Magnesium hydroxide sulfate (Mg6(OH)10(SO4)), hydrate (1:3)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/08/2006-05/02/2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 03/04/07

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-390-9

EC Name:

-

Cas Number:

12508-61-1

Molecular formula:

H16Mg6O17S

IUPAC Name:

hexamagnesium(2+) trihydrate decahydroxide sulfate

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River (UK) Ltd., Margate, Kent.

- Age at study initiation:
Approximately 5-8 weeks old

- Weight at study initiation:
Males: 153-184g
Females: 143-169g

- Fasting period before study:
Not stated.

- Housing:
The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.

- Diet (e.g. ad libitum):
Ad libitum.

- Water (e.g. ad libitum):
Ad libitum.

- Acclimation period:
8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21 ± 2 deg C

- Humidity (%):
55 ± 15%

- Air changes (per hr):
At least 15 per hour.

- Photoperiod (hrs dark / hrs light):
12 hours light/12 hours dark

IN-LIFE DATES: From: Day 1 To: Day 28

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% carboxy methylcellulose.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in 1% Carboxy methylcellulose. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results are given in Appendix 15 and show the formulations to be stable for at least nineteen days. Formulations were therefore prepared weekly and stored at 4ºC in the dark.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Not stated.

- Mixing appropriate amounts with (Type of food):
Not applicable.

- Storage temperature of food:
Not stated.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not stated.

- Concentration in vehicle:
0, 1.5, 15 & 100 mg/ml

- Amount of vehicle (if gavage):
10 ml/kg/day

- Lot/batch no. (if required):
Not applicable.

- Purity:
Not stated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration of MOS-HIGE at Safepharm Analytical Laboratory. The method used for analysis of formulations and the results obtained are given in Appendix 15. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Daily for 28 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5 males and 5 females at each dose level.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of the range-finder test.

- Rationale for animal assignment (if not random):
Random.

- Rationale for selecting satellite groups:
Not applicable.

- Post-exposure recovery period in satellite groups:
Not applicable.

- Section schedule rationale (if not random):
Not applicable.

Positive control:

Not conducted.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

- Cage side observations checked in table No. 1 were included.
Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily, as above.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also performed prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Not applicable.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data
Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Not applicable.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Haematological investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

- Anaesthetic used for blood collection:
No

- Animals fasted:
No

- How many animals:
All test and control animals.

- Parameters examined:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Cresyl blue stained slides were prepared but reticulocytes
were not assessed
Prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

- Animals fasted:
No

- How many animals:
All test and control animals.

- Parameters examined:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Prior to the start of treatment and on Days 5, 9, 16 and 23, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

- Dose groups that were examined:
All test and control animals were tested.

- Battery of functions tested:
Motor Activity.
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The evaluation period was thirty minutes for each animal. The time in seconds each animal was active and mobile was recorded for the overall period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory reactivity.
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Behavioural assessments.
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.

OTHER:

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS PATHOLOGY:
Yes (see table 14)

HISTOPATHOLOGY:
Yes (see table 15)

Other examinations:

None stated.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
All data was summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

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:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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 specified

Details on results:

CLINICAL SIGNS AND MORTALITY:
- Mortality:
There were no unscheduled deaths during the study.

- Clinical observations:
A summary incidence of daily clinical observations is given in Table 1.
No clinically observable signs of toxicity were detected.
An isolated incident of increased salivation detected up to ten minutes after dosing was observed in two males treated with 1000 mg/kg/day on Day 28, with noisy respiration also being evident for one of the males. Noisy respiration was also noted in one female treated with 1000 mg/kg/day on Day 27 only. Observations of this nature are often reported following the oral administration of an unpalatable and/or locally irritant test material formulation and, in isolation, are considered not to be indicative of systemic toxicity.
No such effects were detected for animals of either sex treated with 150 and 15 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN:
Group mean weekly bodyweights and standard deviations are given in Table 6 and are presented graphically in Figure 1 and Figure 2. Group mean weekly bodyweight gains and standard deviations are given in Table 7 (statistically significant differences are indicated). Individual data are given in Appendix 4 and Appendix 5.
No adverse effect on bodyweight development was detected for treated animals when compared to controls.
A statistically significant reduction in bodyweight gain was observed in males treated with 1000 mg/kg/day during Week 1. Males treated with 15 mg/kg/day also showed a reduction in bodyweight gain during Week 4. In the absence of a dose related response or an adverse effect on overall bodyweight gain the intergroup differences were considered to be of no toxicological importance. Females from all treatment groups showed a statistically significant reduction in bodyweight gain during Week 2. In the absence of an adverse effect in overall bodyweight gain the intergroup differences were considered to be of no toxicological significance.

FOOD CONSUMPTION
Group mean weekly food consumptions are given in Table 8 and are presented graphically in Figure 3 and Figure 4.
No adverse effects on dietary intake or food efficiency were detected for treated animals in comparison to controls.

FOOD EFFICIENCY
Weekly food efficiencies are given in Table 9.
No adverse effects on dietary intake or food efficiency were detected for treated animals in comparison to controls.
WATER CONSUMPTION
Group mean daily water consumptions are given in Table 10.
Daily visual inspection of water bottles and daily measurements undertaken during Weeks 3 and 4 revealed a significant increase in water consumption for animals of either sex treated with 1000 mg/kg/day, when compared to controls.
Increased water consumption can often follow the oral administration of an unpalatable and/or locally irritant test material formulation however in the absence of any histopathological correlates to suggest irritancy the intergroup differences were considered to be of no toxicological significance.
No such effects were detected in animals of either sex treated with 150 and 15 mg/kg/day.

OPHTHALMOSCOPIC EXAMINATION
Not examined.

HAEMATOLOGY
Group mean values and standard deviations for test and control group animals are given in Table 11 (statistically significant differences are indicated). Individual data are given in Appendix 6 and Appendix 7.
There were no toxicologically significant changes detected in the haematological parameters measured.
Males treated with 15 mg/kg/day showed a statistically significant increase in clotting time. In the absence of a dose related response or any associated haematological changes the intergroup difference was considered to be of no toxicological importance.

CLINICAL CHEMISTRY
Group mean values and standard deviations for test and control group animals are given in Table 12 (statistically significant differences are indicated). Individual data are given in Appendix 8.
There were no toxicologically significant changes detected in the blood chemical parameters measured.
A statistically significant increase in glucose levels was detected in males treated with 1000 mg/kg/day whilst females from this treatment group showed statistically significant reductions in plasma chloride and phosphorus concentrations and a statistically significant increase in alkaline phosphatase. Males from all treatment groups also showed a statistically significant reduction in phosphorous. In the absence of any histopathological correlates, the intergroup differences were considered to be of no toxicological importance.
No such effects were detected in females treated with 150 and 15 mg/kg/day.

URINALYSIS
Not examined.

NEUROBEHAVIOUR
- Functional Observations:
A summary incidence of behavioural assessments is given in Table 2 and Table 3. Group mean functional test values and standard deviations are given in Table 4. Individual values are given in Appendix 1 and Appendix 2. A summary incidence of sensory reactivity assessments is given in Table 5. Individual responses are given in Appendix 3.

- Behavioural Assessments:
Weekly open field arena observations did not reveal any treatment changes in behaviour.
All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and ages used, and were of no toxicological importance.

- Functional Performance Tests:
There were no treatment-related changes in the functional performance parameters measured.
Statistical analysis of the data revealed no significant intergroup differences.

- Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and ages used, and were of no toxicological importance.

ORGAN WEIGHTS
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 13. Individual data are given in Appendix 9 and Appendix 10.
There were no treatment-related organ weight changes detected.
Statistical analysis of the data revealed no significant intergroup differences.

GROSS PATHOLOGY
A summary incidence of necropsy findings is given in Table 14. Individual data are given in Appendix 11.
No treatment-related macroscopic abnormalities were detected at terminal kill.
One female treated with 150 mg/kg/day incurred damage to the brain during removal at necropsy. One female treated with 15 mg/kg/day also incurred damage to the pituitary during the necropsy procedure. These findings were physical and occurred after termination of treatment and therefore were considered to be of no toxicological significance.

HISTOPATHOLOGY:
A summary incidence of histopathological findings is given in Table 15. Individual data and the grading system are given in Appendix 12.
There were no treatment-related microscopic changes observed.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable.

HISTORICAL CONTROL DATA (if applicable)
Not applicable.

OTHER FINDINGS

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: The oral administration of MOS-HIGE to rats for a period of twenty eight consecutive days at dose levels of 15, 150 and 1000 mg/kg/day did not result in any toxicologically significant effects.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of MOS-HIGE to rats for a period of twenty eight consecutive days at dose levels of 15, 150 and 1000 mg/kg/day did not result in any toxicologically significant effects. The “No Observed Effect Level” (NOEL) was therefore, considered to be 1000 mg/kg/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

Methods.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (1% Carboxy methylcellulose).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Results.

Mortality.

There were no treatment-related deaths during the study.

Clinical Observations.

No clinically observable signs of toxicity were detected.

Behavioural Assessment.

There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.

There were no treatment-related changes in the performance parameters measured.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Bodyweight.

No adverse effect on bodyweight change was detected for treated animals when compared to controls.

Food Consumption.

No adverse effects on dietary intake or food efficiency were detected for treated animals when compared to controls.

Water Consumption.

No toxicologically significant effects were detected.

Haematology.

There were no toxicologically significant changes detected in the haematological parameters measured.

Blood Chemistry

There were no toxicologically significant changes detected in the blood chemical parameters measured.

Organ Weights.

There were no treatment-related effects in the organ weights measured.

Necropsy.

No treatment-related macroscopic abnormalities were detected.

Histopathology.

There were no treatment-related microscopic changes observed.

Conclusion.

The oral administration of MOS-HIGE to rats for a period of twenty eight consecutive days at dose levels of 15, 150 and 1000 mg/kg/day did not result in any toxicologically significant effects. The “No Observed Effect Level” (NOEL) was therefore, considered to be 1000 mg/kg/day.