Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2013-05-06 to 2013-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-01-22
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): TiO2 pg-3
- Physical state: solid, fine crystalline powder, white
- Storage condition of test material: at room temperature

Information taken from the report by Brown, S.C (2014):

Particle size
D50(laser diffraction, 10mg/mL loading)=0.349 µm, particle size is unaffected by 1 week storage (data not shown)
D50(TEM ECD)=132 nm
D50(corrected XSDC)=179 nm

Crystal composition (XRD) and density (He pycnometry)
Rutile= 100%
Anatase= 0%
Crystalline size>100 nm
Density (g/cm³)= 3.976

BET surface area=17.1 m²/g

XPS surface composition (atom%)
Titanium= 8.7
Oxygen= 65.3
Alumina= 12.4
Silica= 3.2
Carbon= 8.3

Isoelectric point (pH)= 3.8

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at the start of the treatment period: 11-12 weeks old
- Weight at the initiation of pairing: males: 306 – 348 g (mean: 323.02 g, ± 20% = 258.42 – 387.62 g); females: 188 – 222 g (mean: 205.44 g, ± 20% = 164.35 – 246.52 g)
- Housing: kept individually in IVC cages (except during the mating period when two females were paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot Nr. 240113)
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice (lot Nr. 0902)
- Water (ad libitum): tap water, sulphuric acid acidified to a pH of approximately 2.8
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 55 ± 10%
- Air changes: 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua ad iniectabilia (water for injection)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tarred plastic vial on a precision balance.
The test item was suspended in the vehicle.
The vehicle was employed based on the test item’s characteristics and testing guideline. The test item and control formulations were prepared freshly on each administration day immediately prior to dosing.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The application volume for all groups was 5 mL/kg bw/day.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured on various gestation days.

VEHICLE
- Batch no.: 2621051-2
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity, stability as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of nominal concentration of the dose formulations of the test item in the vehicle were taken in the first and last week of the study for all doses (8 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose and low dose preparation. Samples were taken in the first and last week of the study (18 samples in total).
Samples for stability analysis were taken from the low dose, medium dose and high dose group in study week 1 at 0 hr and 6 hrs (6 samples in total).
All formulation samples were stored at -20° C.

Results:
Determination of the nominal substance concentrations (in week 1 and last week of the study), stability analysis (0 and 6 hours in week 1) and homogeneity (samples from the top, middle and bottom of container in week 1 and last week of the study) were performed and the results revealed that mean recovery values for TiO2 at all fortification levels obtained by ICP-OES were between 81.1 to 97.1 which complied with the standard acceptance criteria of SANCO/3029/99, which demands that the mean recovery at each fortification level should be in the range of 70% - 110%.
The relative standard deviations per fortification level ranged from 0.52% to 3.16% (overall: 2.38%) for TiO2 analysis in dose formulations. These values meet the guideline requirements of SANCO/3029/99 rev. 4 (RSD ≤ 20%).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused/M/F ratio per cage: females were paired with males as per the ratio of 1:2 (male to female).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
Duration of treatment / exposure:
Gestation day 5 through gestation day 19
Frequency of treatment:
once daily, 7 days per week
Duration of test:
20 days
No. of animals per sex per dose:
24-25 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/day corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: general clinical observations: at least once a day (approximately within half an hour after application); morbidity and mortality: at least once daily.
- Cage side observations checked: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size, changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of the mating, a detailed clinical observation outside the home cage was made.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment. The sperm positive females were weighed during gestation days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes, food consumption of pregnant females was measured on gestation days 0, 5, 8, 11, 14, 17 and 20.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
At the time of termination, each dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
- Organs examined: immediately after the termination, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appeared non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, with cervix
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: the uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The position and number of foetuses in each uterine horn were also recorded.
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, craniofacial examination of the heads of the foetuses used for the soft tissue examination
All foetuses were weighed and sexed based on the anogenital distance.
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Chi-square test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as a statistically significant).
Indices:
no data
Historical control data:
Historical control data is provided for the following:
- mean uterine data
- mean litter weight (g) data
- foetal external examination
- foetal visceral examination
- foetal craniofacial examination
- foetal skeletal examination

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
- none of the females showed signs of abortion or premature delivery prior to the scheduled terminal sacrifice.

- mortality: all sperm positive females survived until the scheduled necropsy.

- clinical signs: no test item-related clinical signs were observed in the study.

- body weight development: statistical analysis of body weight and body weight gain data during the gestation period revealed no statistically significant effect on body weight development in treatment groups when compared with the controls.

- food consumption: no statistically significant effect of TiO2 pg-3 on food consumption was observed in any of the treatment groups when compared with controls and in correlation to the body weight and body weight gain, the food consumption was comparable in all groups.

- statistical analysis of prenatal data revealed no significant effect on prenatal parameters like gravid uterus weight, adjusted maternal weight, number of corpora lutea, number of implantation sites, resorptions (early and late), percent preimplantation loss, or post implantation loss in the treated groups when compared with the controls.

- successful mating resulted in 20/23 pregnancies in the control, 21/23 pregnancies in 100 mg/kg bw/day, 22/23 in 300 mg/kg bw/day and 21/22 in 1000 mg/kg bw/day groups. Nine females were not mated and therefore excluded from the study without any further observations.

- pregnancy rates (Nr. of pregnancies achieved /Nr. of females mated or sperm positive x 100) in treatment groups 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day were 91.30 %, 95.65% and 95.45%, respectively, compared to 86.96 % in control group. The differences in pregnancy rates between the 100 mg/kg bw/day and 300 mg/kg bw/day groups and the control were considered to be due to biological variation and of no toxicological relevance.

- pathology: no macroscopic findings were observed in any female of the control and treatment groups at necropsy.

Please also refer to the field "Attached background material" below.

Effect levels (maternal animals)

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

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
- statistical analysis of prenatal data revealed no significant effect on prenatal parameters like live foetuses, dead foetuses, sex ratio, group mean number of male and female foetuses in the treated groups when compared with the controls.

- no statistically significant or treatment-related effects were observed in any treatment group as compared with the control on group mean litter weight, total litter weight, male litter weight, female litter weight, group mean number of live foetuses, or number of males and number of females.

- no test item related gross external abnormalities were seen in either the control or treatment groups. However, some few and unremarkable abnormalities were observed, generally in single foetuses, and were considered to be incidental in nature and unrelated to treatment.

- skeletal examination revealed a range of abnormalities which were generally of a type or which occurred at an incidence comparable to or lower in treated groups when compared to the control group or were seen only at the 300 mg/kg bw/day or 100 mg/kg bw/day treated groups and were not dose dependent.

- statistically significant increases were observed in the incidences of unossified 4th forelimb phalanx (L) and unossified 5th hindlimb phalanx (R) in the 1000 mg/kg bw/day group, wavy ribs in the 300 mg/kg bw/day and 1000 mg/kg bw/day groups, incomplete ossification on the interparietal and incomplete ossification of the right parietal. In all cases, the incidence rate of these findings were within recent historical control ranges reported in the laboratory. Increases in incidences of wavy ribs and incomplete ossification are considered to be variations and not associated with long term consequences on survival, general growth and development.

- internal examinations of foetal viscera revealed a range of visceral abnormalities in all groups including the control. However, there was a statistically significant increase in incidence of hemorrhagic bladder content in the 1000 mg/kg bw/day group when compared with the controls. The bladder finding is most likely attributable to a dissection artefact during visceral examinations and it is not a developmental variation and therefore not considered to be adverse.
The remaining visceral abnormalities observed in the treated groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls.

- craniofacial examination revealed a range of visceral abnormalities in all groups including controls. Statistical analysis of the data revealed no significant effect in any of the findings when compared with controls.

Please also refer to the field "Attached background material" below.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study, the repeated dose oral administration of TiO2 pg-3 to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg bw/day on gestation days 5 to 19 produced no significant toxicological effects in the females or foetuses or significant developmental effects at any administered dose.
Based on the findings from this study, the NOAEL for TiO2 pg-3 for both maternal and developmental toxicity in the Wistar rat is considered to be 1000 mg/kg bw/day.