Lithium titanium oxide (Li4Ti5O12)

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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A guideline study with GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

Principles of method if other than guideline:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the toxicity of the test item following repeated daily administration by oral gavage to Wistar rats. Reversibility of any treatment-related changes was evaluated following a 14-day Recovery period. The study also included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.
In the Main Groups, male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day 5.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetralithium(1+) 3,3-di{[oxido(oxo)titanio]oxy}-1,5-dioxotrititanoxane-1,5-bis(olate)

EC Number:

619-916-2

Cas Number:

12031-95-7

Molecular formula:

Li4Ti5O12

IUPAC Name:

tetralithium(1+) 3,3-di{[oxido(oxo)titanio]oxy}-1,5-dioxotrititanoxane-1,5-bis(olate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch/Lot number: 0857 VX 037
Molecular formula: Li4Ti5O12
Appearance: White solid powder
Purity: >98%
Expiry date: 30 November 2012
Storage condition: Room temperature (15-25 °C)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:WI rats
Source: Charles River Laboratories, Germany GmbH, Sandhofer Weg 7, D-97633
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose.
At the completion of the study, the spare animals were returned as no replacements with spare animals were performed.
Age of animals: Young adult rats, approximately 10 weeks old at starting and 12 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 348 g – 389 g, Females: 204 g - 287 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 5 days

Husbandry:
Animal health: Only healthy animals were used for the test. Females were nulliparous and nonpregnant.
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.9 – 25.0 °C
Relative humidity: 37 – 66 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
Randomization
All parental/adult (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.1 % in water

Details on oral exposure:

The test item was formulated in 0.1% Carboxymethylcellulose (0.1% CMC) at 6.25, 25 and 100 mg/mL concentrations without correction for purity. Formulations were prepared fresh prior to administration to animals.
Test item or negative control material treated Groups 1-4 main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item formulations were analysed for concentration and homogeneity at the Test Site Seibersdorf Labor GmbH; 2444-Seibersdorf; Austria. Top, middle and bottom triplicate samples were taken from test item formulations on 3 occasions, during the first, fourth and the sixth weeks of the treatment. Two sets to analyse (which was collected in replicates as practical) and one set as a back-up for any confirmatory analyses. Similarly, one sample was taken in duplicate from the vehicle Control Group 1 solution for concentration measurements.
In addition, on the first occasion, two samples were taken from the middle of the formulation vessel from all three dose levels and sent to the test site for method validation. At the Test Site, all samples were digested and diluted, the same way as used in the main study. From the resulting solutions, six different dilutions were prepared giving similar end concentrations for all concentration steps. All resulting 36 solutions were measured against an external concentration. This procedure covered the whole analysis process and proved the independence of dilution. For correctness of the data, a standard addition experiment was applied, at two different concentrations, each carried out as triplicates.
The validation data were calculated using the program VALDATA according to DIN 38402 – part 51 giving the following information: Linearity, calculated calibration line, residual standard deviation, method standard deviation, detection limit, quantification limit.
Description of the analytical method:
An aliquot of the samples was digested with 10 ml Hydrofluoric acid (HF) and 5 ml Hydrochloric acid (HCl). The acids were evaporated until nearly to dryness and again 10 mL HF and 5 mL HCl were added and the first step was repeated. The residue was dissolved in 50 mL HCl and filled up to 50 mL with distilled water. Li and Ti were measured with ICP.
Dose formulations were homogenous. The measured concentrations of Lithium-Titanium-Oxide evaluated for each test item-dose group varied between 90.7 and 107.0% (mean values of each two replicates). No test item was detected in the control samples. These results were within acceptable ranges (85 % - 115%).

Duration of treatment / exposure:

Dosing procedure
An overview of the dosing scheme and the investigations performed is presented in the attachment.
Main animals:
Test item or negative control material treated Groups 1-4 main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.
Dosing of both sexes began after an acclimation period (A) of at least 5 days after the animal arrival; the animals were dosed for 2 weeks before mating, during the mating/post-mating, and were continued up to the day of necropsy.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.
Females were dosed for 14 days pre-mating, for up to 8 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum.

Recovery animals
Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 55.

Frequency of treatment:

Test item or negative control animals were administered daily on a 7 days/week basis.

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 62.5; 250; 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose.
At the completion of the study, the spare animals were returned as no replacements with spare animals were performed.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose selection and route of administration
The dose levels and the vehicle were selected based on available data, formulation and analytical trials and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 11/346-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
The oral route was selected as it is a possible route of exposure to the test item in humans.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological and ophthalmoscopic assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day 4.
Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.
An overview of the dosing scheme and the investigations performed is presented in the attachment.

Sacrifice and pathology:

At termination, necropsy with macroscopic examination was performed.
For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery), animal found dead pre-terminally during the study in control group.

Females showing no-evidence of copulation were sacrificed as practical, 24-26 days after the end of the mating period.
All F1 offspring were terminated on Day 4 post-partum; in order to allow for overnight fasting of dams prior to urine collection onpost-partum day 5, offspring were euthanized on post-natal day/post-partum day 4, and the dams on post-natal day/post-partum day 5.

Other examinations:

See Section 7.8 for reproductive parameters.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

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:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

In the P- and F1-generation, and in the recovery group.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In the P-generation and the recovery group.

Histopathological findings: neoplastic:

no effects observed

Effect levels

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:

other: No toxic effects were observed at the highest dose.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Dose formulations were homogenous. The measured concentrations of Lithium-Titanium-Oxide evaluated for each test item-dose group varied between 90.7 and 107.0% (mean values of each two replicates). No test item was detected in the control samples. These results were within acceptable ranges (85 % - 115%).

Applicant's summary and conclusion

Conclusions:

The NOAEL for Lithium-Titanium-Oxide for parental/adult and F1 effects is 1000 mg/kg bw/day under conditions of this study.

Executive summary:

Lithium-Titanium-Oxide was administered daily to 3 groups of Wistar rats by gavage for at least 28 days to males and ca. 45 day to females according to the OECD test guideline 422 for a combined repeated dose toxicity and reproduction toxicity study. Dosing did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment, in ophthalmological changes, or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, urinalysis and in macroscopic or microscopic changes parameters at dose levels of 62.5, 250, or 1000 mg/kg bw/day during either the treatment or after a 14-Day Recovery period under the conditions of this study.

The NOAEL for Lithium-Titanium-Oxide for parental/adult and F1 effects is 1000 mg/kg bw/day under conditions of this study.