Magnesium potassium titanium oxide

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:

22 November-20 December 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

(1996)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1995)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

436-900-9

EC Name:

-

Cas Number:

39290-90-9

Molecular formula:

Hill Empirical formula: K(0.2-0.7) Mg(0.4) Ti(1.6) O(3.7-3.95) CAS Empirical formula: K(0.2-0.7) Mg(0.4) Ti(1.6) O(3.7-3.95)

IUPAC Name:

Magnesium Potassium Titanium Oxide

Details on test material:

Batch: 0G99
White powder
Expiry date: 21 August 2001
Test substance storage: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Outbred, SPF quality. Source: Charles River Deutschland, Sulzfeld, Germany.
Age of the animals at the start of reatment: 6 weeks.
Identification by earmark and tattoo.
A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Aq. Carboxymethyl cellulose

Details on oral exposure:

A stainless steel stomach tube was used. Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quadruple samples of formulations (5.00 ml) were taken on 11 December 2000.
Groups 1 and 3 (0 and 150 mg/kg bw/day): samples taken from the middle (accuracy)
Groups 2 and 4 (50 and 1000 mg/kg bw/day): samples taken from top, middle and bottom (accuracy and homogeneity)

The samples were stored at room temperature in labelled pots until analysis.

Analysis of the samples was performed under the responsibility of Solvias AG, Elemental and Microanalytical Services, Basel, Switzerland

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150 and 1000 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

Control animals:

yes

Details on study design:

The dose levels were selected on the basis of a 5-day dose range finding study.
A controlled environment was maintained in the room with optimal conditions considered as being approximately 15 air changes per hour, a temperature of 21±3C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.

Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm.) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.

Free access to standard pelleted laboratory animal diet and to tap water.

Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Examinations

Observations and examinations performed and frequency:

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

MORTALITY/Viability: twice daily

CLINICAL SIGNS: once daily,
detailed clinical observations were made in all animals. Once prior to start of treatment and on days 8, 15, 22 and 28, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:

FUNCTIONAL OBSERVATIONS:
During week 4 of treatment, the following tests were performed on all animals:
-hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
-motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system).

BODY WEIGHT: weekly
On days 1, 8, 15, 22 and 28.

FOOD CONSUMPTION: Weekly

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

CLINICAL LABORATORY INVESTIGATIONS:
Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.25 ml), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 ml).

Sacrifice and pathology:

All animals surviving to the end of the observation period were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a 4% formaldehyde solution:
Adrenal glands, Aorta, Brain, Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar
Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina) and aAll gross lesions. Identification marks: not processed.

All organ and tissue samples, as defined below, were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. The following slides were examined by a pathologist:
-all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group;
-all gross lesions of all animals.
All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement

Other examinations:

The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: adrenal glands, brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes and Thymus.

Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduledpost mortemexamination, between 7.30 andThe animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.25 ml), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 ml).

Statistics:

The following statistical methods were used to analyse the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance. Group means were calculated for continuous data and medians were calculated for
discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

References:
- C.W. Dunnett, Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no mortality

Mortality:

no mortality observed

Description (incidence):

no mortality

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Details on results:

MORTALITY:
No mortality occurred during the study period.

CLINICAL SIGNS:
No treatment-related abnormalities were found.
Hunched posture was shown by one high dose male in weeks 3 and 4. This male also showed slightly reduced body weight gain, and reduced weight of the thymus and spleen. The other animals within this group were without such findings. Therefore, this sign was considered to be related to a general deteriorated health status and not to be a sign of toxicity. Scabs, as shown by one high dose female, are commonly noted in rats of this age and strain and housed and treated under the conditions in this study and considered of no toxicological significance. Other animals were without clinical signs.

NEUROBEHAVIOUR:
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with TERRACESS P, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.
Increased motor activity as recorded by the high sensors was noted for one low dose group male. Low sensor readings were comparable to control values and no dose-related response was obtained. Motor activity of one high dose male as measured by the low sensors was slightly increased, but values of the other animals within this group were largely comparable to controls. Therefore, these changes were considered to be of no toxicological significance.

BODY WEIGHTS and FOOD CONSUMPTION:
No treatment related effects.
Body weights and body weight gain among group 3 and 4 males were slightly reduced in week 4. Among group 4 males this was largely due to a reduced weight gain of one animal. On exclusion of the value of this animal no dose-related decrease in body weights in obtained. Moreover, no effects on food consumption were noted. Therefore, the slightly decreased body weights of group 3 males in week 4 were considered to have occurred by chance. Also, the statistically significant decreased body weight gain of group 3 females in week 2 was considered to be of no toxicological relevance since group 4 values in week 2 were comparable to controls.

HAEMATOLOGY:
No treatment-related effects were found.
A (slightly) low platelet value was measured for two group 4 males, one group 2 female and one group 3 female. No microscopic evidence was obtained to support this finding. Values of other animals within the same group were comparable to control values. Mean corpuscular haemoglobin concentration showed a decrease among group 3 females. Since control values were slightly high when compared to historical data and a dose-response relationship was absent, this change was considered to be of no toxicological significance.

CLINICAL BIOCHEMISTRY:
No treatment-related effects were found.
A slightly high aspartate aminotransferase activity value was obtained for one animal (high dose male) and a high urea value was measured for one high dose female. There were no microscopic correlates and values of the other animals within the same group were comparable to controls. Statistically significant decreases of sodium and calcium values among group 3 and/or 4 males occurred in the absence of a clear dose-response relationship and all individual values were within the range of historical control data. Therefore, these changes were considered to be of no toxicological significance.

MACROSCOPIC EXAMINATION:
No treatment-related effects were found.
Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, a reduced size of the thymus, an accesory lobe on the liver and scab formation on the skin. These findings are occasionally seen among rats used in these types of studies and were considered changes of no toxicological significance.

ORGAN WEIGHTS:
No treatment-related effects were found.
The slightly low terminal body weights of group 4 males were largely due to one animal. On exclusion of the value of this animal, no dose related decrease of terminal body weights was obtained. Decreased (relative) spleen and liver weights among group 2 females occurred in the absence of a dose related response. Therefore, these changes were considered not to represent a sign of toxicity.

MICROSCOPIC EXAMINATION:
No treatment-related effects were found.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects at highest dose tested / original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Test substance formulations in 1% aqueous carboxymethyl cellulose formed a homogeneous suspension at the concentrations tested. Values of one group 3 sample and one group 4 sample slightly exceeded the acceptable range of 100±10% of nominal. However, since the other values were within this range and the homogeneity was acceptable, it was concluded that accuracy measurements represented an acceptable level for formulations of this type. A minimal amount of titanium was found in the control group vehicle. There were no indications that this caused any relevant effects in the control group animals. Therefore, this was considered not to be of any influence on the study integrity not the outcome.

Applicant's summary and conclusion

Conclusions:

Terracess P is not classified as toxic (NOAEL: 1000 mg/kg/day)