Magnesium chloride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

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.

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.

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.

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 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.

Positive control:

No

Examinations

Parental animals: Observations and 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.

WATER CONSUMPTION No

OPHTHALMOSCOPIC EXAMINATION: No

Other: See Rep. Dose Tox. Oral-1-2010-RUDR

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

Parameters examined in Parent male:
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.

Litter observations:

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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (described when, e.g. as soon as possible after the last litters in each generation were produced)
- Maternal animals: All surviving animals (described when, e.g. after the last litter of each generation was weaned)

GROSS NECROPSY
- The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.
- Special attention was paid to the organs of the reproductive system for parental male at necropsy.

ORGAN WEIGHTS
- 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.

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.

Reproductive indices:

copulation index and fertility index

Offspring viability indices:

Delivery index and viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

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.

Effect levels (P0)

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 signs

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

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.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

other: Pre and Post natal data, Litter data

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.