12

14

Mean

198.40

202.25

199.70

32.75

SD

10.75

10.78

9.04

7.97

N

10

8

10

9

 

Table 6: Body weight (g) – Females

Day of treatment

Group

-

C (0 mg/kg)

LD (250 mg/kg)

MD (500 mg/kg)

HD (1000 mg/kg)

Gestation

0

Mean

197.50

204.00

198.80

32.75

SD

13.95

9.04

9.38

7.97

N

10

8

10

9

7

Mean

222.70

228.25

221.50

227.00

SD

13.27

7.61

11.21

10.74

N

10

8

10

8

14

Mean

247.78

253.75

248.56

253.00

SD

14.76

9.02

11.71

11.17

N

9

8

9

8

20

Mean

301.70

300.88

305.11

310.00

SD

18.22

23.52

15.46

14.94

N

10

8

9

8

Lactation

0

Mean

233.70

248.25

238.11

239.25

SD

16.08

10.26

16.35

14.04

N

10

8

9

8

4

Mean

242.80

248.25

243.22

248.00

SD

12.73

12.80

17.19

8.82

N

10

8

9

8

 

Applicant's summary and conclusion

Conclusions:
The repeated dose administration of magnesium chloride hexahydrate in deionised water to the male and female Wistar rats at dosages of 250, 500 and 1000 mg/kg bw/day revealed no major toxicological findings. The cause of death of animals during the study could not be determined nevertheless a gavaging error or regurgitation could not be excluded. The deaths were therefore not thought to be treatment releated.
Based on the data generated from the study, the NOAEL is believed to be 1000 mg/kg bw/day for magnesium chloride hexahydrate.

The equivalent NOAEL for magnesium carbonate is therefore 414 mg/kg bw/day.