Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

 Based on the weight of evidence from the available long-term toxicity/carcinogenicity studies in rodents and the relevant information on the toxicokinetic behaviour in rats it is concluded that TiO2 does not present a reproductive toxicity hazard.

Within the scope of the re-evaluation of titanium dioxide (E171) as a food additive by the European Food Safety Authority (EFSA) adopted on the 28thJune 2016, the conduct of a “multigeneration or extended-one generation reproduction toxicity study according to the current OECD guidelines” was recommended. This study was requested in order to establish a health-based guidance value (ADI) for the food additive titanium dioxide (EFSA Journal 2016;14(9):4545).

The preparatory work for the conduct of an extended one generation reproductive toxicity study is currently ongoing, i.e. the selection of an appropriate test item and the decision on a suitable study design and whether to include or exclude additional cohorts. Upon study completion, a robust study summary will also be incorporated in the REACH registration dossier. Further experimental testing for the endpoint toxicity to reproduction within the scope of REACH is therefore not considered to be required.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction

 

Within the scope of the re-evaluation of titanium dioxide (E171) as a food additive by the European Food Safety Authority (EFSA) adopted on the 28thJune 2016, the conduct of a “multigeneration or extended-one generation reproduction toxicity study according to the current OECD guidelines” was recommended. This study was requested in order to establish a health-based guidance value (ADI) for the food additive titanium dioxide (EFSA Journal 2016;14(9):4545).

 

The preparatory work for the conduct of an extended one generation reproductive toxicity study is currently ongoing, i.e. the selection of an appropriate test item and the decision on a suitable study design and whether to include or exclude additional cohorts. Upon study completion, a robust study summary will also be incorporated in the REACH registration dossier. Further experimental testing for the endpoint toxicity to reproduction within the scope of REACH is therefore not considered to be required.

 

During a comprehensive literature search for information on reproduction toxicity of TiO2, seven references were identified representing studies on toxicity to reproduction, conducted either in mice or rats receiving ultrafine or pigment-grade titanium dioxide via oral (gavage), inhalation (whole body), subcutaneous or intravenous administration. After a thorough reliability screening, all of these references were considered of very limited relevance for hazard assessment purposes. The criteria for quality, reliability and adequacy of experimental data under REACH and for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X) are not fulfilled. These references are discussed below, highlighting their findings and the reasons for their exclusion from hazard assessment:

 

NIER (2009) describes a study which evaluated the reproductive and developmental toxicity of titanium dioxide according to OECD 421. Groups of 10 male and 10 female Sprague-Dawley rats were treated with 1,000 mg TiO2/kg bw/day in 1 % methylcellulose via gavage. According to the authors, no treatment-related changes were observed in the parental generation regarding general toxicity and reproductive performance. Also, the authors stated that no treatment-related changes were observed in the F1 generation. The NOAEL of titanium dioxide was considered to be above ,1000 mg/kg bw/day. The reference is however only available as a study summary without raw data and was therefore rated as not assignable (RL=4). Whereas the original study was not obtainable, it was nevertheless assessed and peer-reviewed within the OECD SIDS initial assessment report (OECD CoCAM4, April 2013) and approved by the OECD procedure on Mutual Acceptance oaf Dta (MAD). Thus, the study is considered useful as supporting information.

 

Engler-Chiurazzi et al. (2016) investigated the effects of titanium dioxide nanoparticles on the behavioural and cognitive function of male Sprague-Dawley rats (20 weeks old) whose maternal parents (n = 4) were exposed to the substance via inhalation (whole body) for 4 days/week, 5 hours/day during gestation day 7 up to and including gestation day 20. The concentration level was 10.4 mg TiO2/m³ air (analytical). A vehicle control group was run concurrently. Eleven male rats were taken from four control or four exposed litters to undergo behavioural assessment. According to the authors, prenatal nano-TiO2exposure induced significant working or short-term memory impairments and changes in initial motivation in male pups. In addition, the authors mentioned that gestational nano-TiO2exposure significantly altered visible platform performance (group comparisons on the final trial not significant). The authors did not find any effects on locomotor, balance, affective, anxiety-like, or depressive-like behaviour in male pups following gestational nano-TiO2 exposure via maternal inhalation. Reference memory learning, retention, and perseveration in any phase of the Morris water maze test were not markedly altered in pups. Furthermore, no significant effects for either Working memory incorrect or Reference memory errors were observed. Lastly, the authors did not observe any marked impact on maternal weight, implantation site number or pup number/litter after test item exposure. This reference has several reporting and experimental deficiencies which do not allow a definitive conclusion on the exposure-effect correlation:

-      the number of pregnant rats was too low in this study (4 rats/group). The guideline foresees a total of 20 litters for each dose level in order to have an adequate number of offspring for the evaluation of neurotoxicity. This reduction of pregnant dams, which in turn leads to a low number of offspring available for testing, constitutes a significant reduction of the statistical power to levels below acceptability.

 

-      the number of offspring used for behavioural testing were too low, and it was not clearly stated how many offspring were taken from the respective groups. As a minimum number of animals in each dose group for pre-weaning and post-weaning examinations the guideline recommends 10/sex (1/litter) or 20/sex (1/sex/litter). However, only a total of eleven males was reported for testing behavioural and cognitive function, both for treatment and control groups. This low number of male pups again constitutes a substantial reduction in statistical power.

 

-      Only males were used for behavioural and cognitive testing which does not allow a conclusion for effects in females. It is widely accepted that female animals are usually more susceptible to behavioural and cognitive alterations. In addition, the guideline foresees testing of both genders.

 

-      The guideline foresees that at least three dose level and a concurrent control should be used. However, in this study only one high concentration level (10.4 mg TiO2/m³) was tested. Firstly, the usage of one concentration level precludes the possibility to demonstrate any dose-response and a determination of a No-Observed-Adverse Effect level (NOAEL) or a benchmark dose. Secondly, the concentration of 10 mg/m³ used in this study clearly exceeded the MTD, resulting in extreme stress and impaired breathing to be expected which may well influence foetal development indirectly without being test-item related.

 

-      The guideline foresees, as a minimum, that the test substance or control are given to the mated females from gestation day 6 (implantation) throughout lactation day 21. The pre- and postnatal neurological development phase of the offspring would then be covered by the exposure of the test item by using this dosing regime. In contrast, the test item exposure in this study was limited to the period of gestation (day 6 to day 20), which precludes the pups being exposed during the postnatal neurological development. In addition, the exposure did not occur continuously (4 d/week).

 

-      Observations of the dams with regard to clinical signs, mortality, detailed clinical observations or food consumption were missing. The observations of the offspring on clinical signs, mortality, body weight, and detailed clinical observations are missing. As stated by the guideline, body weight is a good indication of physical development of the pups. By not observing the health conditions of the offspring, it cannot be ruled out that alternative explanations for the results exist.

 

-      The guideline recommends that young adults be tested at postnatal day 60 – 70. In this study, behavioural testing of the male offspring was conducted at 20 weeks of age. Since testing was conducted at 20 weeks of age, it cannot be ruled out that the effects observed during behavioural testing might have been caused by an other factor due to the long lag tome between gestational exposure and testing.

 

-      Finally, the authors did not report any neuropathological examination of the male pups. The guideline states that the purposes of the examinations are to “(i) to identify regions within the nervous system exhibiting evidence of neuropathological alternations, (ii) to identify types of neuropathological alternations resulting from exposure to the test substance; (iii) to determine the range of severity of the neuropathological alterations”. A neuropathological examination of the pups would support and confirm the findings reported to have occurred during the behavioural testing. Due to the lack of this examination in this study, the findings of the authors cannot be further confirmed.

 

Due to the shortcomings presented above, this publication was disregarded for hazard and risk assessment.

 

 

Kubo-Irie et al. (2016) exposed groups of six pregnant ICR mice by subcutaneous injection to alumina-coated titanium dioxide nanoparticles (35 nm) in saline containing 0.05 % Tween-80. Concentrations used were 1, 10, 100, and 1000 µg/mL. A vehicle control group was run concurrently. The mice were treated on gestation day 5, 8, 11, 14, and 17 and they gave birth on gestation day 19. The effects of prenatal exposure to the test item on spermatogenesis in 12-week old mice was investigated. According to the authors, the test item was detected in the seminiferous epithelium of the male offspring and low cellular adhesion and degenerated Sertoli cells were observed in the seminiferous epithelium. These observations were made in all treatment groups. The detrimental function of the Sertoli cells resulted in the formation of abnormal spermatozoa. Lastly, they mentioned that the incident of abnormal sperm were a tendency to increase in line with the concentrations. The study represents a mechanistic investigation with very limited value for hazard assessment purposes. The following reporting and experimental deficiencies were noted in the publication:

 

- the test item was administered via subcutaneous injection. This unphysiological route of exposure is irrelevant for health hazard assessment of industrial chemicals.

 

- 20 pregnant females per treatment group are usually required for the investigation of the effect of the test item, but in this study only six females were used per treatment group. This significantly reduces the statistical power and precludes any meaningful evaluation of the effect of the test item.

 

- the female mice were exposed only on gestational day 5, 8, 11, 14, and 17. The guideline however foresees that the female mice are exposed daily prior to mating, during the mating period, pregnancy and up to the weaning of offspring.

 

- clinical signs, mortality, food consumption, body weight and gross pathology were not recorded for the female mice. Without this kind of information, it is unknown whether the test item had any adverse effect on the female animals, which in turn could have an adverse outcome on the developing offspring.

 

- full examination of offspring are missing (spermatogenesis examined only), it can therefore not be excluded that the findings were secondary to other effects in the offspring.

 

Based on the shortcomings given above, this publication was disregarded for hazard and risk assessment purposes.

 

Morgan et al. (2015) investigated the reproductive toxicity of titanium dioxide nanoparticles (Aeroxide P25; anatase; particle size: 10 nm) in male rats. Forty adult male albino rats were divided into two equal groups. One group was used as control receiving no treatment, whereas the second group received 100 mg/kg bw/day of the test item in distilled water via gavage. Half of the animals per group were killed after 4 weeks of exposure and the remaining animals were killed after 8 weeks of exposure. Frequency of exposure was daily. According to the authors, the results showed that titanium dioxide nanoparticles affected the male reproductive system as evidenced by marked reduction in body weight and relative sex organ weights (testis, epididymis, seminal vesicle and prostate gland). Furthermore, they stated that the sperm motility, concentration and viability percentage were also significantly reduced after exposure to titanium dioxide nanoparticles with increased incidences of sperm morphological abnormalities (deformed head, detached head, curved tail and coiled tail). In addition, serum testosterone levels were also said to be significantly decreased compared with control. Lastly, the investigators stated that marked histopathological alterations in testis, epididymis, seminal vesicle and prostate gland were observed. This reference however exhibits major reporting and experimental deficiencies, which do not allow firm conclusions on any reprotoxic effects, as follows:

 

- the rat strain used in the study was not characterised further, which does not allow any comparison with strain-specific historical control data, thus a meaningful evaluation of findings is not possible.

- although the bodyweight of 180-200 g at the start of the study appears plausible for a young rat (aged 6 -7 weeks), the bodyweight after 4 and 8 weeks of exposure appears grossly implausible: after 4 and 8 weeks the average body weight is given as 271 g and 272 g respectively. For an adult rat after 8 weeks post study initiation (age 14 - 15 weeks), one would expect an average body weight of either 470 - 480 g (Sprague Dawley) or 390 - 410 g (Wistar), for example.

- the body weight gain of 1 g between week 4 and week 8 after study initiation in the control group animals appears grossly implausible: for a young rat one would expect approx. 20 g of weight gain.

- the implausible body weight and the unusual bodyweight gain during the conduct of the study indicate inappropriate housing conditions and a failure of the lab to handle the animals properly, so that effects on relative organ weights cannot be excluded because of this.

- the SD of the testosterone levels appear surprisingly low, since these hormone levels are known to undergo a periodic alteration in the circadian cycle, resulting in a large variation over the day and also between animals; in this study, 

- the variation of the testosterone levels between exposed and control animals are in fact within the normal range of physiological fluctuation.

Based on the shortcomings given above, this publication was disregarded for hazard and risk assessment purposes.

 

Gao et al. (2012) investigated the ovarian injury and gene-expressed characteristics in female CD-1 (ICR) mice (30 mice/group) induced by intragastric administration of titanium dioxide nanoparticles (anatase; self-synthesised) in 0.5 % (w/v) hydroxypropylmethylcellulose at 10 mg/kg bw daily for 90 consecutive days. A vehicle control group was run concurrently. Following the 90-day administration, ten treated females were mated with ten males in order to provide information on fertility. According to the authors, the findings indicated that titanium dioxide nanoparticles can accumulate in the ovary and result in ovarian damage, cause an imbalance of mineral element distribution and sex hormones, decrease fertility or pregnancy rate and oxidative stress in mice. Furthermore, they state that microarray analysis showed that in ovaries from mice treated with TiO2 NPs compared to controls, 223 genes of known function were up-regulated, while 65 ovarian genes were down-regulated. The study represent a mechanistic investigation and is considered not to be of any value for hazard assessment purposes, because of the following deficiencies in reporting and experimental design:

 

- the data of this publication show standard deviations of exactly 5 % for all measured data. Standard deviations of exact 5 % throughout all experiments covering multiple endpoints in an in vivo system are implausible and factually impossible and therefore put into question whether this study represents authentic, reliable research data.

 

- the authors suggest that the test substance caused several adverse effects following oral administration of (assumed) nano-sized titanium dioxide in CD-1 mice. The test item was however a self-synthesised material by the authors, but with a poor description of the generated test substance. Since the test item is poorly described in the publication and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2particles at all were used in these experiments, and (ii) whether the effects can really be attributed to titanium dioxide.

 

- the authors describe a decrease of fertility in the female mice. Normally, 20 females per treatment group are used to investigate this effect of the test item on fertility, but in this study only 10 females were used. This significantly reduces the statistical power and reduces the meaningful evaluation of the effect of the test item.

 

- the guideline foresees that at least three dose levels and a concurrent control should be used. In this study, only one dose level (10 mg/kg bw) was tested. The usage of one dose level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL).

 

Based on the shortcomings given above, this publication was disregarded for hazard and risk assessment purposes.

 

Hougaard et al. (2010, 2011; please refer to Section 7.9.1. Neurotoxicity) exposed time-mated C57BL/6BomTac female mice (22 - 23 mice/group) by inhalation 1 h/day to 42.4 mg/m³ air (analytical data) aerosolised titanium dioxide nanoparticles (UV-titan L 181; rutile; major particle size: ~100 nm (geometric mean number diameter: 97 nm)) on gestation days 8 - 18. A vehicle control group was run concurrently. The developmental neurotoxicity in offspring was investigated as well as maternal inflammatory response. In a follow-up study (Kyjovska et al., 2013), the influence of the maternal airway exposure to titanium dioxide nanoparticles on male reproductive function in the two following generations (F1 and F2) were evaluated. In order to evaluate the reproductive function, F1 offspring were cross-mated with naive CBA/J mice. According to Hougaard et al. (2010, 2011), inhalation of nano-sized coated titanium dioxide induced long-term lung inflammation in time-mated adult mice, and their gestationally exposed offspring displayed neurobehavioral alterations. Furthermore, Kyjovska et al. (2013) stated that maternal particulate exposure did not affect daily sperm production statistically significantly in the F1 generation, although titanium dioxide tended to reduce sperm counts. They also stated that time-to-first deliver the F2 litter increased with decreasing sperm production and there was no effect on sperm production in the F2 generation originating after TiO2 exposure. Furthermore, the authors report statistically significant differences in sperm production between mouse strains. This investigation has several reporting and experimental deficiencies, which do not allow firm conclusions on hazard potential to be drawn, for the following reasons:

 

-      the number of litters was too low in this study (13 - 14/dose). The guideline recommends 20 litters for each dose level in order to have an adequate number of offspring for the evaluation of neurotoxicity. This reduction of litters causes a significant reduction of the statistical power.

 

-      the guideline foresees that at least three dose levels and a concurrent control should be used. In this study, only one high concentration level (42.4 mg/m³ (analytical)) was tested. Firstly, the usage of one concentration level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL). Secondly, the concentration of 42.4 mg/m³ used in this study clearly exceeded the MTD, resulting in extreme stress with impairment of normal breathing behaviour to be assumed, which mas well influence foetal development indirectly.

 

-      the guideline also foresees, as a minimum, that the test substance or control are given to the mated females from gestation day 6 (implantation) throughout lactation day 21. The pre- and postnatal neurological development phase of the offspring would then be covered by the exposure of the test item by this dosing regime. In this study, however, the test item exposure occurred only during gestation (days 8 to 18), which precludes that the pups were exposed during the postnatal neurological development.

 

-      behavioural investigations of the animals were conducted during the light period. Since rats are nocturnal animals, testing the animals during a period where they are not normally active might result in findings which one might not obtain otherwise.

 

-      observations of the dams with regard to food consumption are missing, just as data on clinical signs alsonot being reported. In addition, clinical signs, detailed clinical observations, learning and memory testing (adolescences), and body weight (weanlings) of the offspring are not documented. By not observing the health conditions of the offspring, it cannot be ruled out that alternative explanations for the results exist.

 

-      the authors did not report neuropathological examinations of the offspring. The guideline states that the purposes of the examinations are to “(i) to identify regions within the nervous system exhibiting evidence of neuropathological alternations, (ii) to identify types of neuropathological alternations resulting from exposure to the test substance; (iii) to determine the range of severity of the neuropathological alterations”. A neuropathological examinations of the pups would have supported and confirmed the findings observed during behavioural testing. Due to the lack of this examination in this study, the findings of the authors cannot be verified.

 

-      The guideline foresees that young adults be tested for motor and sensory function at postnatal day 60 – 70. In this study, testing of the offspring was conducted at 4 months of age. Since testing was conducted at 4 months of age, it cannot be ruled out that the effects observed during behavioural testing might have been caused by another factor during this long period between gestational exposure and testing.

 

-      Motor activity testing was too short and missing for the pre-weaning period. Firstly, since motor activity testing was not conducted during the pre-weaning period, there is no possibility to check whether a change in motor activity occurred during the developmental period of the offspring. Secondly, by measuring the motor activity over a shorter period of time than recommended, different results may have been obtained as compared to if the activity had been measured over longer period of time.

 

 

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

 

Zhao et al. (2013) exposed groups of 100 female CD-1 (ICR) mice to titanium dioxide nanoparticles (self-synthesised) via oral (gavage) administration for 90 consecutive days. The dose levels were 2.5, 5, and 10 mg/kg bw. The authors investigated fertility, histopathological changes of ovary, ovary weight, titanium content in ovaries, sex hormones levels, and cytokine expression in the ovaries. According to the authors, the test item exposure led to atretic follicle increases, severe inflammatory response and necrosis in the ovary. Furthermore, the results suggested a reduction in mating rate, pregnancy rate, number of newborn, impaired growth of neonates and altered sex hormone levels. Lastly, the study suggest that cytokine levels were significantly altered. The study represent a mechanistic investigation with no value for hazard assessment purposes with sever reporting and experimental designdeficiencies, as follows:

 

- all data of this publication show a standard deviations of exactly 5 % for all measured data. Standard deviations of exact 5 % throughout all experiments covering multiple endpoints in an in vivo system are implausible and put into question whether this study represents reliable research data.

 

- the results appear to indicated that the test substance caused adverse effects following oral administration of (assumed) nano-sized titanium dioxide in CD-1 mice. The test item was self-synthesised by the authors, but with a poor description of the generated test substance. Since the test item is poorly described in the publication and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in these experiments, and (ii) whether the effects can really be attributed to titanium dioxide.

 

- although 100 animals per group were stated, only 5 females per group were used for the evaluation of TiO2-induced toxicity to reproductive system. Thus, the number of animals per group is too low for an appropriate statistical analysis.

 

Based on the shortcomings given above, this publication was disregarded for hazard and risk assessment purposes.

 

 

Miura et al. (2017) exposed male C57BL/6J mice to titanium dioxide nanoparticles (Aeroxide P25) (0.1, 1, 2, and 10 mg/kg bw) via intravenous injection once per week for 4 consecutive weeks. A vehicle control group (disodium phosphate) was run concurrently. The authors investigated the weight of testicular organs (testes, epididymides and cauda epididymides), sperm motility, sperm head numbers, titanium content in testis, and plasma sex hormones. According to the authors, no differences in testicular organ weights or plasma sex hormones could be observed. Motile percent and progressive percent were reduced by TiO2treatment. Sperm head numbers of the left cauda epididymis were significantly reduced in TiO2NP treated animals. This reference however has the following several reporting and experimental deficiencies, which do not allow reliable conclusions on hazard potential:

 

- the test item characterisation is poorly described, which makes it impossible to verify the identity of the test item.

 

- the test item was administered via intravenous injection. This is considered to be an unphysiological route of exposure and irrelevant for health hazard assessment.

 

- the number of animals per group is not clearly stated which does not allow any judgement on the statistical robustness of the results.

 

- animals were not examined thoroughly, body weight record, food and water consumption, mortality, clinical observation, full histopathology (only male reproductive organs) and full gross pathology are missing.

 

- the dosing regime was once per week for 4 consecutive weeks, whereas the guideline foresees a daily dosing.

 

Based on the shortcomings given above, this publication was disregarded for hazard and risk assessment purposes.

 

Conclusion

There is currently no relevant and reliable guideline compliant study available for the endpoint toxicity to reproduction. One reproductive toxicity screening assay only is available, but only as a brief study summary without raw data and was therefore rated as not assignable (RL=4). This study showed no impairment of male or female fertility. Due to its use within the OECD HPV programme, the study is used as supporting information.

 

Further eight references report mechanistic studies with a focus on organ specific investigations, such as ovarian injury, investigation of male reproductive system or female reproductive system only, behavioural/neurotoxicity testing of offspring, or fertility in offspring of exposed dams. However, the study designs of these eight references are not in accordance with any accepted guideline and are therefore of limited relevance for chemical hazard assessment. The references also lack significance for example due to poor test item characterisation, non-physiological routes of administration, low number of animals available for testing, offspring too old during behavioural testing, one concentration tested only, short exposure duration, wrong dosing regime, implausible body weight documentation, and/or incomplete observation data. It is therefore concluded that all of these references do not fulfil the criteria for quality, reliability and adequacy of experimental data under REACH for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies were therefore included for information purposes only.

 

Within the scope of the re-evaluation of titanium dioxide (E171) as food additive by the European Food Safety Authority (EFSA) adopted on the 28th June 2016, the conduct of a “multigeneration or extended-one generation reproduction toxicity study according to the current OECD guidelines” was recommended. This study was requested in order to establish a health-based guidance value (ADI) for the food additive titanium dioxide (EFSA Journal 2016;14(9):4545). The preparatory work for the conduct of an extended one generation reproductive toxicity study is currently ongoing (status December 2017). Upon study completion, a robust study summary will also be included in the REACH registration dossier. Further experimental testing for the endpoint toxicity to reproduction within the scope of REACH is therefore considered not to be required.

Effects on developmental toxicity

Description of key information

Based on the information from the available six pre-natal developmental toxicity studies via gavage in rats (according to OECD 414, GLP) it is concluded that maternal toxicity up to the highest dose level of 1000 mg/kg bw/d, and reproduction data of dams were not affected by treatment. There was no embryo-toxicity and no effect on the development of foetuses. No external visceral or skeletal malformations were observed and the incidence of variations was not different between treated and control groups. No incidences of skeletal retardations were observed.

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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.
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
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.
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
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.
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
Abnormalities:
not specified
Developmental effects observed:
not specified
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.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-10 to 2014-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 67 days
- Weight at gestation day 0:
0 mg/kg/day: 200 g - 225 g
100 mg/kg/day: 200 g - 225 g
300 mg/kg/day: 200 g - 225 g
1000 mg/kg/day: 200 g - 225 g
- Housing: animals were housed in pairs (when possible) in solid-bottom caging with Bed-o'Cobs® bedding and nestlets as enrichment.
- Diet (ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: yes, but length of period was not stated

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26ºC
- Relative humidity: 30-70%
- Photoperiod: approximate 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
other: sterile water for injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations of the test substance in the vehicle were prepared at least weekly. The stability of the dosing formulations at concentrations bracketing the concentrations administered to the animals was demonstrated for up to 8 days.
The test substance was suspended in sterile water for injection. The dosing formulations were not adjusted for purity; a purity of value of 100% was assumed for the purposes of dose formulation calculations. Dosing formulations were stored at room temperature until used.
The volume administered (5 mL/kg) was based on the most recent body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of pigment grade titanium dioxide (TiO2 pg-1) in water at concentrations of a control, 20, 60 and 200 mg/mL were analyzed for homogeneity and concentration verification. Samples of each formulation were taken 2 times: near the beginning and end of the study. Dose samples at the same concentrations prepared near the end of the study were analyzed for concentration verification. Samples were digested in a chemical microwave using nitric and hydrofluoric acid and then analyzed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).
The analysis results for samples prepared at the beginning of the study show that the test substance was homogeneously mixed as represented by top, middle and bottom samples from each dose preparation. The mean concentrations were 19.6, 64.6, and 198 mg/mL of the dose preparations.
The measured values were 98.0%, 108%, and 99.0% of nominal, all within the acceptable range of 80 to 120% of the respective targeted suspension concentrations of 20, 60, and 200 mg/mL samples.
The analysis results for samples prepared at the end of the study show that the test substance was at the targeted concentrations for each dose preparation. The concentrations were 19.9, 59.9 and 203 mg/mL for the dose preparations. The measured values were 99.7%, 99.9% and 102% of nominal, all within the acceptable range of 80-120% of the respective targeted suspension concentrations of 20, 60, and 200 mg/mL samples.
The test substance was not detected in the control samples.
Details on mating procedure:
- Impregnation procedure: timed mated
Duration of treatment / exposure:
Gestation days 6 - 20
Frequency of treatment:
once daily
Duration of test:
20 days
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:
22 timed mated females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Quarantine and pretest: mortality/moribundity at least once daily; clinical observation on gestation day 4
Testing period: mortality/moribundity twice daily (morning and evening); clinical observations twice daily on gestation days 6 - 20 (during weighing and at least 2 hours post-dosing) and once on gestation day 21.

DETAILED CLINICAL No

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine and pretest: gestation day 4
Testing period: daily on gestation days 6 - 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Quarantine and pretest: gestation day 4

Testing period: gestation days 6, 8, 10, 12, 14, 16, 18, 20, and 21
Food consumption was measured as food consumed per cage, which was then divided by the number of animals in each cage for individual food consumption values. Excess food spillage was recorded.

- 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 21
- Organs examined: a gross external and a visceral examination were performed immediately after euthanasia.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: uteri with no visible implantation sites were placed in a 10% aqueous solution of ammonium sulfide to detect very early resorptions.
Fetal examinations:
- External examinations: Yes, all foetuses classified as live were examined for alterations. External sex was recorded for each live foetus.
- Soft tissue examinations: Yes, approximately half of the live foetuses
- Skeletal examinations: Yes, all live foetuses; The skeletal bodies of all the foetuses and the skulls of half the foetuses (foetuses that were not designated for head examination) were examined for alterations.
- Head examinations: Yes, approximately half of the foetuses
- Body weight of each live foetus was recorded.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- no test substance-related mortality at any level tested; all animals on study survived until scheduled euthanasia.
- no test substance-related clinical observations at any level tested; the observations that were recorded were unremarkable and occurred infrequently.
- no test substance-related effects on maternal body weight parameters at any level tested; data for maternal body weights and weight changes were comparable across all dose levels tested. Mean final body weights (absolute or adjusted) GD 21 were within 2% of the control group mean at every dose level tested.
- no test substance-related effects on maternal food consumption at any level tested. Mean maternal food consumption from GD 6-21 was within 3% of the control group mean for all dose levels tested.
- 100 mg/kg/day: mean food consumption from gestation days 6-8 was slightly increased relative to the control mean. At 300 mg/kg/day, mean food consumption from days 14-16 and 16-18 of gestation were slightly lower than the respective control means. These instances were statistically significant; however, they are considered spurious and unrelated to the test substance. The changes are minimal in magnitude, variably increased or decreased, not dose dependent, and had no impact on cumulative food consumption values (gestation days 6-21) for these groups.
- no test substance-related maternal gross postmortem observations at any level tested. All animals appeared normal at the group at necropsy.
- no test substance-related effects on any reproductive outcome endpoint.
- litter means for numbers of implantation sites, early and late resorptions were all comparable to control group values for every dose level tested.
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter means for numbers of live and dead foetuses as well as for foetal weight and sex ratio were all comparable to control group values for every dose level tested.
-100 mg/kg/day dose level: mean foetal weight was significantly increased relative to the control group mean. This increase was considered to be spurious and unrelated to the test substance because it was minimal in magnitude and not dose dependent. Further, an increase in mean foetal weight is not typically considered to reflect an adverse outcome as a decrease in this endpoint might.
- no test substance-related foetal malformations or variations observed at any dose level tested. The foetal alterations occurred with low frequency across all groups tested and occur with similar frequency in the test facility historical control database.
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
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this study, there was no evidence of either maternal or developmental toxicity at doses up to 1000 mg/kg/day. Therefore, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity is at the limit dose of 1000 mg/kg/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-05-06 to 2013-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
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
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
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/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.
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
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.
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
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.
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
Abnormalities:
not specified
Developmental effects observed:
not specified
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.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-05-05 to 2014-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 62-66 days
- Weight at gestation day 0:
0 mg/kg/day: 201 g - 225 g
100 mg/kg/day: 200 g - 225 g
300 mg/kg/day: 200 g - 225 g
1000 mg/kg/day: 200 g - 225 g
- Housing: animals were housed in pairs in solid-bottom caging with Bed-o'Cobs® bedding and nestlets as enrichment.
- Diet (ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: yes, but length of period was not stated

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26ºC
- Relative humidity: 30-70%
- Photoperiod: approximate 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
other: sterile water for injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations of the test substance in the vehicle were prepared at least weekly. The stability of the dosing formulations at concentrations bracketing the concentrations administered to the animals was demonstrated for up to 8 days.
The test substance was suspended in sterile water for injection. The dosing formulations were not adjusted for purity; a purity of value of 100% was assumed for the purposes of dose formulation calculations. Dosing formulations were stored at room temperature until used.
The volume administered (5 mL/kg) was based on the most recent body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of ultrafine titanium dioxide (TiO2 uf-1) at concentrations of 20, 60 and 200 mg/mL were analysed for homogeneity and concentration verification. Samples of each formulation were taken 2 times: near the beginning and end of the study. Dose samples at the same concentrations prepared near the end of the study were analysed for concentration verification. A 0 mg/mL control sample containing the vehicle only, sterile water, was included and analysed with each set of study samples.
Samples were prepared in a chemical microwave using nitric and hydrofluoric acid and then analysed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).
The analysis results show that the test substance was homogeneously mixed and at the targeted concentrations.
The test substance was not detected in the control samples.
Details on mating procedure:
- Impregnation procedure: timed mated
Duration of treatment / exposure:
Gestation days 6 - 20
Frequency of treatment:
once daily
Duration of test:
20 days
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:
22 timed mated females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Quarantine and pretest: mortality/moribundity at least once daily; clinical observation on gestation day 4
Testing period: mortality/moribundity twice daily (morning and evening); clinical observations twice daily on gestation days 6 - 20 (during weighing and at least 2 hours post-dosing) and once on gestation day 21.

DETAILED CLINICAL No

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine and pretest: gestation day 4
Testing period: daily on gestation days 6 - 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Quarantine and pretest: gestation day 4

Testing period: gestation days 6, 8, 10, 12, 14, 16, 18, 20, and 21
Food consumption was measured as food consumed per cage, which was then divided by the number of animals in each cage for individual food consumption values. Excess food spillage was recorded.

- 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 21
- Organs examined: a gross external and a visceral examination were performed immediately after euthanasia.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: uteri with no visible implantation sites were placed in a 10% aqueous solution of ammonium sulfide to detect very early resorptions.
Fetal examinations:
- External examinations: Yes, all foetuses classified as live were examined for alterations. External sex was recorded for each live foetus.
- Soft tissue examinations: Yes, approximately half of the live foetuses
- Skeletal examinations: Yes, all live foetuses; The skeletal bodies of all the foetuses and the skulls of half the foetuses (foetuses that were not designated for head examination) were examined for alterations.
- Head examinations: Yes, approximately half of the foetuses
- Body weight of each live foetus was recorded.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Indices:
no data
Historical control data:
Historical control data for rat teratology studies was provided for the following parameters:
- corrected maternal weight gain
- mean food consumed
- mean foetal sex ratio
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- no test substance-related mortality at any level tested; all animals on study survived until scheduled euthanasia.
- no test substance-related clinical observations at any level tested; the observations that were recorded were unremarkable and occurred infrequently.
- no adverse test substance-related effects on maternal body weight parameters at any level tested; data for maternal body weights and weight changes were comparable across all dose levels tested.
- 1000 mg/kg/day dose level: the mean body weight gain from days 20 to 21, and the cumulative weight gain (gestation days 6 to 21) calculated using the final body weight corrected for the weight of the products of conception, were significantly higher than for the control group (cumulative interval:1000 mg/kg/day group: 47.78 g (average); control group: 36.86 g (average)). This apparent increase is the result of an atypically low value for the concurrent control group. The test facility historical control mean is 56.5 g and ranges from 41.4 to 68.7 g.
- no adverse test substance-related effects on maternal food consumption at any level tested.
- during gestation days 18 to 20, the mean food consumption values for the 100 and 1000 mg/kg/day groups were significantly higher than for the control group (100 mg/kg/day: 26.6 g; 1000 mg/kg/day: 26.5 g; control group: 23.9 g). The test facility historical control mean for this interval is 28.5 g and ranges from 25.3 g to 31.1 g; therefore, these apparent increases are reflective of an atypically low concurrent control value.
- no adverse test substance-related maternal gross postmortem observations at any level tested.
- one female in the 300 mg/kg/day group was observed to have a large placenta; this is considered to be an incidental finding and was not dose-related.
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- no adverse test substance-related effects on endpoints quantitating intrauterine growth or embryo-foetal survival.
- 1000 mg/kg/day dose level: mean foetal sex ratio and the means for male and female foetuses per litter were statistically significantly different from the control group means.
male foetuses (mean): 7.2; control group: 5.7 historical control group: 5.2 to 7.4
female foetuses (mean): 4.8; control group: 6.7 historical control group: 5.8 to 8.3
mean foetal sex ratio: 60%; control group: 46%; historical control group: 43 to 53%.
These changes in sex ratio are not considered to be indicative of a test substance-related change because foetal sex is determined shortly after conception and well before the onset of dosing on gestation day 6. In the absence of any other effect on determinants of litter size such as post-implantation losses in the form of resorptions or dead foetuses, these statistical changes are spurious.
- no test substance-related foetal malformations or variations observed at any dose level tested. The foetal alterations occurred with low frequency across all groups tested and occur with similar frequency in the test facility historical control database.
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
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this study, there was no evidence of either maternal or developmental toxicity at doses up to 1000 mg/kg/day. Therefore, the no-observed-effect level (NOAEL) for maternal and developmental toxicity was considered 1000 mg/kg/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-14 to 2014-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 62-66 days
- Weight at gestation day 0:
0 mg/kg/day: 200 g - 225 g
100 mg/kg/day: 201 g - 225 g
300 mg/kg/day: 201 g - 225 g
1000 mg/kg/day: 201 g - 221 g
- Housing: animals were housed in pairs in solid-bottom caging with Bed-o'Cobs® bedding and nestlets as enrichment.
- Diet (ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: yes, but length of period was not stated

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26ºC
- Relative humidity: 30-70%
- Photoperiod: approximate 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
other: sterile water for injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations of the test substance in the vehicle were prepared at least weekly. The stability of the dosing formulations at concentrations bracketing the concentrations administered to the animals was demonstrated for up to 8 days.
The test substance was suspended in sterile water for injection. The dosing formulations were not adjusted for purity; a purity of value of 100% was assumed for the purposes of dose formulation calculations. Dosing formulations were stored at room temperature until used.
The volume administered (5 mL/kg) was based on the most recent body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of ultrafine titanium dioxide (TiO2 uf-3) in water at concentrations of a control, 20, 60 and 200 mg/mL were analyzed for homogeneity and concentration verification. Samples of each formulation were taken 2 times: near the beginning and end of the study. Dose samples at the same concentrations prepared near the end of the study were analysed for concentration verification.
Samples were digested in a chemical microwave using nitric and hydrofluoric acid and then analyzed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).
The analysis results for samples prepared at the beginning of the stud show that the test substance was homogeneously mixed and at the targeted concentrations.
The analysis results for samples prepared at the end of the study show that the test substance was at the targeted concentration for the 20 mg/mL concentration and lower than expected at the 60 and 200 mg/mL concentration. The 60 mg/mL concentration found 54.3 and 48.2 mg/mL in the samples sent for analysis. The relative standard deviation is 8.34% indicating that the sample concentrations are not consistent. For the high concentration (200 mg/mL), the relative standard deviation was 5.20% also indicating that the sample concentrations are not consistent. The analyzed concentrations ranged from 83.0 to 89.4% with an average of 86.2% of the target. This finding indicates the samples were not completely homogeneous. The increased density of the test substance affects the mixing and sampling of the samples.
The test substance was not detected in the control samples.
The results from the second set of analyses reveal that the intermediate level formulation (60 mg/mL) concentration was slightly lower than intended and that the intermediate and high level formulations (60 and 200 mg/mL) were less homogeneous that expected. The instance of low concentration and the slight increase in heterogeneity do not adversely affect the study because the ability to interpret the animal endpoint data is not impaired. Even considering an occasional increase in variability of the formulation concentration at either the intermediate or high dose levels, the spacing of the dose levels was adequate for discerning any potential effects of dose if any dose-related outcomes had been present. There were no test substance-related effects at any dose level on either maternal or foetal endpoints.
Details on mating procedure:
- Impregnation procedure: timed mated
Duration of treatment / exposure:
Gestation days 6 - 20
Frequency of treatment:
once daily
Duration of test:
20 days
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:
22 timed mated females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Quarantine and pretest: mortality/moribundity at least once daily; clinical observation on gestation day 4
Testing period: mortality/moribundity twice daily (morning and evening); clinical observations twice daily on gestation days 6 - 20 (during weighing and at least 2 hours post-dosing) and once on gestation day 21.

DETAILED CLINICAL No

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine and pretest: gestation day 4
Testing period: daily on gestation days 6 - 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Quarantine and pretest: gestation day 4

Testing period: gestation days 6, 8, 10, 12, 14, 16, 18, 20, and 21
Food consumption was measured as food consumed per cage, which was then divided by the number of animals in each cage for individual food consumption values. Excess food spillage was recorded.

- 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 21
- Organs examined: a gross external and a visceral examination were performed immediately after euthanasia.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: uteri with no visible implantation sites were placed in a 10% aqueous solution of ammonium sulfide to detect very early resorptions.
Fetal examinations:
- External examinations: Yes, all foetuses classified as live were examined for alterations. External sex was recorded for each live foetus.
- Soft tissue examinations: Yes, approximately half of the live foetuses
- Skeletal examinations: Yes, all live foetuses; The skeletal bodies of all the foetuses and the skulls of half the foetuses (foetuses that were not designated for head examination) were examined for alterations.
- Head examinations: Yes, approximately half of the foetuses
- Body weight of each live foetus was recorded.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- no test substance-related mortality at any level tested; all animals on study survived until scheduled euthanasia.
- no test substance-related clinical observations at any level tested; the observations that were recorded were unremarkable and occurred infrequently.
- no test substance-related effects on maternal body weight parameters at any level tested; data for maternal body weights and weight changes were comparable across all dose levels tested. Mean final body weights (absolute or adjusted) on GD 21 were within 1% of the control group mean at every dose level tested.
- no test substance-related effects on maternal food consumption at any level tested. Mean maternal food consumption from GD 6-21 was within 3% of the control group mean for all of the dose levels tested.
- no test substance-related maternal gross postmortem observations at any level tested. All animals appeared normal at the group at necropsy.
- no test substance-related effects on any reproductive outcome endpoint
- litter means for numbers of implantation sites, early and late resorptions were all comparable to control group values for every dose level tested.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- litter means for live and dead foetuses as well as for foetal weight and sex ratio were all comparable to control group values for every dose level tested.
- no test substance-related foetal malformations or variations observed at any dose level tested. The foetal alterations occurred with low frequency across all groups tested and occur with similar frequency in the test facility historical control database.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this study, there was no evidence of either maternal or developmental toxicity at doses up to 1000 mg/kg/day. Therefore, the no-observed-effect level (NOAEL) for maternal and developmental toxicity was considered 1000 mg/kg/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-15 to 2013-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
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: 287-330 g (mean: 306.7 g, ± 20% = 368.04 – 245.36 g); females: 174-223 g (mean: 203.99 g, ± 20% = 244.79 – 163.19 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. 1531)
- 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
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 suspension 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 the 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 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 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.

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 the quantification and one for the 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 the sample number 13 (LD, week 1) for which recoveries were 125.8 and 122.8 (‘a’ and ‘b’, respectively). This increase in the concentration was noticed in one sample of the LD group meant for stability analysis for 0 hours. This isolated result was considered to be due to sampling error.
In this case the formulations 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 the analytical samples were collected from the same stock as that used for the dose administration. The formulation sample was assumed to have not been subjected to a proper homogenization procedure before the collection of sample number 13. This was evident from the sample numbers 4, 14, 21, 22 and 23 (LD, week 1) for which the recoveries were 90% to 103.3%. The sample numbers 4, 14, 21, 22 and 23 were collected from the same formulation preparation as that of sample number 13. As there were no toxicity findings seen at any of the tested dose levels, this concentration increase in an isolated analytical sample of the LD group 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
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/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.
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 (if drinking water study): 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 statistically significant).
For the abnormality “hindlimb talus- unossified” the statistical analyses was performed by combining all unilateral and bilateral findings.
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
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: no deaths recorded during the course of the study in any of the groups.

- clinical observations: no clinical signs of toxicological relevance noted during the study.

- 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 the 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 prenatal parameters including number of corpora lutea, number of implantation sites, early and late resorptions, or pre- and post-implantation loss.
However, there was a slight decrease in the uterus weight, number of implantation sites and number of live foetuses in the 100 mg/kg bw/day and 1000 mg/kg bw/day groups in comparison to the control. In the absence of statistical significance, dose related response and the values being within the historical control data range, the changes were considered to be incidental.
There was an increase in pre implantation loss in the 100 mg/kg bw/day and 1000 mg/kg bw/day groups. As the dose administration begins from gestation day 5, the changes noted for pre implantation loss is not associated with the treatment.

- there were no macroscopic findings noted in any of the animals of the control or the test item treated groups at necropsy.

Please also refer to the field "Attached background material" below.
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- no treatment related changes noted for prenatal parameters including live foetuses, number of dead foetuses, number of male and female foetuses, or sex ratio.

-there was a slight decrease in the number of male foetuses in the 1000 mg/kg bw/day group, which was due to the absence of male foetuses in 2/ 24 females in the 1000 mg/kg bw/day group. As this was observed in two isolated females and also was within the historical control data range, the finding was considered incidental and not 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 effects 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 decrease in the total and female litter weights noted in the 100 mg/kg bw/day and 1000 mg/kg bw/day groups. In the absence of the statistical significance and a dose related response and in addition, all mean values being within historical control ranges, the changes were not considered to be associated with treatment.
Furthermore, there was also a decrease (-13.75%) in the mean male litter weight noted in the 1000 mg/kg bw/day group in comparison to the control, which corresponded to the decreased number of male foetuses in the 1000 mg/kg bw/day group. But this finding was due to the absence of male foetuses in 2/ 24 females in the 1000 mg/kg bw/day group. As this was noted in two isolated females and also taking into account the data being within the historical control data range the finding was considered to be incidental and not associated with the treatment.

- no test item related gross external abnormalities were seen in either the control or treatment groups. There were no statistically significant changes noted. However, There were abnormalities like hematoma of the neck, flank and tail in the control and hematoma of the forelimb in the 1000 mg/kg bw/day group. These were observed in a single isolated foetus of a single female of the control and/ or 1000 mg/kg bw/day groups and were considered spontaneous in origin. Micrognathia was observed in a single foetus of a single female of the 100 mg/kg bw/day group and was considered to be spontaneous in origin.

- skeletal examination foetuses 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 14th full rib-bilateral in the 100 mg/kg bw/day and 1000 mg/kg bw/day groups. The percent foetal incidence of 14th full rib- bilateral was within the historical control data range and there was no dose related response observed. This abnormality was considered to have no relevance to treatment.

- internal examinations 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 the controls. There were no abnormalities of toxicological relevance. The statistical analysis of data showed no significant changes.

Please also refer to the field "Attached background material" below.
Dose descriptor:
NOAEL
Effect level:
100 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
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In this prenatal developmental toxicity study, the repeated dose oral administration of TiO2 uf-2 to pregnant female Wistar rats at doses of 100, 300 and 1000 mg/kg bw/ day from gestation day 5 through gestational 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) for TiO2 uf-2 for both maternal and developmental toxicity in the Wistar rat is considered to be 1000 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
6 guideline- and GLP-conform studies available, the overall quality of the database is therefore high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity

 

Holalagoudar (2014) evaluated ultrafine and pigment-grade titanium dioxide in two separate developmental toxicity studies according to OECD 414 (2001) and under GLP. Groups of 24 -25 mated female Wistar rats were given titanium dioxide or titanium dioxide nanoparticles in aqua ad iniectabilia (water for injection) at dose levels of 100, 300, and 1000 mg/kg bw/day via gavage once daily during gestation day 5 through gestation day 19. A vehicle control group was run concurrently. Animals were sacrificed on day 20 of gestation and were investigated for general toxicity including examination of ovaries and uterine content. Pups were evaluated for viability (number of alive and dead foetuses), sex distribution and body weights. Foetuses were inspected externally and subjected to skeletal and soft tissue examinations and evaluated for malformations, variations and retardations. There were no adverse test substance-related effects on any maternal or foetal data collected. Therefore, the no-observed-effect level (NOAEL) for maternal and developmental toxicity was considered to be 1,000 mg/kg/day.

 

Munley (2014) evaluated ultrafine and pigment-grade titanium dioxide in three separate developmental toxicity studies according to OECD 414 (2001) and under GLP. Groups of 22 time-mated female Crl:CD(SD) rats were treated with titanium dioxide or titanium dioxide nanoparticles in sterile water for injection at dose levels of 100, 300, and 1000 mg/kg bw/day via gavage once daily during gestation days 6 – 20. A vehicle control group was run concurrently. Animals were sacrificed on day 21 of gestation and were investigated for general toxicity including examinations of ovaries and uterine content. Alive foetuses were inspected externally and subjected to skeletal and soft tissue examinations. Furthermore, alive foetuses were evaluated for sex distribution and body weight. There were no adverse test substance-related effects on any maternal or foetal data collected. Therefore, the no-observed-effect level (NOAEL) for maternal and developmental toxicity was considered to be 1,000 mg/kg/day.

 

Takawale (2014) evaluated pigment-grade titanium dioxide in a developmental toxicity study according to OECD 414 (2001) and under GLP. Groups of 24 -25 mated female Wistar rats were treated with the test item in aqua ad iniectabilia (water for injection) at dose levels of 100, 300, and 1000 mg/kg bw/day via gavage once daily during gestation days 5 through 19. A vehicle control group was run concurrently. Animals were sacrificed on gestation day 20 and were investigated for general toxicity including examinations of ovaries and uterine content. Pups were evaluated for viability (number of alive and dead foetuses) sex distribution and body weight. Furthermore, they were inspected externally and subjected to skeletal and soft tissue examinations. There were no adverse test substance-related effects on any maternal or foetal data collected. Therefore, the no-observed-effect level (NOAEL) for maternal and developmental toxicity was considered to be 1,000 mg/kg/day.

 

Furthermore, during a comprehensive literature search for data on developmental toxicity of TiO2, nine references were identified representing studies with information on developmental toxicity in rats or mice receiving either ultrafine orpigment-grade titanium dioxide via oral, inhalation, intraperitoneal or intravenous administration. After a thorough reliability screening, these references were considered of limited relevance for hazard assessment purposes. The criteria for quality, reliability and adequacy of experimental data under REACH for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X) are not fulfilled. These references are discussed briefly below, highlighting their findings and the reasons for their exclusion from health hazard assessment:

 

NIER (2009) describes a study which evaluated the reproductive and developmental toxicity of titanium dioxide according to OECD 421. A group of 10 male and 10 female Sprague-Dawley rats was treated with 1000 mg TiO2/kg bw/day in 1 % methylcellulose solution via gavage. According to the authors, no treatment-related changes were observed in the parental generation regarding general toxicity and reproductive performance. Also, the authors stated that no treatment-related changes were observed in the F1 generation. The NOAEL of titanium dioxide was considered to be above 1,000 mg/kg bw/day. The reference is only available as study summary record without raw data and was therefore rated as not assignable (RL=4). The original study record was not obtainable, however the study was assessed and peer-reviewed within the OECD SIDS initial assessment report (OECD CoCAM4, April 2013) and approved by the OECD procedure on Mutual Acceptance of Data (MAD). Thus, the study is considered useful as supporting information.

 

Stapleton et al. (2013) investigated the effect of ultrafine titanium dioxide on the maternal and foetal microvascular function. Starting on gestation day 10, 10 pregnant Sprague Dawley rats were exposed to the test item at 11.3 ±0.039 mg/m³ via inhalation for 5 hours/day. The average duration of exposure was 8.2 ±0.85 days (varying between 5 - 13 days). A vehicle control group was run concurrently (n = 6). At gestation day 20, isolation of the microvascular tissue and arteriolar reactivity of the uterine premyometrial as well as of foetal tail arteries were carried out. According to the authors, test item exposures led to significant maternal and foetal microvascular dysfunction which presented as robustly compromised endothelium-dependent and -independent reactivity to pharmacological and mechanical stimuli. Isolated maternal uterine arteriolar reactivity was consistent with a metabolically impaired profile and hostile gestational environment, impacting foetal weight. The foetal microvessels isolated from exposed dams were suggested to demonstrate significant impairments to signals of vasodilation specific to mechanistic signalling and shear stress. However, this reference has several reporting and experimental deficiencies which do not reliable conclusions on developmental toxicity:

 

-      the number of pregnant rats was too low in this study (treatment: n = 10; control group: n = 6): in comparison, the guideline foresees a total of approx. 20 pregnant females for each dose level and control group. This reduction of pregnant dams represents a significant reduction of statistical power and relevance of findings.

 

-      the exposure regime was not the same for all exposed animals. The duration of exposure varied between 5 – 13 days. This regime makes it impossible to compare the data between the various females.

 

-      the guideline foresees that at least three dose level and a concurrent control should be used. In this study, only one concentration level of 11.3 ±0.039 mg/m³ was tested. The usage of one concentration level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL).

 

-      the age of dams was not stated. Furthermore, data on clinical observations, body weight measurements (during dosing), food consumption, and gross pathology of dams are missing. Lastly, uterine weight, no. of corpora lutea, no. and % of live and dead foetuses, no. and % of pre- and post implantation losses and resorptions are not reported. Without this kind of information, it is unknown whether the test item had any adverse effect on the pregnant animals, which in turn could have an adverse outcome on the developing offspring.

 

-      the sex of the offspring was not reported. This precludes the possibility to determine whether the test item had any sex-specific effect.

 

-      the skeletal examination and soft tissue examination (exception microvascular level) of the foetuses are missing. These examinations are foreseen by the guideline.

 

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

 

Stapleton et al. (2015) investigated the effect of ultrafine titanium dioxide on the uterine and coronary microvascular function of female offspring after gestational exposure. Pregnant Sprague-Dawley rats (n = 6) were exposed to test item aerosols (10.6 mg/m³) from gestational day 6 to gestation day 20. A control group (n = 7) was run concurrently, which received filtered air. Exposure duration was 5 hours/day, 4 days/week. Pups were delivered, and the progeny grew into adulthood. Microvascular reactivity, mitochondrial respiration and hydrogen peroxide production of the coronary and uterine circulations of the female offspring were evaluated. According to the authors, the study demonstrated microvascular dysfunction coinciding with mitochondrial inefficiencies in both the cardiac and uterine tissues of adult offspring exposed to the test item in utero, which may represent initial evidence that prenatal test item exposure produces microvascular impairments that persist throughout multiple developmental stages. This reference has several reporting and experimental deficiencies, as follows:

 

-      the number of pregnant rats was too low in this study (n = 6). The guideline foresees a total of approx. 20 pregnant females for each dose level and control group. This reduction of pregnant dams causes a significant reduction of the statistical power.

 

-      exposure was not daily from implantation to the day prior to caesarean section (4 days/week from GD6 to GD 20). The guideline foresees that normally the pregnant animals should be exposed to the test item from implantation to caesarean section.

 

-      the guideline foresees that at least three dose level and a control should be used. In this study, only one concentration level (10.6 mg/m³) was tested. The use of one concentration level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL).

 

-      clinical observations, body weight, food consumption and gross pathology of dams were not examined/recorded. Furthermore, dams were sacrificed after weaning but examination of uterine content was not conducted (except: implantation). Without this kind of information, it is unknown if the test item had any adverse effect on the pregnant animals, which in turn could have an adverse outcome on the developing offspring.

 

-      Only female progeny were examined, which does not allow a conclusion for effect in males.

 

-      The external, skeletal, and soft tissue examinations of the offspring were missing. These examinations are foreseen by the guideline.

 

Based on the shortcomings given above, this publication was disregarded for health hazard assessment.

 

Hathaway et al. (2017) investigated the effect of ultrafine titanium dioxide on the cardiomyocytes of foetuses. A group of 13 pregnant Sprague-Dawley rats were exposed to titanium dioxide (P25) nano-aerosols (approx. 10 mg/m³) for 7 – 8 non-consecutive days, beginning at gestational day 5 – 6. A control group was run concurrently. The effects on cardiomyocytes of foetuses were evaluated on gestational day 20. According to the authors, foetal cardiomyocytes showed a decrease in basal respiration and an increase in proton leak in the experimental group following inhalative administration of the test item to pregnant rats. Furthermore, they stated that for the proton leak, qPCR of UCP2 revealed increased mRNA levels in the experimental group compared with the control, with qPCR of fatty acid metabolism constituents PPAR-γ and CPT1A (P = 0.023) exhibiting a similar increase. This reference has the following several reporting and experimental deficiencies, precluding it from being useful for hazard assessment purposes, as follows:

 

-      test item information is incomplete: the purity and stability of the test item are missing.

 

-      the number of pregnant rats was too low in this study (n = 13). The guideline foresees a total of approx. 20 pregnant females for each dose level and control group. This reduction of pregnant dams causes a significant reduction of the statistical power.

 

-      test item administration was not daily (7 - 8 non-consecutive days between gestation day 5 or 6 and gestational day 20) as foreseen by the guideline.

 

-      the guideline foresees that at least three dose level and a control should be used. In this study, only one concentration level (approx. 10 mg/m³) was tested. The use of one concentration level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL).

 

-      the age of the parental animals was missing. Furthermore, examinations of the maternal parent are missing (clinical signs, mortality, food consumption, gross pathology missing, corpora lutea count, no. of implantations, no. and % of live and dead foetuses, resorptions, and no. and % of pre- and post-implantation losses missing). Without this kind of information, it is unknown whether the test item had any adverse effect on the pregnant animals, which in turn could have an adverse outcome on the developing offspring.

 

-      the examinations of the foetuses are missing (sex ratio, % of live offspring, body weight, external examinations, skeletal examinations, soft tissue examinations missing). These examinations are foreseen by the guideline.

 

-      The vehicle is not described, which precludes the possibility to determine if an appropriate vehicle was used.

 

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

 

 

Elbastawisy and Almasry (2014) performed a study to determine the potential for histomorphological alterations occurring in maternal and neonatal pulmonary distal airspaces of Wistar rats after maternal administration of ultrafine titanium dioxide. A group of 15 pregnant rats were treated with 5000 mg/kg bw/day of the test item suspended in phosphate-buffered saline. The test item was given orally by gavage once daily from days 6 to 12 of gestation. A control group of 15 pregnant rats receiving the vehicle alone was run concurrently. On gestation day 21, the dams were sacrificed. Maternal and neonatal lungs were collected and processed for energy-dispersive X-ray (EDX) and histological analysis. The mean linear intercept and airspace wall thickness were measured by a stereological procedure with image analysis to assess alveolarisation. According to the authors, titanium dioxide was detected in maternal and neonatal lungs after oral intake by pregnant rats. They stated that the pulmonary response manifested as inflammatory lesions and delayed saccular development in neonates. This reference has the following severe reporting and experimental deficiencies:

 

- test item was insufficiently characterised, which makes it impossible to verify the identity of the test item.

 

- furthermore, the results presented in this publication are implausible. No publication has thus far shown systemic availability of titanium dioxide (whether pigment-grade or nano-sized) via the oral route. Furthermore, there is the potential that the titanium dioxide particles translocated via aspiration or mis-gavage to the lungs.

 

- the number of pregnant female per group is too low (n = 15). The guideline foresees a total of approx. 20 pregnant females for each dose level and control group. This reduction of pregnant dams causes a significant reduction of the statistical power.

 

- dosing was not conducted up to sacrifice of the dams.

 

- maternal and foetal examination were incomplete, which makes it difficult to determine whther the effects observed were truly caused by the test item.

 

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

 

Further references were identified, representing in vivo mechanistic investigations in rats and mice, following repeated oral administration of nano-sized titanium dioxide. These studies are primarily academic research papers, focussing on isolated organs, tissues or biomolecules, which respond to exposure with titanium dioxide. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to the low number of animals used, missing dose response relationship, contradictory results in comparison with highly reliable guideline studies. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data under REACH for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given below were therefore included in the IUCLID for information purposes only:

Ghareeb, A. et al. (2015): the experimental design is insufficiently documented, rat strain used in the study not further characterised, precluding a comparison with strain-specific historical control data, number of test animals/group is not stated, documentation of the results is imprecise (important raw data is missing, grading and localisation of findings were not specified), non-physiological route of administration via intraperitoneal injection (single dose) does not comply with relevant guidelines.

 

Mohammadipour, A. et al. (2014): The investigated experimental parameters are not useful for the fulfilment of data requirements under REACH for hazard assessment purposes. The test material used in this study is not sufficiently characterised and the experimental design (e.g. only one dose administered) is not in accordance to any guideline. The repetitions were not sufficient to reach a statistically relevant level and moreover, the number of animals used in the tests were too low. Regarding the data analysis, it is unclear, whether only a positive variation is stated in the graphs.

 

Yamashita, K. et al. (2011): This publication is not relevant for human health risk assessment due to the irrelevant route of test item application. Furthermore, the publication is not relevant for hazard assessment since data on particle characterisation are poorly described. In addition, the number of animals is not clearly stated, one dose level only was tested, and exposure duration was too short. Lastly, the following parameters were not examined/recorded: clinical observations, mortality, food consumption, full examination of uterine content (resorption only), macroscopic examination of the dams, and no full foetal examination (body weight only).

 

Hong, F. et al. (2017): The publication is not relevant for human health risk assessment since the test item was self-synthesised and data on particle characterisation are poorly described. The number of animals per group is too low for an appropriate statistical analysis. Besides of these major restrictions, the following information is not included: mating cages, dosing volume, clinical observation, maternal mortality rate, food consumption, macroscopic examination of the dams, weight of gravid uteri including cervix, number of corpora lutea, sex ratio of foetuses, examination of the reproductive tract of the foetuses for signs of altered development.

 

Conclusion

 

Based on the information from the available six pre-natal developmental toxicity studies via gavage in rats (according to OECD 414 and under GLP) it is concluded that maternal toxicity up to the highest dose level of 1,000 mg/kg bw/d and reproductive performance of dams were not affected by treatment. There was no embryo-toxicity and no effect on the development of foetuses. No external visceral or skeletal malformations were observed and the incidence of variations was not different between treated and control groups. No incidences of skeletal retardations were observed.

 

One reproductive toxicity screening assay is available, but only as a brief study summary without raw data and was therefore rated as not assignable (RL=4). This study showed no developmental toxicity in the foetuses. Due to its use within the OECD HPV programme, the study is used as supporting information.

 

Further eight references report mechanistic studies with a focus on organ-specific investigations, such as uterine and coronary microvascular function, histomorphological alterations in maternal and neonatal pulmonary distal airspaces, effect of ultrafine titanium dioxide on the cardiomyocytes of foetuses. The study designs of these eight references are not in accordance with any accepted guideline and are therefore of limited relevance for chemical hazard assessment. The references lack significance due to poor test item characterisation, non-physiological route of administration, low number of animals available for testing, one concentration tested only, short exposure duration, wrong dosing regime, and/or incomplete observation data. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies were included for information purposes only.

 

Justification for classification or non-classification

Effects on developmental toxicity:

Based on the weight of evidence from the available long-term toxicity/carcinogenicity studies in rodents and the relevant information on the toxicokinetic behaviour in rats, it is concluded that titanium dioxide does not present a reproductive toxicity hazard. There is evidence from the animal chronic toxicity/carcinogenicity studies in rats and mice that the intake of high amounts of titanium dioxide or inhalation to high concentrations of titanium dioxide was not associated with adverse effects on the reproductive organs.

The results of a toxicokinetic study demonstrate that no relevant systemic absorption occurs via the oral exposure route as indicated by the titanium concentrations in whole-blood and urine which were below the limit of quantification (<0.04 mg/l). Tissue titanium concentrations in liver, kidney and muscle were below the limit of detection (<0.1 - <0.2 mg/kg wet weight) indicating no substantial accumulation of titanium in the body. Furthermore, any metabolism of the inorganic material whatsoever can be excluded.

For the reasons presented above, conducting a two-generation reproduction toxicity study would therefore not provide any further insights in the toxicity of titanium dioxide. Because of the lack of absorption and systemic distribution, any quantitatively relevant exposure of reproductive organs in male and female mammalian species to titanium dioxide is unlikely, so that any specific effects on reproduction are not to be expected. Therefore, it is scientifically not justified to conduct a two-generation reproduction study in rats which complies with the 3R-rules and the principles of animal welfare. For the reasons presented above, no classification for reproductive toxicity is required.

 

Effects on fertility:

In six guideline and GLP compliant studies, titanium dioxide did not show any effects on the developing foetuses of rats when administered up to the limit dose of 1000 mg/kg bw/day. The test items covered titanium dioxide in the crystalline forms rutile and anatase in pigment and ultrafine grade. In conclusion, based on the data observed for titanium dioxide in six developmental toxicity studies in rats, a classification as reproductive toxicant is not justified due to the complete absence of any adverse effects.