Lithium 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:

27 May-24 June 2003

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:

2003

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:

Lithium Potassium Titanium Oxide

EC Number:

445-620-6

Cas Number:

39318-30-4

Molecular formula:

Hill Empirical formula: K(0.5-0.7) Li(0.27) Ti(1.73) O(3.8-3.95) CAS Empirical formula: K(0.5-0.7) Li(0.27) Ti(1.73) O(3.8-3.95)

IUPAC Name:

Lithium Potassium Titanium Oxide

Details on test material:

Batch: 2D79A
White powder, crystal structure
Expiry date: 11 April 2004
Specific gravity: 3.4
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:

TEST ANIMALS
- Animals: Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Earmark and tattoo. Males/females. Females were nulliparous and non-pregnant
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks.
- Weight at study initiation:
- Fasting period before study:
- Housing: 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). 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.
- Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
- Water: Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.
A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health


ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 19.5 – 23.8°C), a relative humidity of 30 – 70% (actual range: 34 – 81 %) and 12 hours artificial light and 12 hours darkness per day. Temporary deviations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal level of 70% for relative humidity.
Based on laboratory historical data these conditions were considered not to have affected the study integrity.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Stainless steel stomach tube. Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight. Formulations were placed on a magnetic stirrer during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 4 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

Test substance formulations in propylene glycol formed a homogeneous suspension at the concentrations tested. Analyses of group 2 samples revealed some higher percentages in one top sample and/or in one bottom sample. The cause for these differences remains unclear. Since the duplo samples showed results which were comparable with the remaining values and with the group 4 analyses, it was concluded the homogeneity was sufficient. Analysis of the accuracy of dose preparations revealed values within the range of 85 –110% (Li), 89 – 114% (Ti) and 85 – 110 % (K) of target. Although some values were outside the range of 90 – 110%, the mean accuracy values were within these limits. Therefore, these values were considered to represent an acceptable level of accuracy for formulations of this type.

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

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for at least 28 days, 7 days per 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:
other: formulations in propylene glycol

No. of animals per sex per dose:

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

Randomisation: by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Control animals:

yes

Details on study design:

The dose levels were selected on the basis of a 5-day dose range finding study.






- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

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: at least 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 . 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.
-motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system).

BODY WEIGHT: 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 (0.5 ml), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml). The following parameters were determined:

Parameter Abbreviation Unit

Haematology a
Erythrocytes count RBC 1012/l
Haemoglobin HGB mmol/l
Haematocrit HCT l/l
Mean corpuscular volume MCV fl
Mean corpuscular haemoglobin MCH fmol
Mean corpuscular haemoglobin concentration MCHC mmol/l
Platelet count PLT 109/l
Red cell distribution width RDW %
Total leucocytes count WBC 109/l
Differential leucocyte count %WBC
neutrophils (NEUT), lymphocytes (LYMPHO), monocytes (MONO),
eosinophils (EOS), basophils (BASO)

Clotting Potential b
Prothrombin time PT s
Partial thromboplastin time APTT s

Clinical Biochemistry c
Alanine aminotransferase ALAT U/l
Alkaline phosphatase ALP U/l
Aspartate aminotransferase ASAT U/l
Bilirubin, total µmol/l
Chloride mmol/l
Cholesterol, total mmol/l
Creatinine µmol/l
Glucose mmol/l
Phosphorus INORG.PHOS mmol/l
Protein, total g/l
Protein, albumin g/l
Urea mmol/l
Calcium mmol/l
Potassium mmol/l
Sodium mmol/l

a Instrumentation: Sysmex K-1000 (Goffin Meyvis). Differential leucocyte count determined manually by microscope.
b Instrumentation: STA Compact (Roche Diagnostics).
c Instrumentation: Olympus AU400 (Goffin Meyvis).

Sacrifice and pathology:

At the end of the observation period all animals 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 all gross lesions. Identification marks: not processed.

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

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. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement

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:

effects observed, treatment-related

Description (incidence and severity):

No mortality

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality during the study period.
A hunched posture was observed among all females dosed at 1000 mg/kg/day in weeks 2/3. In view of its occurrence in high dose females only, this sign was considered to be related to treatment.

Scabs, brown staining of the fur (neck), piloerection, and chromodacryorrhoea and a broken tail were incidentally noted. Based on the incidental occurrence, the lack of an effects at higher levels and/or the prevalence in both the control group and (to a lesser extent) the highest dose group, these findings were considered signs of no toxicological significance. No clinical signs were noted in the control males, males treated at 50 and 1000 mg/kg/day and in females treated at 50 and 150 mg/kg/day.


BODY WEIGHT AND WEIGHT GAIN
A reduced weight gain was observed for females at 150 and 1000 mg/kg/day throughout the study, the average weight gain deficit at termination being 23% and 14% relative to female controls, respectively. Although the effect was not clearly related to the dose level, these changes were considered to be related to treatment.
During the study statistical significance was attained in most instances for lower body weight gain in females at 150 and 1000 mg/kg/day, and for absolute weight gain in females at 150 mg/kg/day on day 28 only. Higher weight gains than in controls were recorded throughout the study for males at 1000 mg/kg/day.

The weight gains observed for males and females at 50 mg/kg/day and for males at 150 mg/kg/day were considered to be within control range.


HAEMATOLOGY
White blood cell counts (WBC) were increased in males at 1000 mg/kg/day. The finding was not associated with significant changes in the distribution of leukocyte types, and may have been secondary to stressful conditions. Males at 150 mg/kg/day showed significantly lower haemoglobin (Hb) and haematocrit (HCT) values.

Lower white blood cell counts found in female rats treated at 50 and 150 mg/kg/day. Individual increases of neutrophil counts with concurrently reduced lymphocyte counts were noted among the dose groups without a treatment related distribution. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance. Also, the slightly lower partial thromboplastin time for females at 150 mg/kg/day was considered of no toxicological significance considering the direction of change (i.e. a decrease).


CLINICAL CHEMISTRY
In males and females at 1000 mg/kg/day alanine aminotransferase (ALAT) activity was slightly but significantly elevated. In addition, females at 1000 mg/kg/day showed significantly increased alkaline phosphatase (ALP) activity.

Sodium concentration was depressed in males and females at 1000 mg/kg/day, and in females at 150 mg/kg/day (the latter without statistical significance). Furthermore, inorganic phosphate concentration was elevated in males at 1000 mg/kg/day, and potassium concentration was increased in females treated at 150 and 1000 mg/kg/day. Urea concentration was also higher in males and females at 1000 mg/kg/day, although the changes were not significant in statistical terms.

Lower calcium, total protein and albumin values in males at 150 mg/kg/day and lower bilirubin values in females at 150 mg/kg/day were considered not to be toxicologically relevant in the absence of similar effects at 1000 mg/kg/day.


NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals. The variation in motor activity did not indicate a relation with treatment.


ORGAN WEIGHTS
Absolute and relative spleen weights were increased in males treated at 1000 mg/kg/day. Additionally, relative spleen weights in males at 150 mg/kg/day showed an increase, although without statistical significance.

The increased absolute thymus weights in females at 50 mg/kg/day and relative thymus weights in females at 50 mg/kg/day and 1000 mg/kg/day, were considered not to be a sign of toxicity in the absence of a dose relationship and the lack of corroborative findings at microscopic examinations.


OTHER FINDINGS
MACROSCOPIC EXAMINATION:
Necropsy did not reveal any toxicologically relevant alterations.

Incidental findings among control and treated animals included pelvic dilation of the kidneys, cysts on the kidneys, foci on the clitoral gland and on the thymus, enlargement of the thymus and mandibular lymph nodes, discolouration of the thymus, mandibular and popliteal lymph node, watery fluid in the uterus, and a tail fracture. These findings are occasionally seen among rats used in these types of studies. Further, in the absence of a treatment-related distribution and/or their incidental occurrence they were considered changes of no toxicological significance.


MICROSCOPIC EXAMINATION:
There was and increase in severity of hemopoiesis, primarily erythropoiesis, in the spleen of 150 mg/kg/day and 1000 mg/kg/day treated males.

No treatment related abnormalities were seen in the other males or in the females. The remainder of the microscopic findings recorded was within the range of background pathology encountered in rats of this strain and age.

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the the subacute 28-day oral toxicity test results with Terracess L by daily oral gavage, Terracess L is not classified as toxic by oral administration in the rat ( NOAEL: 50 mg/kg bw/day).