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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-15 to 2013-08-29
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
Reference Type:
publication
Title:
Developmental toxicity studies with 6 forms of titanium dioxide test materials (3 pigment-different grade & 3 nanoscale) demonstrate an absence of effects in orally-exposed rats
Author:
Warheit DB, Boatman R, Brown SC
Year:
2015
Bibliographic source:
Regulatory Toxicology and Pharmacology 73, 887-896

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-2
- Physical state: solid, fine white crystalline powder
- 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)=1.734 µm, particle size is unaffected by 1 week storage (data not shown)
D50(TEM ECD)=165 nm
D50(corrected XSDC)=162 nm

Crystal composition (XRD) and density (He pycnometry)
Rutile= 100%
Anatase= 0%
Crystalline size>100 nm
Density (g/cm³)= 4.107

BET surface area= 7.1 m²/g

XPS surface composition (atom%)
Titanium= 18
Oxygen= 63.8
Alumina= 8.4
Silica= 0.4
Carbon= 6.1

Isoelectric point (pH)= 6.4

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: 285-323 g (mean: 304.5 g, ± 20% = 243.62 – 365.42 g); females: 184-225 g (mean: 201.14 g, ± 20% = 161.71 – 242.57 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. 1426)
- 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 formulation was prepared freshly on each administration day immediately prior to dosing.
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed for 2-3 minutes.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspensions thoroughly before every dose administration.
Application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured (measured weekly).

VEHICLE
- Batch no.: 26210S1-2
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 the 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 the analysis of 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 the stability analysis were taken from low, medium and high dose groups in study week 1 at 0 hr and 6 hrs (6 samples in total).
All formulation samples were stored at -20° C.

The analytical method for the determination of titanium in dose formulation samples was successfully validated according to SANCO/3029/99 rev. 4 (11/07/00).
The determinations were performed by ICP-OES using two independent emission wavelengths highly specific for titanium, one wavelength for quantification and one for confirmation.
The requirements of SANCO/3029/99 rev. 4 (11/07/00) regarding linearity, precision (repeatability), accuracy (recovery) and specificity were fulfilled.
The mean recovery values for titanium at all fortification levels obtained by ICP-OES comply 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%.
Test substance concentration, stability and homogeneity in dosing formulations were obtained by ICP-OES.

Results:
The analytical results obtained for the individual dose groups were consistent with the analysis of the % of nominal of the test item for the concentration, stability and homogeneity analyses, with the exception of homogeneity sample numbers 18 (300 mg/kg bw/day, week 1) and 31 (100 mg/kg bw/day, last week) for which the recoveries were higher. The mean recovery noted for sample number 20a (300 mg/kg bw/day, week 1) was 43.1%. These variations were considered to be caused by a sampling error.
In this case the samples need to be vortexed for approximately 2 minutes before sampling. The dose formulation sample preparation was made every day freshly before the dose administration. On the day of analytical sample collection the formulation samples were prepared in excess and analytical samples were collected from the same stock as that used for the dose administration. The formulation samples were assumed to have not been subjected to proper homogenization procedure before the collection of sample numbers 18, 20 and 31. This was evident from the % recoveries of the nominal concentration and the stability analysis of sample numbers 3, 11, 12 of 300 mg/kg bw/day group and 4, 8, 13 and 14 of 100 mg/kg bw/day group (collected from the same formulation preparation as that of the sample numbers 18, 20 (300 mg/kg bw/day group) and 31 (100 mg/kg bw/day group), respectively, which had a consistent and an acceptable recovery. As there were no toxicity findings seen at any of the tested dose levels including the HD group, this concentration increase and decrease in isolated analytical samples of the 100 mg/kg bw/day and/or 300 mg/kg bw/day groups had no impact on the validity and integrity of the study.
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
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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/d 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: once a day; morbidity and mortality: twice daily, except during holidays and weekends where the observation was made 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 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 or death during the study, 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 statistically significant).
For abnormality “pelvic girdle ilium bone offset” the statistical analyses was performed by combining all unilateral and bilateral findings.
For abnormality “cervical vertebral centra- unossified” the statistical analyses was performed by combining all the unossified cervical vertebral centra (vertebral centra 1 to 7).
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: none of the animals died due to treatment in the study. However, one female animal in the 100 mg/kg bw/day group showed signs of paraplegia during the initial days of dose administration. This animal was euthanized on gestation day 10. This finding was considered to be incidental and not related to treatment.

- clinical observations: no clinical signs of toxicological relevance noted in any of the animals of the treated groups in comparison to the control.

- body weight development: no treatment related effect noted for body weight and body weight change in the treated groups in comparison to the controls. No treatment related changes noted for terminal and adjusted maternal body weight in the treated groups in comparison to the controls. The statistical analysis of the body weight data showed no statistical significance.

- food consumption: no treatment related effect noted for food consumption in treated groups in comparison to the controls. The statistical analysis of the food consumption data showed no statistically significant changes between the treated and the control group.

- no treatment related changes noted for the prenatal parameters including the number of corpora lutea, number of implantation sites, early and late resorptions, or pre- and post-implantation loss.
However, there was an increase in the number of early resorptions in the 300 mg/kg bw/day group, which also accounted for the increased total resorption in the 300 mg/kg bw/day group. There was no statistically significant or dose-related response noted for the increase in early resorptions and these were not considered treatment related.


- no effects on the pregnancy rate of the animals. The rates in the control and treated groups were as follows: control group: 80%; 100 mg/kg bw/day group: LD 92%; 300 mg/kg bw/day group: 83.33% and 1000 mg/kg bw/day: 83.33%.

- pathology: there were no macroscopic findings considered to be related to the treatment in any of the animals of the control and or test item treated groups at necropsy. However, there was a fluid distended uterus observed in one female of the 1000 mg/kg bw/day group. This animal was non pregnant and the fluid distension could be the normal physiological change of uterus during normal oestrus cycle. Also considering the finding was reported in a single female of the 1000 mg/kg bw/day group this was not considered to be related to the treatment.

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:
- no treatment related changes noted for the prenatal parameters including the live foetuses, number of dead foetuses, number of male and female foetuses, or sex ratio.
In addition, there was a slight decrease in the number of female foetuses in the 1000 mg/kg bw/day group, but considering the slightly higher number of male foetuses and total live foetuses in the 1000 mg/kg bw/day group being comparable to the control it was assumed that decreased numbers of female foetuses were compensated by a slightly higher number of male foetuses. In addition the mean values for the numbers of male and female foetuses were within the historical control data range. Hence, the finding was not considered to be associated with treatment. Because foetal sex is determined shortly after conception and well before the onset of dosing on gestation day 5, such changes in sex ratio are not considered to be indicative of a test substance-related effect.
The statistical analysis of data showed no statistically significant changes between the treated and the corresponding control group.
- no effect of treatment noted for the litter data including mean litter weight, total litter weight and male and female litter weight. However, there was a slight but and not statistically significant decrease in female litter weight noted in the 1000 mg/kg bw/day group (-12.96%). Given that all litter weight data reported were within the historical control data range, the finding was not considered to be associated with the treatment.

- no external abnormalities considered to be of toxicological relevance noted in any of the treated groups. The statistical analysis showed no significant changes. However, there were a few abnormalities noted in a few isolated foetuses of the control, 300 mg/kg bw/day and 1000 mg/kg bw/day groups. The abnormalities were gastroschisis in the control, micrognathia in the 300 mg/kg bw/day and 1000 mg/kg bw/day groups and small upper jaw in the 300 mg/kg bw/day group. The single foetus with micrognathia and small upper jaw in the 300 mg/kg bw/day group was used for visceral examination and could not be verified by skeletal examination.
The foetuses with micrognathia in the 1000 mg/kg bw/day group were checked during skeletal examination and only 1 of 2 foetus was confirmed with micrognathia and the other looked normal. These abnormalities were observed in a single foetuses of a single isolated female animal from either control or treated groups and therefore were considered to be spontaneous in their origin and unrelated to the treatment.

- skeletal examination revealed a range of abnormalities in the control and treated groups that were either within historical control ranges recorded for this laboratory; were significantly lower than the corresponding control values; or were seen only in the 300 mg/kg bw/day or 100 mg/kg bw/day dose groups and were not dose dependent.
There was statistically significant increase in the foetal incidences for unossification of vertebral cervical centrum in the 1000 mg/kg bw/day groups. However, the percent litter incidence in the 300 mg/kg bw/day and 1000 mg/kg bw/day groups was lower than in the concurrent control group. This variation, from the developmental perspective was of minimal significance and is normal in the foetuses of this strain of rats with C-section on gestation day 20. Therefore, this abnormality was not considered to be an adverse effect related to the treatment.

- internal examinations of the foetal viscera revealed a range of visceral abnormalities in all groups including the control. There were no abnormalities of toxicological relevance.

- craniofacial examination revealed a range of abnormalities in all groups including controls. These abnormalities either did not differ significantly from control values or did not show dose-related responses and were thus considered to have no relevance to treatment.

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-2 to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg bw/ day from gestation day 5 through gestation day 19 produced no adverse toxicological effects in the females or foetuses or significant developmental effects at any administered dose.
Based on the findings from this study, the NOAEL (No-Observed-Adverse-Effect-Level) of TiO2 pg-2 in the Wistar rat for both maternal toxicity and developmental toxicity is considered to be 1000 mg/kg bw/ day.