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Administrative data

Endpoint:
developmental toxicity
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:
other: OECD 422
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

Constituent 1
Chemical structure
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
Details on test animals or test system and environmental conditions:
Animals:
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 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%).
Details on mating procedure:
Main animals
Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 8 days. A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.
Recovery animals:
Recovery animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.
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.
Frequency of treatment:
Test item or negative control animals were administered daily on a 7 days/week basis.
Duration of test:
Until post-natal Day 5.
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:
An overview of the dosing scheme and the investigations performed is presented in the attachment.

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.

Examinations

Maternal examinations:
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable.
The duration of gestation was recorded and was calculated from Day 0 of pregnancy.
Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach.

Ovaries and uterine content:
Not examined.
Fetal examinations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal) and on post-natal day 4, with accuracy of 0.01g.
All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death.
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.
Indices:
Parental Females
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non mated females
- Female mating index
- Female fertility index
- Gestation index
- Duration of pregnancy (days)
- Number of Corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups Day 0 and 4
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)

Offspring
- Mean pup body weight (per pup within the group and per litter) on postnatal Day 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4
- Survival Index of pups on postnatal Days 0 and 4
- Sex ratio % (on postnatal Days 0 and 4)

Historical control data:
No data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No adverse effects were detected at the highest dose.
An overview of the results obtained is presented in the attachment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No structurally abnormal pups were detected, even at the highest dose used.
An overview of the results obtained is presented in the attachment.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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.

No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and of gestation, delivery and post-partum/lactation period until post-partum Day 5.

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs or development.

 

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