Magnesium chloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Only limited data are available on effects of magnesium on the reproductive performance of male and female mice and rats. The
results of studies examining the effects of magnesium deficiency in rats substantiated the importance of magnesium for proper
reproductive function and foetal development or immune competence of pups, and the detrimental effects of magnesium deficiency
during gestation and lactation.

Additional information

 

 

Effects on developmental toxicity

Description of key information

 Developmental toxicity/teratogenicity toxicity studies have been conducted with magnesium chloride hexahydrate via the oral route. The results have been corrected to magnesium chloride anhydrous.

Two key studies are available:

- Rudragowda et al., 2010 (OECD 422) on Wistar rat indicates no adverse effects and the NOAEL is over 1000 mg/kg bodyweight for MgCl2 hexahydrate (eq. 466 mg/kg/bw for Magnesium Chloride anhydrous).

- Usami et al. 1994 (OECD 414) on Wistar rat indicates no adverse effect and the NOAEL is over 800 mg/kg/day (eq 373 mg/kgbw/day for MgCl2 anhydrous).

 

One Developmental toxicity/teratogenicity toxicity study has been conducted with magnesium chloride via the oral route. 

 - NIER 2009 on spragues dawley rat indicates no adverse effects on reproductive fonction and the NOAEL is over 1000 mg/kg bodyweight for MgCl2.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009

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)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD TG 421

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Lot No.: 12031JH
- Appearance: White powder
- Affinity: Hydrophobic
- Storage condition: Room temperature (15.9~27.1 degree C

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS]
- Source: ORIENTBIO INC., Korea
- Sex, number, age and body weight range at receipt: Male, 60 rats, 8 weeks old, 252.0~278.8 g, Female, 60 rats, 7 weeks old, 171.9~192.6 g
- Sex, number, age and body weight range at the start of administration: Male, 52 rats, 9 weeks old, 311.0~348.4 g, Female, 52 rats, 8v weeks old, 196.0~232.1 g
- Housing: Stainless wire mesh cages, 260W x 350D x 210H (mm), Polycarbonate cage 260W x 420L x 180H 9mm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 7 days

[ENVIRONMENTAL CONDITIONS]
- Temperature (°C): 16.5~23.0 degree C
- Relative humidity (%): 43.4~77.3%
- Air changes (per hr): 10~15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle, 7AM to 7PM via automated timer
- Number of animals per cage: One animal (during pre- and post mating periods)One male and one female (during mating period)
- Intensity of illumination: 150~300 Lux
- Cage washing: Stainless wire mesh cages and feeders were replaced once every two weeks and twice a week for water bottles and once a week for polycarbonate cages were replaced. These were washed with the cage washer and sterilized by an autoclave.

Route of administration:

oral: gavage

Details on exposure:

[PREPARATION OF DOSING SOLUTIONS]: The required amounts of test substance were weighed on a balance (CP323S, BP3100S, Sartorius, Germany) and placed in a bottle. A small amount of vehicle, water for injection, was added and mixed using a vortex mixer until dissolved.

[DIET PREPARATION]
- Type: Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Lot No.: 2918C 050409 MA, 2918C 060909 MA
- Feeding: The diet was placed in feeders and provided ad libitum.
- Analysis of diet: Dietary analysis of all batches used for the study was provided by the supplier. The results were confirmed to meet the allowable standard of this facility.

[VEHICLE]
- Name: Water for injection
- Lot no.: GBA9001, GBA9002, GBA9003
- Storage condition: Room temperature
- Justification for use and choice of vehicle: The test substance was dissolved in water for injection

Details on mating procedure:

- M/F ratio per cage: 1(M)/1 (F)
- Length of cohabitation: 14 days
- Proof of pregnancy: a vaginal plug twice a day, AM and PM
- Evaluation of non-pregnant: Animals did not show signs of parturition such as development of mammary gland and abdominal expansion until Day 26 of gestation.
- Final diagnoses of pregnancy: observation of uterine implantation sites at necropsy
- Mating results
* Mating index (%)=(Number of females mated/number of females placed with males)x100
* Mating period=mating confirmation-mating initiation (based on dosing day)
* Gestation period=Day 0 of delivery-Day 0 of gestation (based on dosing day)
* Male fertility index (%)=(Number of males impregnating a female (number of males that became sire)/umber of males with successful copulation)x100
* Female fertility index (%)=(Number of females pregnant/number of females with successful copulation)x100
* Gestation index (%)=(Number of females that delivered live pups/number of pregnant females)x100

Duration of treatment / exposure:

- Male: for a total of 42 days (two weeks each prior to, during and post mating)
- Female: for two weeks prior to mating, throughout gestation and for 4 days after delivery

Frequency of treatment:

once a day

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

No. of animals per sex per dose:

- Control (G1): 0 mg/kg bw/day, male-13 rats, female-13 rats
- Low dose (G2): 250 mg/kg bw/day, male-13 rats, female-13 rats
- Mid dose (G3): 500 mg/kg bw/day, male-13 rats, female-13 rats
- High dose (G4): 1,000 mg/kg bw/day, male-13 rats, female-13 rats

Control animals:

yes, concurrent vehicle

Details on study design:

In the repeated dose 28-day oral toxicity study with a recovery period for 14-day of Magnesium chloride in rats (Biotoxtech Study No.: B08007), soft stool and salivation were observed in animals dosed with 1,000 mg/kg, and the NOAEL for females was determined as 500 mg/kg. Based on the results, 1,000 mg/kg bw/day was selected as the high dose level for this study and sequentially divided by a geometric ratio of 2 to produce two additional lower doses of 500 and 250 mg/kg bw/day as mid and low dose levels. The control group received vehicle only (water for injection) at the same dose volume as the dosed groups.

Maternal examinations:

[Clinical signs]
All animals were observed for mortality, general condition and gross evidence of clinical signs and symptoms. Females were also observed for signs of abortion and premature birth.

[Body weights]
Body weights of males were recorded just prior to dosing on Day 0 (the first day of dosing), once a week throughout the dosing period, on the day prior to necropsy and on the day of necropsy (fasted body weights).Body weights of females were recorded just prior to dosing on Day 0 (the first day of dosing), once a week throughout the dosing period (except during mating) on Days 0, 7, 14 and 20 of Gestation, on Days 0 and 4 of Lactation, on the day prior to necropsy and on the day of necropsy. Fasted body weights taken on the day of necropsy were presented but were not included in statistical analyses.

[Food consumption]
Food consumption of males were recorded just prior to dosing on Day 0 (the first day of dosing), once a week during period (except during mating) and on the day prior to necropsy.Food consumption of females were recorded just prior to dosing on Day 0 (the first day of dosing), once a week throughout the dosing period (except during mating) on Days 1 and 4 of Lactation and on the day prior to necropsy. Residual feed were recorded. Food consumption was not recorded during mating.Individual food consumption was calculated by subtracting residual feed from the amount presented.

[Necropsy]
Necropsies were conducted after completion of dosing for males and on Day 5 after delivery or 26 days after conformation of mating for females. All surviving animals were fasted for at least 18 hours prior to necropsy and sacrificed by exsanguinations from the abdominal aorta under isoflurane anesthesia. Complete gross postmortem examinations were conducted on all animals including the external and internal surfaces. All grossly visible abnormalities were recorded.

[Organ weights]
The following organs were properly trimmed and weighed at necropsy. Organs were weighed and organ-to body weight ratios calculated.
- Testes - Lt./Rt.
- Epididymes - Lt./Rt.
- Ovaries - Lt./Rt.
- Uterus

[Histopathology]
At necropsy, the following organs and tissues were harvested and preserved in 10% neutral buffered formalin. Testes and epididymes were fixed in Bouim's solution, and eyes with optic nerves were fixed in Davidson's fixative.
- Brain
- Pituitary
- Thymus
- Lungs with bronchi
- Trachea
- Thyroid
- Esophagus
- Heart
- Liver
- Spleen
- Kidneys
- Adrenals
- Stomach
- Duodenum
- Jejunum
- Ileum
- Cecum
- Colon
- Rectum
- Testes
- Epididymes
- Prostate
- Ovaries
- Uterus
- Submandibular lymph node
- Spinal cord (cervical, Thoracic and lumbar)
- Mesenteric lymph node
- Eye & etc.
- Femur and sternum
- Urinary bladder
- Sciatic nerve
All preserved organs or tissues were trimmed, processed, embedded in paraffin, sectioned, and stained with Hematoxylin & Eosin (H&E).Histopathological examinations were conducted as follows:-Ovaries, testes and epididymes of control and high dosed groups- Lungs of dead animals during the study- All gross, macroscopic lesions

Fetal examinations:

* The following parameters were examined in F1 offspring:
- Mean litter size
- Live birth index (%)=(Number of live pups on postnatal Day 0/number of implantations)x100
- Viability index on postnatal Day 0 (%)=(Number of live pups on postnatal Day 0/number of pups delivered)x100
- Viability index on postnatal Day 4 (%)=(Number of live pups on postnatal Day 4/of live pups on postnatal Day 0)x100

* Body weights of pupsBody weights of pups were recorded on postnatal Day 0 and 4.

* External examination and sex ratio of pupsAll pups were examined on delivery day and on postnatal Day 4 for external findings. And, the sex of each pups were confirmed by the distance between anus and genital (relatively short for females and long for male).Pups were euthanized by CO2 gas after external examination on postnatal Day 4 and were calculated as follows:
- Sex ratio=Number of male pups/number of female pups (on postnatal Day 0)

Statistics:

* The following parameters were examined in F1 offspring:
- Mean litter size
- Live birth index (%)=(Number of live pups on postnatal Day 0/number of implantations)x100
- Viability index on postnatal Day 0 (%)=(Number of live pups on postnatal Day 0/number of pups delivered)x100
- Viability index on postnatal Day 4 (%)=(Number of live pups on postnatal Day 4/of live pups on postnatal Day 0)x100

* Body weights of pupsBody weights of pups were recorded on postnatal Day 0 and 4.

* External examination and sex ratio of pupsAll pups were examined on delivery day and on postnatal Day 4 for external findings. And, the sex of each pups were confirmed by the distance between anus and genital (relatively short for females and long for male).Pups were euthanized by CO2 gas after external examination on postnatal Day 4 and were calculated as follows:
- Sex ratio=Number of male pups/number of female pups (on postnatal Day 0)

Details on maternal toxic effects:

[CLINICAL SIGNS AND MORTALITY]
In the 1,000 mg/kg bw/day dosed group, deaths were observed in one male on Day 36 of administration, in two females on Day 2 of administration and in one female on Day 3 of Parturition. Soft stool, diarrhea and salivation that showed immediately after dosing were observed in all dead animals, and those dead animals died within 15 minutes after dosing.In the control group, no clinical signs were observed in males and females, except soft stool in one female and diarrhea in two females on Day 1 of administration.In the 250 mg/kg bw/day dosed group, soft stool and salivation were observed occasionally or sporadically in a few males from Day 34 of administration. Soft stool was observed in one female on Day 7 and 8 of administration, and it was observed in one female prior to mating. It was observed in a few females during post-gestation and lactation periods.In the 500mg/kg dosed group, soft stool was observed continuously, salivation was observed immediately after dosing and diarrhea was observed sporadically in all males and females throughout the study. In the 1,000 mg/kg bw/day dosed group, soft stool, diarrhea and salivation were observed in all males and females continuously and showed to be dose-dependent.

[BODY WEIGHT]
Body weights of males dosed with 1,000 mg/kg bw/day were statistically significantly decreased (p<0.05 or p<0.01) on Days 20, 27, 34 and 41 of administration compared to the control group. Also, in the male 500 mg/kg bw/day and female 1,000 mg/kg bw/day dosed groups, body weights showed decreasing tendency compared to the control group.

[FOOD CONSUMPTION]
During the study, no significant differences in food consumption were noted in the all dosed groups compared to the control group.

[MATING AND GESTATION]
Each of 13, 13, 13 and 11 males and females dosed with 0 (control), 250, 500 and 1,000 mg/kg bw/day mated. The mating periods were 2.3, 2.9, 2.6 and 2.2 days, the gestation periods were 21.9, 22.2, 22.0 and 21.8 days, the mating indices were 100%, and the fertility indices of sexes were 84.6, 92.3, 92.3 and 90.9%, respectively. There were no statically significant differences in any dose group.

[PARTURITION AND EXAMINATION OF NEONATE]
Pregnant females dosed with 0 (control), 250, 500 and 1,000 mg/kg bw/day were 11, 12, 12 and 10, respectively and they showed normal deliveries. The mean litter size was 14.7, 14.9, 13.8 and 13.8, the gestation indices were 100%, the pre-implantations loss rates were 9.8, 14.9, 14.0 and 12.8%, and the post-implantations loss rates were 8.8, 6.4, 6.6 and 9.4%, respectively. Also, the live birth indices were 91.2, 93.7, 93.4 and 90.7%, the viability indices were 99.4, 97.5, 97.8 and 99.3% on postnatal Day 0 and 98.9, 98.5, 99.0 and 96.9% on postnatal Day 4, and the sex ratios were 0.8, 1.0, 0.9 and 0.8, respectively. There were no statistically significant differences in any dose group.

[ORGAN WEIGHTS]
No statistically significant differences in absolute and relative organ weights of testes and epididymes for males and in ovaries and uterus for females were observed in all dosed groups compared to the control group.

[NECROPSY]
No abnormal macroscopic findings were observed in the control and all dosed groups, except cyst of spleen in one female dosed with 250 mg/kg bw/day.Also, no abnormalities were observed in dead animals throughout the study.

[HISTOPATHOLOGY]
No abnormalities were noted in testes, epididymes and ovaries of the control and 1,000 mg/kg bw/day dosed groups. Cyst of spleen in one female with 250 mg/kg bw/day, which was found at necropsy, was determined as cyst of capsular in spleen. No abnormalities were observed in lungs of dead animals throughout the study.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

[VIABILITY]
In the control, 250, 500 and 1,000 mg/kg bw/day dosed groups, the number of dead pups were 2 3, 2 and 4 pups on postnatal Days 1 through 4 postpartum.

[CLINICAL SIGNS]
Edema of whole body was observed in one pup in the 1,000 mg/kg bw/day dosed group on postnatal Day 0, but the other abnormal signs were not observed in the control and all dosed groups.

[BODY WEIGHT]
On postnatal Day 0 and 4, no significant differences in body weight were noted in the all dosed groups compared to the control group.

[External Examinations of pups]
No abnormal signs were observed in pups in the control and all dosed groups on postnatal Days 0 and 4, except edema of whole body in one pup (0.8%) in the 1,000 mg/kg bw/day dosed group on postnatal Day 0.

Table 1. Clinical signs of F1 in developmental toxicity screening test of SD rats (Group summary) Sex Clinical signs Group G1 G2 G3 G4 Dose (mg/kg bw/day) 0 250 500 1,000 Male . No. of animals 73 91 82 63 No abnormalities detected . 71 91 81 62 Abnormal detected . 2 0 1 1 - Death . 2 0 1 1 Female . No. of animals 90 93 88 76 No abnormalities detected . 90 90 87 73 Abnormal detected . 0 3 1 3 - Death . 0 3 1 3 - Edema of whole body . - - - 1 Table 2. Body weights of F1 in developmental toxicity screening test of SD rats (Group summary) Group/ Dose (mg/kg bw/day) . Postnatal day Male Female 0 4 0 4 G1 0 Mean (g) 6.66 10.70 6.25 10.04 S.D. (g) 0.38 1.28 0.42 1.25 N 11 11 11 11 G2 250 Mean (g) 6.62 10.51 6.32 10.00 S.D. (g) 0.34 1.06 0.29 0.98 N 12 12 12 12 G3 500 Mean (g) 6.63 10.98 6.24 10.40 S.D. (g) 0.45 0.81 0.38 0.54 N 12 12 12 12 G4 1000 Mean (g) 6.74 11.06 6.34 10.71 S.D. (g) 0.48 0.89 0.47 0.89 N 10 9 10 9 Table 3. External findings of F1 in developmental toxicity screening test of SD rats (Group summary) Group/ Dose (mg/kg bw/day) . No. of dams Postnatal day 0 Postnatal day 4 No. of Pups External findings No. of Pups External findings Edema (Whole body) G1 0 Total 11 163 0 160 0 Incidence . . (0.0) . (0.0) G2 250 Total 12 184 0 176 0 Incidence . . (0.0) . (0.0) G3 500 Total 12 170 0 164 0 Incidence . . (0.0) . (0.0) G4 1000 Total 10 139 1 118 0 Incidence . . (0.8) . (0.0) Incidence (%) = Sum of data in the parenthesis/Total No. of dams x 100

Conclusions:

There were no effects on the sex ratio, external findings, body weights and clinical signs of pups up to postnatal Day 4, so there were no dosing related on pups. Edema of whole body was observed in one pup dosed with 1,000 mg/kg bw/day incidentally. In conclusion, the No Observed Adverse Effect Level (NOAEL) was considered to be 1,000 mg/kg bw/day for pups.

Executive summary:

The purpose of this study was to evaluate the developmental toxicity of the test substance, Magnesium chloride (CAS No.: 7786 -30 -3), when administered orally to male and female rats at dose levels of 0 (control group), 250, 500 and 1,000 mg/kg bw/day. Males were dosed once daily for a total of 42 days (two weeks each prior to, during and post mating), and females were dosed were dosed once daily for two weeks prior to mating, throughout gestation and four days after delivery. Edema of whole body was observed in one pup in the 1,000 mg/kg bw/day dosed group on postnatal Day 0, but the other abnormal signs were not observed in the control and all dosed groups. On postnatal Day 0 and 4, no significant differences in body weight were noted in the all dosed groups compared to the control group. No abnormal signs were observed in pups in the control and all dosed groups on postnatal Days 0 and 4, except edema of whole body in one pup (0.8%) in the 1,000 mg/kg bw/day dosed group on postnatal Day 0. Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 1,000 mg/kg bw/day for pups.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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.

Route of administration:

oral: gavage

Details on 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 are 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 was 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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Animals were paired in the ratio of 1:1 (male to female).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- In case of unsuccessful mating, re-mating of females with proven males of the same group will be considered.
- Further matings after two unsuccessful attempts: no
- After successful mating, each pregnant female was individually caged.

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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 an 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.

Maternal examinations:

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.

Fetal examinations:

STANDARDISATION OF LITTERS
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live Pups were identified by writing actual numbers on the back with the help of permanent marker. In addition to the observations on parent animals, abnormal behaviour of the offspring, if any, was recorded.

PARAMETERS EXAMINED
- Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter were carefully examined for gross abnormalities.

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.

Clinical signs:

no effects observed

Details on maternal toxic effects:

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 were not assumed to be related to systemic toxicity, but rather appeared 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.

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, these 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.

Precoital Interval, Duration of gestation and fertility Index
Statistically no significant difference was observed on precoital interval when compared with their corresponding controls. All pregnancies resulted in normal births.
Successful mating resulted in 10/12 pregnancies in the control, 8/10 pregnancies in LD groups, 10/11 pregnancies in MD and 8/11 pregnancies in HD groups.
Slightly but not significantly reduced fertility indices (No. of pregnant females/No. of copulated females X 100) were observed in HD (72.73%) and LD groups (80%) as compared to MD groups (90.91%) and Control (83.33%).

Reproductive indice
There were no significant differences observed for any of the reproductive indices (copulation index, fertility index, delivery index and viability index) between the treatment and control groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Details on embryotoxic / teratogenic effects:

Viability:
The survival of the pups from PND 0 to PND 4 remained unaffected due to treatment in all treatment groups.(Pre and post natal data)

Pre and post natal data:
Group mean number of corpora lutea, No. of implantation sites, number of live pups born on PND 0, percent pre implantation loss and post implantation loss remained unaffected to treatment when compared with control group values. There was no statistical significant difference observed between any of the treatment groups compared to corresponding controls.

Litter data:
No treatment related effect was observed on the total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

Conclusions:

Under the condition of this study, a NOAEL value of 1000 mg/kg/day (MgCl2, 6H2O by oral gavage to rat) was derived for reproduction/developmental toxicity. 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 bodyweight for Magnesium chloride hexahydrate in males and females in Wistar rats

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No information on analytical verification

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Wistar rats obtained from Nippon Charles River, Kanagawa, Japan were used.
The nulliparous female and male animals were 10 and 11 weeks old, respectively.
Pregnant animals were kept individually in aluminum cages.

The animals were maintained under controlled environmental conditions of 25 °C ± 2 °C temperature, 55 ± 5% relative humidity, and 12-hours light-dark cycle, (light phase: 6:00 ~ 18:00) and received solid food and tap water ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The concentration of the test substance solution was set at 5 mL/kg/day at each dosage level.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Details on mating procedure:

Nulliparous females were mated overnight with males. Females revealing spermatozoa in the vaginal smear in the following morning were considered to be pregnant and used in the experiment. The day, when sperms were found in the vaginal smear, was designated as day 0 of gestation.

Duration of treatment / exposure:

From day 6 through 15 of pregnancy (10 days)

Frequency of treatment:

By gavage (one by day)

Duration of test:

20 day of gestation

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

The pregnant rats were sacrificed on day 20 of pregnancy and their fetuses were examined for malformation.
For the control group, 5 mL/kg/day distilled water was administered to the animals in the same manner as for the dose groups.

Maternal examinations:

CAGE SIDE OBSERVATION: YES
The general condition of the animals was observed every day.

BODYWEIGHT: Yes
Body weight was measured at day 0, 1, 3, 6, 9, 12, 15, and 17 of pregnancy.

FOOD INTAKE: YES
Food intake was measured at day 0, 1, 3, 6, 9, 12, 15, and 17 of pregnancy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- Weight : Yes all per litter
- External examinations: Yes all per litter
- Soft tissue examinations: Yes all per litter according to a gross sectioning technique (abdominal cavity) and a microdissection technique (thoracic space)
- Skeletal examinations: Yes half per litter (prepared for staining with alizarin red)
- Head examinations: Yes half per litter (according to a gross sectioning technique)

Statistics:

Pregnant animals or one uterus were taken as sample units. For frequency data, Fisher's direct exact probability test was applied to test the significance of differences between the control group and the magnesium chloride hexahydrate groups. With regard to observation data, statistical analysis and Scheffé's method were applied when no variance differences between the groups were found according to Bartlett's equal variance test. For measured data and count data with variance differences between the groups, we used the H test (Kruskal-Wallis test) and Scheffé's method.

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:not examined

Details on maternal toxic effects:
There were no differences between the groups with regard to general condition and death. Furthermore, no significant differences between the control group and magnesium chloride hexahydrate groups were observed with respect to body weight and food consumption.

Dose descriptor:

NOAEL

Effect level:

> 800 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Regarding the number of corpora lutea, number of implants, number of living fetuses, sex ratio, fetal weight and mortality of implants/fetuses, no significant differences between control group and the magnesium chloride hexahydrate groups were observed.
One to 4 fetuses with gross malformations were found in each group. However, regarding the incidence rate, there was no significant difference between control group and magnesium chloride hexahydrate groups.
In the 800 mg/kg/day group, one fetus had skeleton malformations, however, with regard to the incidence rate; there was no significant difference between control group and the magnesium chloride hexahydrate groups. Furthermore, no significant differences between control group and magnesium chloride hexahydrate groups were observed as far as the incidence rate of skeletal variations is concerned. Regarding the incidence rate of fetuses with lumbar rib and additional rib bones, there were no significant differences. No significant differences were found in the number of ossification centers, metacarpal and metatarsal bones as well as sacral and tail vertebrae. The aforementioned numbers were examined as indicators for the progress of ossification.
Four to 6 fetuses in each group showed malformations, however, no significant difference were observed between control group and magnesium chloride hexahydrate groups.

Dose descriptor:

NOAEL

Effect level:

> 800 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Fetal growth in pregnant rats treated with MgCl2, 6H2O

	Dose (mg/kg/day)
	Control	200	400	800
Nb of litters	22	22	22	22
Nb of corpera lutea	373	370	361	362
Mean +/- SD	17.0 +/-1.6	16.8 +/-2.2	16.4 +/-2.1	16.5 +/-1.5
Nb of implants	364	340	345	345
Mean +/- SD	16.5 +/-1.7	15.5 +/-3.2	15.7 +/-1.9	15.7 +/-1.8
Implantation rate (%)	97.7 +/-4.5	91.4 +/-12.2	95.9 +/-6.6	95.4 +/-8.3
Nb of live fetuses	346	326	324	332
Mean +/- SD	15.7 +/-1.5	14.8 +/-3.5	14.7 +/-1.8	15.1 +/-1.8
Sex ratio male/female	1.28	1.15	1.23	1.38
Fetal weight (g)
Male	3.95 +/-0.22	3.98 +/-0.29	3.87+/-0.21	3.98 +/-0.22
Female	3.73 +/-0.25	3.75 +/-0.23	3.72 +/-0.20	3.81 +/-0.17
Nb of dead implants	18	14	21	13
Early death	18	14	21	13
Late death	0	0	0	0
Mortality (%)	4.8 +/-4.6	4.8 +/-5.8	5.9 +/-6.1	3.6 +/-5.7

 Mean +/- SD is shown

Gross malformation in the fetuses from pregnant rats treated with MgCl2, 6H2O

	Dose (mg/kg/day)
	Control	200	400	800
Nb of litters	22	22	22	22
Nb of fetuses examined	346	326	324	332
Nb of litters with malformed fetuses	3	1	3	1
Nb of fetuses with malformation	3	1	4	1

Skeletal variations the fetuses from pregnant rats treated with MgCl2, 6H2O

	Dose (mg/kg/day)
	Control	200	400	800
Nb of litters	22	22	22	22
Nb of fetuses examined	174	168	165	165
Nb of fetuses with malformation	0	0	0	1
Nb of fetuses with variation	76	71	89	84
Lumbar rib	14	13	18	16

Visceral variations the fetuses from pregnant rats treated with MgCl2, 6H2O

	Dose (mg/kg/day)
	Control	200	400	800
Nb of litters	22	22	22	22
Nb of fetuses examined	170	157	159	166
Nb of litters with malformed fetuses	4	4	5	4
Nb of fetuses with malformation	5	4	6	5

Conclusions:

under the condition of this study, It is obvious that magnesium chloride hexahydrate has no teratogenic effect when administered orally to rats from day 6 through 5 of pregancy at dose 800 mg/kg/day.

Executive summary:

Teratogenicity of magnesium chloride hexahydrate (MgCl2·6 H2O) was examined in rats. Magnesium chloride hexahydrate dissolved in distilled water was given to pregnant Wistar rats by gavage once a day from day 6 through 15 of pregnancy at doses of 0, 200, 400 and 800 mg/kg/day. The pregnant rats were sacrificed on day 20 of pregnancy and their fetuses were examined for malformation. Magnesium chloride hexahydrate caused no increased incidences of fetal malformation, and no toxic signs in the pregnant rats and the fetuses.

It was concluded that magnesium chloride hexahydrate has not teratogenicity in rats when given by gavage. The no observed adverse effect level was estimated to be over 800 mg/kg/day for both pregnant rats and rat fetuses.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Three key studies are available: Rudragowda et al., 2010 (OECD 422), NIER 2009 (OECD 421) and Usami et al., 1994 (OECD 414).

The study of Rudragowda et al. 2010 is based on the data generated from a 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 bodyweight for Magnesium chloride hexahydrate (eq. 466 mg/kg/bw for Magnesium Chloride anhydrous) in male and female Wistar rats. This study indicates no adverse effects on reproductive organs or tissues.

The study of NIER 2009 was performed according the OECD 421 on magnesium chloride, the results of this study is that an orally adminitration of magnesim chloride in rat does not indicate any reproductive toxicity in parent animals or developmental toxicity in pups at the maximum tested dose of 1000 mg/kg bw/day.

The other dose repeated studies indicate no adverse effects on reproductive organs or tissues for different exposure times:

• Tanaka H. et al., 1993 (male and female B6C3F mice, 90 days)


• Takizawa T. et al., 2000 (male and female Fischer rat, 90 days)


• Kurata Y. et al., 1989 (male and female B6C3F mice, 96 weeks)

 

The study of Usami et al., 1996 is a teratogenicity study on Wistar rats. Magnesium chloride hexahydrate was given to pregnant Wistar rats by gavage once a day from day 6 through 15 of pregnancy. The pregnant rats were sacrificed on day 20 of pregnancy and their fetuses were examined for malformation. Magnesium chloride hexahydrate caused no increased incidences of fetal malformation, and no toxic signs in the pregnant rats and the fetuses. The no observed adverse effect level was estimated to be over 800 mg/kg/day (eq 373 mg/kgbw/day for MgCl2 anhydrous) for both pregnant rats and rat fetuses.

Justification for classification or non-classification

On the basis of studies results, the MgCl2 was not classified for reproduction toxicity under the CLP regulation 1272/2008.

Furthermore, Magnesium is essential mineral nutrients for mammals including humans. Based on evaluation of a wealth of human medical and nutritional data Anonymous, 2001 [SCF opinion]), it is concluded that magnesium, does not pose any hazard for reproduction and/or developmental toxicity.