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Developmental toxicity / teratogenicity

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developmental toxicity
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
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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 reproductive toxicity.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Reference substance name:
Magnesium chloride hexahydrate (see endpoint summary for justification of read-across)
Magnesium chloride hexahydrate (see endpoint summary for justification of read-across)
Constituent 2
Reference substance name:
EC Number:
Cas Number:
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

Details on test animals or test system and environmental conditions:
- 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

- 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
Details on 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.

- 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:
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).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight and into the following day
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of gestation
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:
Duration of test:
Males: 28-29 days
Females: Maximum 54 days
Doses / concentrations
Doses / Concentrations:
0, 250, 500 and 1000 mg/kg bw/day
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.


Maternal examinations:
- 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.

- 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.

- 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 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.

- 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

- 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)

- 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

- 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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
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.
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.

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.

Statistically no significant difference was observed on precoital interval when compared with their corresponding controls. All pregnancies resulted in normal births.
Slightly but not significantly reduced fertility index (No. of pregnant females/ no. of copulated females x 100) was observed in HD (72.73%) and LD groups (80%) as compared to MD groups (90.01%) and control (83.33%).
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.

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.

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

In the female and male reproductive organs, no histopathological lesions considered to be test item related were noted.
Female reproductive organs showed physiological postpartum morphology in most animals. The number of large corpora lutea in the ovaries was essentially similar in all study groups. Two control females, two females of LD group, one female of MD group and 3 females of HD group were found not to be pregnant at terminal sacrifice.
In males, minimal bilateral degeneration of the seminiferous epithelium in the testis, associated with intraluminal cellular debris in the epididymis, was noted in one male each in LD and HD. As a dose response was lacking, this was not considered to be test item related.
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 (maternal animals)

Dose descriptor:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No embryotoxic or teratogenic effects were observed.

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.
Based on the data generated from the study, the NOAEL for reproduction/ developmental toxicty 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.