Titanium dioxide

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

adopted June 25, 2018

Deviations:

no

Principles of method if other than guideline:

A non-guideline 14-day palatability study was conducted prior to the Extended one generation reproductive toxicity (EOGRTS) to obtain information on the palatability of several treatment levels of Titanium dioxide E171-E when given to rats daily via the diet. 5 animals/sex/dose were treated with 0, 300 and 1000 mg Titanium dioxide E171-E/kg bw/day for 14 days. Clinical signs, mortality, body weight and food and drinking water consumption were recorded. At the end of the study all animals were inspected macroscopically. The results of this study are reported in the results and discussion of the parental generation.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 08.05.2017

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : extended pre-mating period of 10 weeks since several publications reported testicular toxicity (impairment of spermatogenesis) or effects on sperm integrity and function as well as effects on the oestrus cycle.
- Basis for dose level selection : based on available toxicity data and the results of a 14-day palatability study conducted prior to the main study (as reported in this robust study summary).
- Inclusion of extension of Cohort 1B : Cohort 1B animals were mated to establish the F2 generation. This was done to investigate publications reporting several reproductive and developmental effects induced by titanium dioxide.
- Termination time for F2 : based on available data all F2 pups were dissected on PND 4-8; the date of dissection of F2 pups would have been postponed to PND 21 to allow further examinations. However, no adverse effects were observed, thus all F2 pups were dissected as planned (on PND 4-8).
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B : the full range of neurodevelopmental toxicity was evaluated to investigate publications reporting several neurotoxic effects induced by titanium dioxide.
- Inclusion of developmental immunotoxicity Cohort 3 : the full range of developmental immunotoxicity was evaluated to investigate publications reporting several immunotoxic effects induced by titanium dioxide.
- Route of administration : Ingestion via the food is the most relevant route of exposure for a food additive such as Titanium dioxide E171-E.


Determination of aberrant crypt foci (ACF): the EFSA panel recommended that in order to investigate the potential for neoplastic lesions (observations made in a publication of Bettini et al., 2017), biomarkers for putative preneoplastic lesions in the colon should be examined as additional parameters in the requested extended one-generation reproductive toxicity study as recommended by EFSA in 2016.

Test material

Test material information

Constituent 1

Reference substance name:

Titanium dioxide

EC Number:

236-675-5

EC Name:

Titanium dioxide

Cas Number:

13463-67-7

Molecular formula:

O2Ti

IUPAC Name:

dioxotitanium

Test material form:

solid: particulate/powder

Details on test material:

CAS no.: 13463-67-7
EC no.: 236-675-5
Molecular formula: TiO2
Molecular weight: 79.866 g/mol
Storage conditions: >5°C and air humidity 20-80 %
Crystal structure: Anatase
Coating: none

In an industry-wide survey over all manufacturers and importers of E171, the form with the highest content of particles below 100nm was selected as test item. The data on number-based particle size distribution is given under "Confidential details on test material".

Test animals

Species:

rat

Strain:

other: CD/Crl:CD(SD)

Remarks:

used for the 14-day palatability study and EOGRTS

Details on species / strain selection:

The rat is a commonly used rodent species for such studies and required by the guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

14-day palatability study:
The same strain and supplier of rats as decribed below were used for the 14-day palatability study. All animals had a adapation period of 5 days and were housed under the same conditions (e.g. diet, water, housing conditions) as decribed below.

EOGRTS:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Servives; Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Health statsus: healthy virgin animals
- Age at study initiation: males and females: 71 days
- Weight at study initiation: males: 353.5 - 446.9 g; females: 198.8 - 285.4 g
- Housing: With the exception of the mating period, the males and females were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week.
- Diet: certified commercial diet (ssniff® R/Z V1320, ssniff Spezialdiäten GmbH, 59494 Soest, Germany; ad libitum (with the exception of the night before the day of blood withdrawal for laboratory examination)
- The titanium content in the diet, in the bedding material and any enrichment material (rat huts, wooden chewing sticks) were analysed under GLP prior to initiation of the study using a validated ICP-OES method.
- Water: tap water; ad libitum
- Acclimation period: 6 days of adaptation

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C±3°C (maximum range)
- Humidity: 55%±15% (maximum range)
- Air changes: 15-20 per hr
- Photoperiod: The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION

14-day palatability study:
The test item formulations were freshly prepared once a week. To maintain a constant dose level in relation to the animal's body weight, the concentration in the diet was adjusted based on the mean group food consumption per sex. The test item concentration was adjusted weekly using the food consumption values from the previous week. The appropriate amount of test item was weighed into a glass container. Some of the test item and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test item was distributed in the diet. Afterwards, the premix was added to the diet, mixed for 15 minutes and then transferred to a closable bucket. Each bucket was labelled according to group, sex, and dose.

EOGRTS
- Rate of preparation of diet (frequency): once a week
- Preparation: The appropriate amount of test item was weighed into a glass container. A portion of the test item and diet was mixed with an impact mill (Grindomix GM300, Retsch GmbH, Haan, Germany) to produce a premix. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG, Ludwigshafen, Germany) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled with group number and dose. To maintain a constant dose level in relation to the animals’ body weight, the concentration of the test item in the diet was adjusted based on the mean group food consumption per sex. The concentration was adjusted weekly using the food consumption values from the previous week.

Details on mating procedure:

- M/F ratio per cage: 1/1, Sexually mature male and female rats were paired randomly monogamously, i.e. 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or two weeks had elapsed.
- Length of cohabitation: two weeks
- Proof of pregnancy: The females were examined each morning for the presence of sperm. The day of conception (day 0 of gestation) was considered to be the day on which sperm was found.

Additional information:
Females without a positive mating sign were separated from its male partner after 2 weeks without further opportunity for mating. After a pseudo gestation period of approx. 24 test days these females were laparotomized and their non-pregnancy status was confirmed by Salewski Staining.

For the establishment of the F2 Generation, males and females of the same dose group were paired (1:1 pairing), sibling mating was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

14-day palatability study:
No analytical verification of test substance the test item-diet mixtures was performed.

EOGRTS:
For the determination of the test item concentration in the exposure diet, samples of approximately 10 g were taken and stored at - 20°C ±10 % or colder until
shipment for analysis. The samples were taken on the day of preparation of the test item-diet mixtures for analysis of concentration and homogeneity and 7 days after the preparation from the left over in the bucket for analysis of stability.

Sampling for F0 Generation
For the F0 Generation samples for analysis were taken from the weekly test item diet preparations at the start of dosing (test week 3) and from the test weeks when the animals were sacrificed (test week 20 for the male animals (sacrifice during test week 20) and test week 19 for the female animals (sacrifice during test week 19 and 20)).

Sampling for F1 Generation
For the F1 Generation samples for analysis were taken from the weekly test item diet preparations from test day 1 of the F1 Generation study and from the test
weeks when the different cohorts of the F1 Generation were sacrificed.

The samples were labelled with the study number, species, type of sample, concentration, generation and cohort number, group number test week, test day, location (top/middle/bottom), and date.

Final results on analytical verification of doses/concentrations were not available at the time of preparation of this robust study summary, but will be given in an update.

Duration of treatment / exposure:

14-day palatability study:
14 days

EOGRTS:
Parental generation (F0)= 18-19 weeks (10 weeks of pre-mating, 2 weeks mating, 3 weeks pregnancy, 3 weeks lactation) (test day 132-136)
F1 Cohort 1A= approx. 12-13 weeks (PND 86 to 96)
F1 cohort 1B= approx. 17-18 weeks (PND 120 to 136, at PND 4-8 of F2 generation)
F1 cohort 2A= approx. 12-13 weeks (PND 84 to 90)
F1 cohort 2B= approx. 3 weeks (PND 21 to 23)
F1 cohort 3= approx. 8-9 weeks (PND 53 to 61)
F2 Generation= PND 4 to 8
Surplus pubs= on PND 4 or PND 22

Frequency of treatment:

ad libitum, via diet

Details on study schedule:

- F1 parental animals not mated until PND 94.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Mean actual dose received (based on food consumption)
Parental generation: males 103.82 mg/kg bw/day; females 109.24 mg/kg bw/day
Cohort 1A: males 100.33 mg/kg bw/day; females 105.27 mg/kg bw/day
Cohort 1B: males 104.39 mg/kg bw/day; females 103.46 mg/kg bw/day
Cohort 2A: males 101.54 mg/kg bw/day; females 116.38 mg/kg bw/day
Cohort 3: males 88.2 mg/kg bw/day; females 97.2 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mean actual dose received (based on food consumption)
Parental generation: males 311.78 mg/kg bw/day; females 319.31 mg/kg bw/day
Cohort 1A: males 304.64 mg/kg bw/day; females 319.58 mg/kg bw/day
Cohort 1B: males 314.12 mg/kg bw/day; females 327.92 mg/kg bw/day
Cohort 2A: males 307.76 mg/kg bw/day; females 311.80 mg/kg bw/day
Cohort 3: males 270.0 mg/kg bw/day; females 286.9 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mean actual dose received (based on food consumption)
Parental generation: males 1050.04 mg/kg bw/day; females 1084.90 mg/kg bw/day
Cohort 1A: males 1066.35 mg/kg bw/day; females 1011.11 mg/kg bw/day
Cohort 1B: males 1045.59 mg/kg bw/day; females 1089.04 mg/kg bw/day
Cohort 2A: males 1028.08 mg/kg bw/day; females 993.33 mg/kg bw/day
Cohort 3: males 956.4 mg/kg bw/day; females 1042.8 mg/kg bw/day

No. of animals per sex per dose:

14-day palatability study:
5 animals/sex/dose

EOGRTS:
Parental generation (F0): 24 males/24 females
Cohort 1A: 20 males/20 females
Cohort 1B: 20 males/20 females
Cohort 2A: 10 males/10 females
Cohort 2B: 10 males/10 females
Cohort 3: 10 males/10 females

Control animals:

yes, plain diet

Details on study design:

14-palatablity study:
- Rationale for animal assignment: Following the initial health check approximately upon delivery, the animals were weighed and allocated based on the body weight by means of a computerised randomisation program to the test groups; only healthy animals were used. Animals of the extremes of the weight distribution and/or any animal showing signs of illness during the period between allocation and the start of treatment were excluded and replaced by spare animals.

EORGTS:
- Dose selection rationale: The dose levels were selected by the Sponsor based on available toxicological data and the results of a palatability study conducted prior to the main study.
- Rationale for animal assignment:
F0- generation: The animals were allocated to the test groups at the last day of the pre-dosing period on test day 14 by using a Provantis10-generated randomization based on the body weights of the animals. Only those female animals were used for randomization that passed the estrous cycle test. The body weight range did not exceed 20 % of the mean weight for each sex at the time of selection.
F1- generation: When the pups from those dams that were the first to have descendants, had reached post-natal day 21, the pups that were used for the establishment of the adult F1 Generation were randomly selected from all litters that were available for their respective group of the F0 Generation. The randomization procedure was performed using Provantis4. The selected pups were separated from the surplus pups after they have reached post-natal day 21. When all selected pups have reached post-natal day 21, the F1 Generation started with test day 1.
- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

none

Examinations

Parental animals: Observations and examinations:

14-day palatabillity study:
Clinical signs and mortality were recorded as described below. Body weight of each rat was recorded at the time of group allocation, on the first treatment day and daily thereafter throughout the experimental period. The quantity of food left by each animal was recorded on a daily basis throughout the experimental period. The food intake per animal (g/animal/day) was calculated using the total amount of food given to and left by each animal in each group on completion of a treatment day. The drinking water consumption was monitored daily by visual appraisal throughout the study. Individual test item intake was calculated on a daily and weekly basis throughout the experimental period based on concentration in the diet, individual food intake and body weight.


EOGRTS:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
Mortality was recorded twice daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Any signs of illness or reaction to treatment were recorded for each individual animal. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
This more detailed examination started for the F0 main study animals on test day 14 (one day before the start of treatment) to allow for within-subject comparisons. Thereafter the examination was performed weekly until termination.
These observations were made outside the home cage in a standard arena and at the same time of the day, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern), as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition and bizarre behaviour (e.g. self-mutilation, walking backwards). Because of the postulated adverse effects (in the public domain) on the gastrointestinal tract, the clinical observations also included the status or condition of the faeces (i.e., whether dry pellets or loose diarrhoea) and any evidence of faecal blood.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed on the first day of dosing (test day 15), weekly thereafter and at sacrifice. Additionally, the female animals were weighed on gestation days 0, 7, 14 and 21 as well as on lactation days 1, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured in weekly intervals for the F0 main study animals.
The actual test item-intake for each test week is given in mg per kg body weight and day. The actual test-item intake was calculated based on the relative food consumption per day and the nominal test item concentration that was used in the respective test week.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was measured in weekly intervals for the F0 main study animals.

HAEMATOLOGY: Yes
Whole blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from 10 animals/sex/group fasted overnight at necropsy (TW 20) (in EDTA anticoagulant for haematological investigations and in Citrate anticoagulant for coagulation tests). The following parameters were investigated:
Differential blood count (relative- Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes), Differential blood count (absolute), Erythrocytes (RBC), Leucocytes (WBC), Haematocrit value (PCV or HCT), Haemoglobin content (HGB), Platelets (PCT or PLT), Reticulocytes (Reti), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (aPTT).

CLINICAL BIOCHEMISTRY: Yes
Whole blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from 10 animals/sex/group fasted overnight at necropsy (TW 20) (in Li-Heparin anticoagulant for clinical chemistry tests). The following parameters were investigated:
Sodium, potassium, calcium, chloride, albumin, total bilirubin, total cholesterol, glucose, total protein, blood urea (BUN), creatinine, alanine aminotransferase (ALAT/GPT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT/GOT), bile acids, lactate dehydrogenase (LDH), low-density lipoprotein (LDL), high-denssity lipoprotein (HDL), sodium/potassium ratio, globulin, albumin/globulin ratio, BUN/creatinine ratio.

THYROID HORMONES: Yes
Blood samples were taken from the scheduled animals under isoflurane anaesthesia. The blood was processed for serum. T4, T3 and TSH were analysed in serum samples of 10 animals/sex/dose (same animals as selected for laboratory examinations).

DETERMINATION OF SEXUAL HORMONES: Yes
Blood samples were taken from the scheduled animals under isoflurane anaesthesia at sacrifice for the determination of sexual hormones. A sufficient amount of blood was withdrawn to obtain at least 530 μL serum. Estradiol, testosterone and estrone were determined in 10 animals/sex/dose using commerical ELISA kits. Animals were fasted overnight.

Ti ANALYSIS IN BLOOD AND URINE: Yes
Lithium-heparin stabilised whole blood and urine samples for Ti level analysis were collected from non-fasted main study animals of the F0 Generation. For these examinations, the remaining 10 animals from each group were used that were not used for blood sampling for laboratory examinations and hormone level analysis. For urine sampling the adult animals were placed in URIMAX funnel cages for 5 h. Blood and urine samples from the adult animals were collected at sacrifice.

URINALYSIS: Yes
Urine samples were collected from animals fasted overnight. The urine was collected for 16 hours in a URIMAX funnel cage. The collection of urine was terminated immediately before the start of blood withdrawal for the haematological and clinical chemical examinations as well as the hormone level determinations. The following parameters were measured: Volume, pH, specific gravity. The following tests were also performed using qualitative indicators (Combur®Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration: Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin, Nitrite.
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters: Epithelial cells, Leucocytes, Erythrocytes, Organisms, further constituents, crystalluria. The colour and the turbidity of the urine were examined visually.

CIRCADIAN CYCLE OD HORMONE LEVELS (in satellite animals):
Hormone analysis of Beta-estradiol, Testosterone, T3, T4 and TSH was performed twice, pre-dose and at the end of the pre-mating period (after 10 weeks) using the F0 Satellite animals of groups 1 and 4 (30 animals per sex and group; 120 in total) throughout a 24-hour cycle. Blood sampling was performed every 2 hours during the 24-hour period of the cycle. The examination was performed at pre-dose (starting at 10.00 a.m. on test day 14 and ending on test day 15 at 8.00 p.m.) and after 10 weeks of treatment (starting at 10 a.m. on test day 84 and ending on test day 85 at 8.00 p.m.) before start of mating of the F0 satellite animals. In order to reduce the total amount of blood collected from each animal within the 24-hour period, each group (control group and high dose group) was split into six subgroups of 5 male and 5 female animals. Blood withdrawal from the 5 animals of each subgroup was performed twice during the cycle period (24 hours) with an interval of 12 hours between each time point of blood withdrawal. At each time point approx. 1.2 mL blood was collected from the tail-vein, sufficient enough to obtain at least 410 μL serum from each animal. The serum was aliquoted for the determination of each hormone. The aliquots were stored at - 20°C ± 10 % until analysis.

Oestrous cyclicity (parental animals):

Estrous cycle monitoring of F0 parental animals:
- 14 days pre-exposure period to select 96 main study animals and 80 satellite animals with regular estrous cycles (4 to 5 days per cycle) (main and satellite animals)
- During 10 weeks of pre-mating until evidence of mating (main study animals only)
- On the day of sacrifice.

Estrous cycle monitoring of Cohort 1B parental animals:
- Starting from the time of pairing until mating evidence was detected
- On the day of sacrifice

Estrous cycle monitoring of Cohort 1A animals:
- Start after onset of vaginal patency until first appearance of cornified cells
- Two weeks starting around PND 75
- On the day of sacrifice

Sperm parameters (parental animals):

Parameters examined in all males of the parental generation (F0) and cohort 1A:
The right epididymis and the right testis were used for sperm count and the examination of sperm viability and sperm morphology. The examinations were performed according to the methods described by L. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).

Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of the F0 and F1 Cohort 1A males of groups 1 and 4 following H-E and PAS staining.

Organ weights of both epididymides and testicles of all parental (F0) and cohort 1A animals were recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (for F0 generation)
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible);
After counting on PND 4, the litters were adjusted to 10 pups per litter (5 pups per sex and litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®. Selective elimination of pups e.g. based upon body weight is not appropriate and was not performed. In case of unequal gender distribution, a partial litter size adjustment was performed (e.g. 6 male and 4 female pups).
Culled pups were used for blood collection for the determination of thyroid hormone levels on PND 4 and PND 22.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring (besides the offspring indices): presence of nipples/areolae in male pups (F1 only), balano-preputial separation or vaginal opening (and body weight at that time point) (F1 only), anogenital distance (AGD), pup weight on the day of AGD, external and internal examination, body weight gain, thyroid hormones (T4 on PND 4 and T3,T4 and TSH on PND 22 of F1 pups only), pup organ weights (absolute) on PND 22 (from surplus F1 pups)

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and F1 pups sacrificed on PND 4 (culled F1 Pups) were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development. Those F1 Pups that were not selected for the F1 cohorts were sacrificed on PND 22 and dissected. They were examined macroscopically for external and internal gross abnormalities. From up to 10 pups per sex and group organs were weighed and preserved in 7% buffered formalin.

OBSERVATIONS AND EXAMINATIONS
COHORT 1A, 1B, 2A, 2B and 3

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (all F1 cohorts and satellite animals)
Mortality was recorded twice daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Any signs of illness or reaction to treatment were recorded for each individual animal. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (Cohort 1B only)
This more detailed examination started after weaning until termination.
These observations were made outside the home cage in a standard arena and at the same time of the day, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern), as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition and bizarre behaviour (e.g. self-mutilation, walking backwards). Because of the postulated adverse effects (in the public domain) on the gastrointestinal tract, the clinical observations also included the status or condition of the faeces (i.e., whether dry pellets or loose diarrhoea) and any evidence of faecal blood.

BODY WEIGHT: Yes
- Time schedule for examinations: Live pups were weighed during the lactation period on their post-natal days 1, 4, 7, 14 and 21. After weaning they were weighed weekly. The female animals of Cohort 1B were additionally weighed on their gestation days 0, 7, 14 and 21. The F2 Pups were weighed on their post-natal days 1 and 4 (sacrifice).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured in weekly intervals for all animals of all cohorts starting shortly after weaning (except cohort 2B, since they were sacrificed after weaning).
The actual test item-intake for each test week is given in mg per kg body weight and day. The actual test-item intake was calculated based on the relative food consumption per day and the nominal test item concentration that was used in the respective test week.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was measured in weekly intervals for all cohort 1B animals (starting shortly after weaning until sacrifice).

HAEMATOLOGY: Yes
Whole blood samples were taken from all Cohort 1A animals from the retrobulbar venous plexus under isoflurane anaesthesia from 10 animals/sex/group fasted overnight at necropsy (PND 87 to 96) (in EDTA anticoagulant for haematological investigations and in Citrate anticoagulant for coagulation tests). The following parameters were investigated:
Differential blood count (relative- Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes), Differential blood count (absolute), Erythrocytes (RBC), Leucocytes (WBC), Haematocrit value (PCV or HCT), Haemoglobin content (HGB), Platelets (PCT or PLT), Reticulocytes (Reti), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (aPTT).

CLINICAL BIOCHEMISTRY: Yes
Whole blood samples were taken from all cohort 1A animals from the retrobulbar venous plexus under isoflurane anaesthesia from 10 animals/sex/group fasted overnight at necropsy (PND 87 to 96) (in Li-Heparin anticoagulant for clinical chemistry tests). The following parameters were investigated:
Sodium, potassium, calcium, chloride, albumin, total bilirubin, total cholesterol, glucose, total protein, blood urea (BUN), creatinine, alanine aminotransferase (ALAT/GPT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT/GOT), bile acids, lactate dehydrogenase (LDH), low-density lipoprotein (LDL), high-denssity lipoprotein (HDL), sodium/potassium ratio, globulin, albumin/globulin ratio, BUN/creatinine ratio.

THYROID HORMONES: Yes
Blood samples were taken from all Cohort 1A animals (fasted) and F1 surplus pups (on PND 4 (T4 only) or 22, both non-fasted) under isoflurane anaesthesia. The blood was processed for serum. T4, T3 and TSH were analysed in serum samples of 10 animals/sex/dose (same animals as selected for laboratory examinations).

DETERMINATION OF SEXUAL HORMONES: Yes
Blood samples were taken from the scheduled animals under isoflurane anaesthesia at sacrifice for the determination of sexual hormones. A sufficient amount of blood was withdrawn to obtain at least 530 μL serum. Estradiol, testosterone and estrone were determined in 10 animals/sex/dose of Cohort 1A, 1B, 2A and 2B using commerical ELISA kits. Animals were fasted overnight.

Ti ANALYSIS IN BLOOD AND URINE: Yes
Lithium-heparin stabilised whole blood and urine samples for Ti level analysis were collected from 10 non-fasted animals/sex/dose of Cohort 1A, 1B and F2 pups. For these examinations, the remaining 10 animals fromeach group were used that were not used for blood sampling for laboratory examinations and hormone level analysis. For urine sampling the adult animals were placed in URIMAX funnel cages for 5 h. Blood and urine samples from the adult animals were collected at sacrifice. For the F2 pups urine was directly collected from the urinary bladder before necropsy.

URINALYSIS: Yes
Urine samples were collected from Cohort 1A animals fasted overnight (on PND 87 to 96). The urine was collected for 16 hours in a URIMAX funnel cage. The collection of urine was terminated immediately before the start of blood withdrawal for the haematological and clinical chemical examinations as well as the hormone level determinations. The following parameters were measured: Volume, pH, specific gravity. The following tests were also performed using qualitative indicators (Combur®Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration: Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin, Nitrite.
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters: Epithelial cells, Leucocytes, Erythrocytes, Organisms, further constituents, crystalluria. The colour and the turbidity of the urine were examined visually.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Neurological screening of Cohort 2A animals
Auditory startle response (on PND 22/23), observation screening and functional tests (males on PND 60 to 64 and females on PND 58 to 63). The observational screening included the following parameters: righting reflex, Body temperature, Salivation, Startle response, Respiration, Mouth breathing, Urination, Convulsions, Piloerection, Diarrhoea, Pupil size, Pupil response, Lacrimation, Impaired gait, Stereotypy, Toe pinch, Tail pinch, Wire manoeuvre, Hind-leg splay, Positional passivity, Tremors, Positive geotropism, Limb rotation, Auditory function, Posture, Palpebral closure, Vocalization, Arousal, Bizarre behaviour, Ease of removal/handling, Muscle tone, Approach response, Touch response
The functional tests included measurement of grip strength and locomotor activity

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
The assessment of potential developmental immunotoxicity of cohort 3 animals included the determination of anti KLH-IgM antibodies in the blood serum using ELISA and teh examination of the splenic lympocyte subpopulations via flow cytometry (cohort 1A and 3).

KLH-assay:
All animals of cohort 3 and satellite animals nos. 941 to 960 (positive control animals) were used to determine the IgM values in serum after a single i.v. bolus injection of 1500 µg/animal KLH (keyhole limpet haemocyanin, in PBS) into the tail vein on PND 53 to 61. Positive control animals received an additional single oral administration on the same day of KLH injection of 40 mg/kg bw of CPA (Cycloposphamide) solved in 0.5% Methyl Cellulose. 5 days after injection, animals were sacrificed and sufficient blood was withdrawn from the animals to receive 2 aliquots of at least 150 μL serum each to determine the IgM values with a Rat Anti-KLH IgM ELISA kit.

Phenotypic analysis of spleen cells (cohort 1A and 3):
The analysis of the subpopulation of lymphocytes in the spleen was performed for all animals of Cohort 1A, Cohort 3 and the satellite animal nos. 941 to 960.
At necropsy, the spleen of the mentioned animals was split in 2 parts. The part of the spleen not preserved for histopathology (histopathology of the spleen was only performed for Cohort 1A) was minced using a mechanic dissociator to prepare single cell suspensions. The prepared spleen samples were used for the determination of the following lymphocyte subpopulations via flow cytometry: helper T cells (CD4+), suppresor/cytotoxic T cells (CD8+), T cells (CD3+), B cells (CD45RA+) and NK cells (CD161+)

Postmortem examinations (parental animals):

14-day palatability study:
On test day 15 (approximately 2 hours after removal of the diet), all animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal. The urinary bladder was examined externally and by palpation. The gastro-intestinal tract was
examined as a whole, and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded. The organs and carcasses were discarded.


EOGRTS:

SACRIFICE
- Male animals: All surviving animals on test day 135, 136 (after weaning of the F1 animals)
- Maternal animals: All surviving animals on test day 132 to 135 (after weaning of the F1 animals)
Satellite animals used for ACF determination: shortly after weaning of the F1 satellite animals
Satellite animals used for hormone cycle blood sampling: after the last blood sampling

GROSS NECROPSY
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system and GI tract, particularly whether for example the caecum was dilated. During necropsy the number of implantation sites was recorded in the female animals. Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
The weights of the organs were recorded from 20 males and female animals of the F0 Generation. With the exception of the thyroid weight (determined after fixation), all organs wereweighed before fixation. Paired organs were weighed individually and identified as left or right.
Organs weights recorded from brain, epidodymis, heart, kidney, liver, lymph node (mesenteric and cervical), ovary, spleen, testicle, thyroid, thymus, prostate, semincal vesicles with coagulating glands, pituitary, uterus including cervix and oviducts.

Histopathology:
The histopathology was performed on the preserved organs of all F0 animals of group 1 and 4 (control and high dose). Since no relevant findings were obserd in group 1 and 4, no further histopathological examination of the low and mid dose animals was required.
The organs listed below were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section. In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined.
Eye with optic nerve, epididymis, adrenal gland, bone, bone marrow (os femoris), brain (cerebrun, cerebellum, pons), gross lesions, heart, intestine (small and large), kidney and ureter, liver, lungs, lymph node (cervical and mesenteric), mammary gland, muscle (skeletal), testicle, nerve (sciatic), oesophagus, ovary, pituitary, prostate, semincal vesicles with coagulating glands, spinal cord, spleen, stomach, thyroid, thymus, trachea, urinary bladder, uterus, vagina

Bone marrow:
During dissection fresh bone marrow was obtained from the os femoris (3 airdried smears / animal) form 10 male and 10 female animals from all groups of the F0 Generation. The same animals were used as those that were selected for the laboratory examinations and the hormone level determinations. The myeloid:erythroid ratio was determined from animals of groups 1 and 4 by cell differentiation (counting of 200 nuclei-containing cells).

Evaluation of aberrant crypt foci (ACF):
10 male and 10 female F0 Satellite animals per group (80 animals in total) were used for evaluation of aberrant crypt foci (ACF). As gestation and lactation are companied by profound physiological changes, the F0 animals for ACF determination were paired and sacrificed after weaning (male and female parental F0 animals). This is necessary to allow correlation of results from ACF determination in the satellite animals with results of regular histopathologic examination of the gastrointestinal tract of parental F0 main study animals that were sacrificed after weaning. Following a randomization scheme, the animals were euthanized by carbon dioxide (CO2) inhalation and exsanguinated by cutting the aorta abdominalis. The colon was taken, opened longitudinally with scissors, and gently rinsed with 0.9% NaCl solution to remove the colon contents. The colon was cut into pieces of suitable size to fit into histological cassettes; the pieces were placed on numbered pieces of filter paper of the same size. The most cranial piece was put onto filter paper number 1, all following pieces on filter papers with ascending numbers going towards the
rectum. Filter papers of an individual animal were placed in one cassette in numerical order with the paper number 1 (the most cranial portion) located at the bottom. The colon portions had to be as flat as possible to facilitate later processing and evaluation. To make sure that the colon portions were pressed flat and do not bulge or roll in the closed cassette, rolls of filter paper were added to the cassette if necessary. The closed tissue cassettes were labelled with study number and animal number and immersed in 5 % buffered formalin. Prior to transfer to the Investigating Scientist the samples were coded to ensure blinded analysis. As the examination of aberrant crypt foci is not a standard method, this part of the
study is not in compliance with GLP.

Postmortem examinations (offspring):

SACRIFICE
- Cohort 1A on PND 86 to 96
- Cohort 1B on PND 120 to 136 (after sacrifice of the F2 pups)
- Cohort 2A on PND 84 to 90
- Cohort 2B on PND 21 to 23
- Cohort 3 on PND 53 to 61
- F2 generation on PND 4 to 8
- Surplus pups (F1 generation, culled): PND 4
- Surplus pups (not selected for the cohorts) on PND 22

GROSS NECROPSY

Surplus F1 pups and the F2 pups:
External inspection for gross abnormalities: Dead pups and F1 pups sacrificed on PND 4 (culled F1 Pups) were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
External and internal inspection for gross abnormalities:
F1 Pups: Those F1 Pups that were not selected for the F1 cohorts were sacrificed on PND 22 and dissected. They were examined macroscopically for external and internal gross abnormalities. From up to 10 pups per sex and group organs were weighed and preserved in 7% buffered formalin.
F2 Pups: The F2 pups that were terminally sacrificed between postnatal days 4 and 8 were dissected and macroscopically examined for external and internal gross abnormalities.

Adult animals:
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system and GI tract, particularly whether for example the caecum was dilated. During necropsy the number of implantation sites was recorded in the female animals. Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS

Organ weights:
Surplus F1 pups:
For the F1 Pups that were sacrificed on PND 22 preservation and / or weighing of organs from up to 10 pups per sex and group was scheduled. Organs preserved and weighed were brain, gross abnormalities, heart, intestine (small and large, large were preserved only), kidney, liver, spleen, lungs, mammary gland, stomach, thymus.

Adult animals:
The weights of the organs listed below were recorded from 20 males and female animals from Cohort 1A (the listed lymph nodes were only weighed from 10 males and females of Cohort 1A). From all male and female animals of Cohort 2A and 2B only the brain weight was determined. With the exception of the thyroid weight (determined after fixation), all organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.
Organs weights recorded from brain, epidodymis, heart, kidney, liver, lymph node (mesenteric and cervical), ovary, spleen, testicle, thyroid, thymus, prostate, semincal vesicles with coagulating glands, pituitary, uterus including cervix and oviducts. From Cohort 2A and 2B only the brain weight was determined.

Histopathology of cohort 1A animals (group 1 and 4):
The histopathology was performed on the preserved organs of all Cohort 1A animals of group 1 and 4 (control and high dose). Since no relevant findings were obserd in group 1 and 4, no further histopathological examination of the low and mid dose animals was required.
The organs listed below were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section. In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined.
Eye with optic nerve, epididymis, adrenal gland, bone, bone marrow (os femoris), brain (cerebrun, cerebellum, pons), gross lesions, heart, intestine (small and large), kidney and ureter, liver, lungs, lymph node (cervical and mesenteric), mammary gland, muscle (skeletal), testicle, nerve (sciatic), oesophagus, ovary, pituitary, prostate, semincal vesicles with coagulating glands, spinal cord, spleen, stomach, thyroid, thymus, trachea, urinary bladder, uterus, vagina.
Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea were performed on one ovary of the F1 Cohort 1A females of groups 1 and 4.

Bone marrow:
During dissection fresh bone marrow was obtained from the os femoris (3 airdried smears / animal) form 10 male and 10 female animals from all groups of Cohort 1A. The same animals were used as those that were selected for the laboratory examinations and the hormone level determinations. The myeloid:erythroid ratio was determined from animals of groups 1 and 4 by cell differentiation (counting of 200 nuclei-containing cells).

Neurohistopathology of Cohort 2A and 2B animals:
Haematoxylin-eosin and lyxul fast blue cresyl violet staining on the following organs of cohort 2A animals (group 1 and 4): Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum).
Haematoxylin-eosin staining on the following organs of cohort 2A animals (group 1 and 4): Eye with optic nerve and retina (left and right), Muscle (skeletal), Nerve (sciatic), Spinal cord (3 sections)
Haematoxylin-eosin staining on the following organs of cohort 2B animals (group 1 and 4): Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum).

Histopathological evaluation included:
- alterations in the gross size or shape of the olfactory bulbs, cerebrum or cerebellum;
- alterations in the relative size of various brain regions, including decreases increases in the size of regions resulting from the loss or persistence of normally transient populations of cells or axonal projections (e.g., externa germinal layer of cerebellum, corpus callosum);
- alterations in proliferation, migration, and differentiation, as indicated by areas of excessive apoptosis or necrosis, clusters or dispersed populations of ectopic,disoriented or malformed neurons or alterations in the relative size of various layers or cortical structures;
- alterations in patterns of myelination, including an overall size reduction or altered staining of myelinated structures.
- evidence of hydrocephalus, in particular enlargement of the ventricles, stenosis of the cerebral aqueduct and thinning of the cerebral hemispheres;
- morphometric (quantitative) evaluation: linear or areal measurement of the following major brain regions: cerebral cortex, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum.

Statistics:

14-day palatability study:
Due to the limited number of characters in this field please refer to the field "any other information on materials and methods incl. tables".

EOGRTS:

DATA AQUISITION:
The following data were captured or calculated by the departmental computerized system (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd):
Clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters.
Raw data not fully compatible with the computerized system (e.g. data from the ELISA experiments, neurological screening or pup data) were maintained on paper according to the appropriate SOPs.
Data maintained on paper (e.g. data from the ELISA experiments, the neurological screening or pup data) was entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.

STATISTICS:
Parametrical data
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd) using
the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05) (see the decision tree on the following page).

Non-parametrical data
Bone marrow: statistical evaluation of the myeloid / erythroid ratio using the Chi2 test with StatXact 4.0.1 software.

Reproductive indices:

Reproducte indices for the F0 females and females of Cohort 1B:
Female fertility index (%)
Gestation index (%)
Pre-coital time
Gestation length

Offspring viability indices:

Implantation sites
- number per dam
- distribution in the uterine horns (implantation sites left or right)
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (alive and dead)
- on post-natal day 4
- on post-natal days 7, 14 and 21 (only F0 females)
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on post-natal day 4
- on post-natal days 7, 14 and 21 (only F0 females)
Number of stillbirths
- per group
- per dam
Number of pups with malformations
- per group
- per dam

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the intermediate and high dose level (300 or 1000 mg Titanium dioxide E171/kg b.w./day) test item-related changes were noted for the male and female animals in the form of pale faeces caused by the white colour of Titanium dioxide E171-E in the diet. This observation was considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

14-day palatability study:

CLINICAL SIGNS:
None of the male and female rats treated with nominal dose levels of 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet for 14 consecutive days died during the course of the study. Changes in behaviour or external appearance did not occur. The faeces of all animals were of a normal consistency and did not reveal any further abnormalities.

BODY WEIGHT AND BODY WEIGHT GAIN:
No test item-related influence was noted on the body weight and the body weight gain of the male and female rats treated with nominal dose levels of 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet for 14 consecutive days compared to the control animals.

FOOD AND DRINKING WSTER CONSUMPTION:
No test item-related influence was noted on the food consumption of the male and female rats treated with nominal dose levels of 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet for 14 consecutive days compared to the control animals. The data did not reveal an influence of Titanium dioxide E171-E on the palatability of the diet: At the exposure level of 300 mg Titanium dioxide E171-E/kg b.w./day via the diet, the food consumption of the male and female animals ranged from 8% or 2% below to 11% or 24% above the values of the control group. There was no clear tendency and no statistically significant difference compared to the control group. In the high exposure level group of 1000 mg Titanium dioxide E171-E/kg b.w./day, the food consumption of the male and female animals ranged from 5% or 8% below to 11% or 16% above the value of the control group. There was no doseresponse- relationship compared to the low dose level group and no statistically significant difference compared to the control group. The visual appraisal of the drinking water consumption revealed no differences between the control and the test item-treated animals.

TEST ITEM INTAKE:
The test item-diet mixtures containing nominal dose levels of 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day and the calculation of the test item intake did not reveal any noteworthy effect on the palatability of the feed for the male and female rats after the 14-day treatment period. The mean intake of Titanium dioxide E171-E/kg b.w./day via the diet over 14 days was 305 mg/kg b.w./day (males) or 348 mg/kg b.w./day (females) at the low exposure level and 988 mg/kg b.w./day (males) or 1088 mg/kg b.w./day (females) at the high exposure level of the test item in the diet. Therefore, the calculated test item intake was 102% or 99% (of the nominal = theoretical values) for the male animals and 116% or 109% for the female animals after the 14-day treatment period. During the 14-day treatment period of the test item-diet mixture containing nominal dose levels of 300 mg Titanium dioxide E171-E/kg b.w./day the mean test item intake ranged from 253 to 358 mg/kg b.w./day for the male rats and from 258 to 462 mg/kg b.w./day for the female rats. Treatment with the higher nominal concentration of 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet led to a test item intake in the range from 831 to 1120 mg/kg b.w./day for the males and 799 to 1322 mg/kg b.w./day for the females. Fluctuations noted on the test item intake during the 14-day treatment period, in particular at the nominal high dose level, are considered to be not test item-related as variations also occurred for the control animals (group 1) on the food consumption. Furthermore, the adjustment of test item concentration in the diet for the 2nd treatment week must be taken into account.

GROSS NECROPSY:
At necropsy on test day 15, no macroscopic changes were noted in the male and female rats treated with nominal dose levels of 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day via diet for 14 consecutive days.



EOGRTS:

For the assessment of general toxicity 24 males and females per group were examined. However, as 20 dams with live born pups were received for each litter (after exclusion of one low dose, two mid dose and two high dose non-pregnant females, discussed below), no necropsy was perfromed for the remaining dams (four control, three low dose, two mid dose and two high dose females). Hence, the number of implantation sites was not determined, and the animals were excluded from the evaluation of the reproductive parameter. However, pups from the excluded animals were used for the F1 generation.
For laboratory examinations (such as haematology and clinical biochemistry) only 10 animals/sex/dose were used. During necropsy and for the determination of organ weights and histopathology 20 animals/sex/dose were used.

GENERAL TOXICITY


CLINICAL SIGNS:
No test item-related changes in behaviour and the external appearance were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).
Non-test item-related changes in the external appearance were noted for one female animal of the low dose group (no. 93 with pale eyes on lactation days 1 and 2) and one female animal of the high dose group (no. 170 with piloerection during an elongated littering period on gestation days 23 to 25). As these observations were only noted for one animal each on 2 or 3 test days, they were considered to be spontaneous.

MORTALITY:
No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals.

DETAILED CLINICAL OBSERVATIONS:
No observations were noted during the detailed clinical observations for the animals of the control group and the animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT AND BODY WEIGHT GAIN:
No test item-related changes in body weight and body weight gain were noted for the male and female rats between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

FOOD CONSUMPTION:
No test item-related difference in food consumption was noted between the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171- E/kg b.w./day).

WATER CONSUMPTION:
No test item-related difference in drinking water consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A slight but statistically significantly increased drinking water consumption was noted at the low dose level between test days 29 and 36 (8.3 % above the control value for the female animals, p ≤0.05). This slight and temporary increase was considered to be spontaneous.

TEST ITEM INTAKE:
The actual mean test item intake during the whole study was in the range of the nominal values for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). No significantly lower actual values in comparison to the nominal values were noted
for the male and female animals. However, for the female animals higher actual values than the nominal values were noted in test week 16 (last week of gestation period). As no signs of toxicity were noted in the whole study, the temporary higher actual dose levels were not relevant, moreover, as the mean values for the whole study period were in the range of the nominal values as mentioned above.

HAEMATOLOGY:
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg
Titanium dioxide E171-E/kg b.w./day). However, statistically significant changes in comparison to the control group were noted at the low dose level for the haemoglobin (HGB) concentration and the haematocrit (HCT) value for the male animals. However, as the differences were only marginal and no dose response-relationship was noted, the differences were considered to be spontaneous.

CLINICAL BIOCHEMISTRY:
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). However, statistically significant changes in comparison to the control group were noted for the LDL cholesterol concentration, the alkaline phosphatase (aP) and the chloride concentration. However, as no dose response-relationship was noted and the differences were observed in one sex only, they were considered to be spontaneous.

URINALYSIS:
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

SEXUAL HORMONE LEVELS:
MALES:
No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Increased estrone levels in comparison to the control group were noted at the intermediate and the high dose level, statistically significant only at the intermediate dose level. However, though the mean values were increased in comparison to the control group, the individual values from 10 of 10 animals of the intermediate dose group (range between 8.7 and 14.0 pg estrone/mL) and from 9 of 10 animals of the high dose group (range between 7.4 and 14.4 pg estrone/mL) were in or only marginally above the range of the control group (between 5.8 and 13.5 pg estrone/mL). One increased value was only noted for the high dosed animal no. 168 (36.4 pg estrone/mL). Hence, as this increased value was only noted for 1 animal of 20 animals of the high dose group, the observation was considered to be spontaneous. For the purpose of an assessment of the relevance of any minor alterations in hormone levels, a 24-hour circadian cycle was monitored in male and female animals at two different time points in satellite animals. As expected, these measurements document that during a 24 h day, all investigated hormones are subject to substantial variation.

FEMALES:
No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

THYROID HORMONE LEVEL:
MALES:
No test item-related differences for the examined thyroid hormone levels (T3, T4 and TSH) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).
FEMALES:
No test item-related differences for the examined thyroid hormone levels were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A slight but statistically significantly increased T3 level in comparison to the control group was noted at the high dose level. However, all mean and all individual values of the control group and the treatment groups were inside the range of the mean values from the examined time points that were measured during the examination of the 24 hour cycle of T3 concentration. Hence, the slight increase was considered to be spontaneous.

CIRCADIAN CYCLES OF HORMONE LEVELS:
The results clearly document that (gender-specific) sexual and thyroid hormones are subject to substantial variation during the day. Overall, no test itemrelated effect on hormone levels were noted between the control group and the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day) for the examined time points of the daily circadian cycle after 10 weeks of dosing.

Ti ANALYSIS IN BLOOD AND URINE:
The analysis of titanium levels in blood and urine samples taken throughout the study is not yet finalised but will be provided in an update of this robust study summary.

BODY WEIGT AT AUTOPSY:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the body weight at autopsy for the male and female animals. A slightly increased body weight at autopsy was noted for the female animals of the low dose group (9.1 % above the control value; statistically not significant). As no statistical significance and no dose-response relationship were noted, this observation was considered to be spontaneous.

GROS NECROPSY:
MALES:
No observations were noted for the male animals of the low and the intermediate dose group (100 or 300 mg Titanium dioxide E171-E/kg b.w./day) and no test itemrelated observation was noted for the male animals of the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day). The following observations were noted for the males of the control and the high dose group.
Group 1: Adrenal gland right and left: small (no. 16)
Group 4: Duodenum: Mucosa with white layer (no. 155)
The observation in animal no. 155 of the high dose group (mucosa with white layer) was confirmed by microscopy (fine granular deposit) and caused by the high concentration of the test-item in group 4. It was not associated with any pathological observation and considered to be of no toxicological relevance.

FEMALES:
No test item-related observations were noted for the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171/kg b.w./day).
The following isolated observations were considered to be spontaneous:
Group 1: Cervix: thickened, glazed induration, approx. 8 mm in diameter (no. 47)
Group 2: Pituitary: Haematoma, 2 mm diameter (no. 75)
Mammary gland: second left, solid, filled with light green mass, approx. 15x15x10 mm, 1.22 g (no. 92)
Group 3: Uterus: slightly thickened, glazed (no. 130)
Stomach: scattered haemorrhagic foci, approx. 1 - 5 mm in diameter (no. 136)
Group 4: Ovary, left: cystic with clear fluid, approx. 4 mm in diameter (no. 174)

BONE MARROW:
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day).

ORGAN WEIGHT:
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

ACF SCORING:
The analysis of sections of the entire colons showed that the administration of Titanium Dioxide E171-E to rats over a time period of 122 days via the diet did not induce the development of any aberrant crypt foci (ACF) in this species.

HISTOPATHOLOGY:
No test item-related changes were observed for the examined male (n = 20) and female (n = 20) animals of the high dose group (1000 mg Titanium Dioxide E171-E/kg b.w./day) during the microscopic examination.
The histopathological examination performed on one testicle and one epididymis of the examined males (n = 20) of groups 1 and 4 (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects.




REPRODUCTIVE FUNCTION/PERFORMANCE

ESTROUS CYCLE DURING EXPOSURE:
No test item-related differences were noted for the mean length and the mean number of estrous cycles per dam during the pre-mating period between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). None of the females showed a complete estrous cycle during the mating period from test day 85 until a positive mating sign was noted.

ESTROUS CYCLE AT NECROPSY:
A stage of diestrus was most commonly noted for the animals of the control group and the treatment groups. The proportion of the different stages at necropsy between the control group and the treatment groups were nearly equal.

SPERMIOGRAM:
Sperm number: No test item-related difference was noted between the rats of the control group and the rats treated with 100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.
Sperm motility: No test item-related differences were noted between the rats of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.
Sperm morphology: The examination of spermatozoa from the epididymal cauda revealed no increased number of spermatozoa with a malformation in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171/kg b.w./day) in comparison to the control group. In detail, the examination of 4000 spermatozoa (200 per animal) from each test group revealed 11 spermatozoa with a malformation in the control group, 6 in the low dose group and 5 in the high dose group. In all cases, the observed malformation was in the form of a banana-like sperm head.

FEMALE FERTILITY:
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). The observation of one non-pregnant female (no. 76) at the low dose group and 2 non-pregnant females each at the intermediate (nos. 125, 144) and the high dose group (nos. 173, 177) was considered to be spontaneous. A fertility index of 90 % is in the range of the historical control data.

GESTATION INDEX:
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg
b.w./day). One female of the intermediate dose group (no. 126) only delivered stillbirth (2 stillbirths), leading to a gestation index of 95% at the intermediate dose level. However, the singular observation of one female with only stillbirths was considered to be spontaneous.

PRE-COITAL TIME:
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

GESTATION LENGTH:
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171/kg b.w./day).

PRE-NATAL DEVELOPMENT F1-GENERATION:

BIRTH INDICES AND POST-IMPLANTATION LOSS:
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

VIABILITY INDEX:
Pre- and post-cull period:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.

SEX RATIO:
No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT OF PUPS:
No test item-related influence on the body weight of pups was noted for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).
Two runts were noted at the high dose level (1000 mg Titanium dioxide E171-E/kg b.w./day). This is regarded to be spontaneous.

LITTER WEIGHT:
No test item-related influence on the litter weight was noted for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

NUMBER OF LIVE PUPS:
No test item-related or statistically significant differences were noted for the mean number of live pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) during the lactation period.

THYROID HORMONES:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171E/kg b.w./day) were noted for the T4 level on lactation day 4 and the T3, T4 and TSH levels on lactation day 22 for the male and female pups.

EXTERNAL AND INTERNAL EXAMINATION:
External examination (pups terminally sacrificed on lactation day 4 or 22 or found dead):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day after terminal sacrifice on lactation day 4 (culled pups), on lactation day 22 or for the pups that died (stillborn or found dead) during the lactation period.
Internal examination (pups terminally sacrificed on lactation day 22):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic examination of the inner organs or tissues of the control pups and the pups from the dams treated with 100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day after terminal sacrifice on lactation day 22.

PUP ORGAN WEIGHTS (ABSOLUTE):
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects were observed

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1B:
At the high dose level 1000 mg Titanium dioxide E171-E/kg b.w./day, test itemrelated changes were noted for all male and female animals in the form of pale faeces consecutively from test day 11 until necropsy. This observation was caused by the incorporated white colour of Titanium dioxide E171-E via the diet and was not considered to be of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P1)

COHORT 1B

As recommended by the corresponding guideline OECD 443 20 animals/sex/dose of Cohort 1B were used for pairing and the establishment of the F2 generation. However, due to the elevated number of non-pregnant females in the mid (3) and in the high dose (3) group, a re-check was performed of the vaginal smears of the non-pregnant females. This additional check showed that no sperm could be found in the vaginal smears from five of six non-pregnant females (only for the non-pregnant female no. 625 the re-check confirmed the sperm detection during the first examination). Hence, the positive mating signal was incorrect for five females and it cannot be ruled out that the animals would have become pregnant during a longer mating period. Therefore, the erroneously considered inseminated non- pregnant females were excluded from the study (two mid dose and three high dose females). Further, no positive mating sign was noted for one low dose female (no. 591) after 14 days of mating. This female was laparotomized on test day 106 and excluded from the study. One mid dose female was not pregnant although a positive mating sign was noted (no. 625, as mentioned above). This non-pregnant female was considered in the calculation of reproductive performance indices but not for other paramters such as general toxicity.
Overall, the reduced number of pregnant females is not considered to be test item-related but a result of technical failure of the testing facility and thus does not invalate the study.


GENERAL TOXICITY

MORTALITY:
No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals.

CLINICAL SIGNS:
No changes in behaviour, the external appearance and the consistency of the faeces were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

DETAILED CLINICAL OBSERVATION:
No observations were noted during the detailed clinical observations for the animals of the control group and the animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT AND BODY WEIGHT GAIN:
MALES: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Slight but statistically significantly (p ≤ 0.05) decreased body weights in comparison to the control group were noted on test days 1, 15, 22, 29 and 36 (at maximum 12.6 % below control on test day 1) for the intermediate dose group. As the difference between the intermediate dose group and the control group was only slight, nearly disappeared during the further course of the study and no decreased body weight was noted for the male animals of the high dose group, the difference between the animals of the intermediate dose group and the control animals at the beginning of the study was considered to be spontaneous.
FEMALES: No differences in body weight and body weight gain were noted between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

FOOD CONSUMPTION:
No test item-related difference in food consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Some minor changes were noted but not considered to be test item-related since no dose response was observed and the changes were only temporary/spontaneous.

WATER CONSUMPTION:
No test item-related difference in water consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Some minor changes were noted but not considered to be test item-related since no dose response was observed and the changes were only temporary/spontaneous.

TEST ITEM INTAKE:
The actual mean test item intake during the whole study was in the range of the nominal values for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

SEXUAL HORMONES:
MALES: No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Decreased estradiol levels were noted at the intermediate and the high dose level in comparison to the control group, statistically significant at the intermediate dose level. However, though the estradiol concentrations of the intermediate and the high dose group of Cohort 1B were decreased in comparison to the control group of Cohort 1B, they were not unusually low, as the mean values of the intermediate group and the high dose group of Cohort 1B were still above the range of the mean values of the circadian cycle and the values that were noted for the F0 Generation and Cohort 1A and Cohort 2A. Hence, unusually increased estradiol concentrations were noted for the control group and the treatment groups of Cohort 1B when compared to the values of the circadian cycle and the values from the F0 Generation and from Cohort 1A and Cohort 2A. These unusually increased estradiol concentrations were more increased in the control group of Cohort 1B as in the intermediate and the high dose group of Cohort 1B, hence, the decrease that was noted between the control group and the intermediate and the high dose group can be considered as spontaneous and of no toxicological relevance.

FEMALES: No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Statistically significantly increased testosterone levels were noted at the intermediate and the high dose level. However, all values were in the range of or slightly below or above the values of the circadian cycle. It is also worth mentioning that the testosterone concentrations of the Cohort 1B control animals were slightly lower than for the control groups of the F0 Generation and Cohort 1A and 2A. Hence, these variations of the female testosterone levels were considered to be spontaneous.

SEXUAL MATURATION:
MALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of balanopreputial gland cleavage and no differences were noted for the body weight at the time point of balanopreputial gland cleavage.
FEMALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of vaginal opening and no differences were noted for the body weight at the time point of vaginal opening.

BODY WEIGHT AT NECROPSY:
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

GROSS NECROPSY:
MALES: No observations were noted for the male animals of the low and the intermediate dose group (100 or 300 mg Titanium dioxide E171-E/kg b.w./day) and no test itemrelated observations were noted for the male animals of the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day). The following isolated observation was considered to be spontaneous:
Group 4: Testes and epididymides (bilateral): small (no. 609).

FEMALES: No test item-related observations were noted for the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). The following isolated observations were considered to be spontaneous:
Group 1: Kidney (bilateral: very soft consistency (no. 554)
Group 4 Uterus: dilatation (no. 669 NV: prenatal loss of 8 implants)

ORGAN WEIGHTS:
MALES: No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Changes that were noted for the left testis and the liver of the male animals were considered to be spontaneous since no dose response was noted and and no test item-realted changes during histopathology were observed.

FEMALES: No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).



REPRODUCTIVE PERFORMANCE/FUNCTION- COHORT 1B

ESTROUS CYCLE- MATING PERIOD
The obtained stages of the estrous cycle during the mating period did not show any sign of an impaired mating behaviour as a stage of estrus (E) or a stage of proestrus (P) that were not followed by a positive mating sign. On nearly all days of the mating period a stage of diestrus (D) was noted. A stage of proestrus (P) was noted on a few days and such a stage of proestrus (P) was in nearly all cases followed by a positive mating signal on the following day. Exceptions were only noted for animal no. 591 (group 2) and no. 640 (group 3). A stage of estrus (E), that was not followed by a positive mating signal (failed mating opportunity), was noted for 2 animals of the intermediate dose group (nos. 637 and 640) on one day each. These single observations can be considered as spontaneous.

ESTROUS CYCLE AT NECROPSY:
No test item-related changes in estrous cycle at necropsy were noted in the control group or in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

FEMALE FERTILIY:
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). All females of the control group, the low dose group and the high dose group that were mated with a male partner and showed a positive mating sign (sperm detection) became pregnant, leading to a fertility index of 100% for the control group and the low and the high dose group. Only one female with a positive mating sign did not become pregnant (no. 625 of the intermediate dose group). This single occurrence was considered to be spontaneous. The fertility index of all treatment groups was in the range of the historical control data.

GESTATION INDEX:
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). One pregnant female of the high dose group (no. 669) did not litter but showed a prenatal loss of all 8 implants, leading to a gestation index of 94%. However, this single occurrence was considered to be spontaneous.

PRE-COITAL TIME:
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). However, 2 females with an elongated pre-coital time of 14 test days were noted in the control group and 3 females with an elongated pre-coital time of 13 or 14 test days were noted in the intermediate dose group. The observation of 3 females with an elongated pre-coital time in the intermediate dose group was considered to be spontaneous, as 2 females with an elongated pre-coital time we noted in the control group and none in the high dose group.

GESTATION LENGTH:
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BIRTH INDICES AND POST-IMPLANTAION LOSS:
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A slightly reduced birth index and consequently a slightly increased post implantation loss in comparison to the control group were noted at the high dose level for the group value (calculated from the whole numbers of implantation sites and pups per group) and the group mean value (mean value from the individual values of implantation sites and pups of each female per group). The group values for the birth index were 89.27% (high dose group) in comparison to 93.59 % for the control group.
For the post-implantation loss, the group value for the high dose group was 10.73 % in comparison to 6.73% for the control group. The reduced birth index and the increased post-implantation loss at the high dose level were due to the high dosed animal no. 669 with a total loss of all its 8 implantation sites, resulting in a birth index of 0.0% and a post-implantation loss of 100% for animal no. 669. However, as the post-implantation loss of all other remaining females of the high dose group was in the range of the animals of the control group, the slightly decreased birth index and the slightly increased post-implantation loss that were noted at the high dose level were considered to be spontaneous.

VIABILITY INDEX:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the viability index (difference between the number of live born pups (pups alive on lactation day 0/1) and the number of live pups on lactation day 4).

SEX RATIO:
No test item-related influence on the male to female ratio was noted for any treatment group (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT OF PUPS (until PND 4):
No test item-related influence on the body weight of pups was noted for any treatment group (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) on lactation days 1 and 4. One runt each was noted in the control group and the low and the intermediate dose groups. No runt was noted at the high dose level.

LITTER WEIGHT:
No test item-related influence on the litter weight was noted for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

NUMBER OF LIVE LITTER:
No test item-related or statistically significant differences were noted for the mean number of live pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) on lactation days 1 and 4.

EXTERNAL AND INTERNAL EXAMINATION OF THE PUPS:
External examination (pups terminally sacrificed between lactation days 4 to 8 or found dead):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the F2 pups from the control group and the F2 pups from the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) after terminal sacrifice between lactation days 4 to 8 or for the pups that died (stillborn or found dead) during the lactation period (PND 1 to 4).
Internal examination (pups terminally sacrificed between lactation days 4 to 8):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic examination of the inner organs or tissues of the F2 pups from the control group and the F2 pups from the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) after terminal sacrifice between lactation days 4 to 8.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects were observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A:
At the high dose level (1000 mg Titanium dioxide E171-E/kg b.w./day) test itemrelated changes were noted for all male and female animals in the form of pale faeces caused by the white colour of Titanium dioxide E171 in the diet from test day 11 until sacrifice. This observation was not considered to be of toxicological relevance.

COHORT 2A:
At the high dose level (1000 mg Titanium dioxide E171-E/kg b.w./day) test itemrelated changes were noted for all male and female animals in the form of pale faeces caused by the white colour of Titanium dioxide E171-E in the diet from test day 11 until sacrifice on test days 64, 65 or 66. This observation was not considered to be of toxicological relevance.

COHORT 3 :
At the high dose level (1000 mg Titanium dioxide E171-E/kg b.w./day) test item related changes were noted for all animals in the form of pale faeces caused by the white colour of Titanium dioxide E171-E in the diet from test day 11 until sacrifice on test days 32, 33, 36 or 37 (postnatal days 53 - 61). This observation was not considered to be of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

For Cohort 1A, 2A, 2B and 3

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For Cohort 1A, 2A and 3.
Cohort 2B was sacrificed directly after weaning, but no changes were observed until weaning.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

For Cohort 1A, 2A and 3.
Cohort 2B was sacrificed directly after weaning, no food consumption was recorded.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

For Cohort 1A (other Cohorts were not analysed)

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

For Cohort 1A (other Cohorts were not analysed)

Urinalysis findings:

no effects observed

Description (incidence and severity):

For Cohort 1A (other Cohorts were not analysed)

Sexual maturation:

no effects observed

Description (incidence and severity):

For Cohort 1A, 2A and 3.
Cohort 2B was sacrificed directly after weaning, no sexual maturation was recorded.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

For Cohort 1A
For Cohort 2A and 2B only brain weights were recorded.
Organ weight of Cohort 3 were not recorded.

Gross pathological findings:

no effects observed

Description (incidence and severity):

For Cohort 1A, 2A, 2B and 3

Histopathological findings:

no effects observed

Description (incidence and severity):

For Cohort 1A only.
Full histopatholocial examination was not conducted on Cohort 2A, 2B and 3 animals.
Neurohistopathology was conducted on Cohort 2A and 2B animals, no changes were observed.

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Cohort 2A only

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 3 only

Details on results (F1)

ANOGENITAL DISTANCE OF F1 PUPS
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

NIPPLE RETENTION OF F1 PUPS:
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).


COHORT 1A

For the assessment of general toxicity 20 animals/sex/dose were used. However, for laboratory examinations (such as haematology, clinical biochemistry and lymphocyte typing) only 10 animals/sex/dose were used. For necropsy, histopathology and the determination of organ weights all 20 animals/sex/dose were used.

MORTALITY:
No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals.

CLINICAL SIGNS
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT AND BODY WEIGHT GAIN:
MALES: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). However, a slightly reduced body weight was noted for the male animals of the intermediate dose group during the whole study period from test day 1 (10.4% below the control) until test day 64 (5.6% below the control). The difference was statistically significant on test days 36 and 64 (6.1% or 5.6% below the control; p ≤ 0.05). However, as the difference between the intermediate and the control group slightly decreased during the course of the study and only marginal differences were noted between the high dose group and the control group, the differences in body weight that were noted between the intermediate and the control group were considered to be spontaneous.
FEMALES:
No differences in body weight and body weight gain were noted between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

FOOD CONSUMPTION:
MALES: No test item-related difference in food consumption was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A slightly increased food consumption was noted at the high dose level between test week 1 and test week 6 (p ≤0.05/0.01). At maximum 14.0% above the control in test week 1. This slight increase was considered to be spontaneous.
FEMALES: No test item-related difference in food consumption was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

TEST ITEM INTAKE:
The actual mean test item intake during the whole study was in the range of the nominal values for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

HAEMATOLOGY:
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). However, statistically significant changes in comparison to the control group were noted for the concentration of neutrophilic granulocytes of the male animals, which were considered to be spontaneous, as all values were still in the range of historical control data.

CLINICAL BIOCHEMISTRY:
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). However, slightly increased sodium / potassium ratios were noted for the female animals at the intermediate and the high dose level, which were due to decreased potassium levels (statistically not significant). However, as the difference was only small, no dose-response relationship was noted and no changes were noted for the male animals, the increased sodium / potassium ratio at the intermediate and the high dose level was considered to be spontaneous. Furthermore, all values were in the range of historical control data, and no changes were seen in the sodium levels.

LYMPHOCYTE TYPING IN SPLEEN:
No test item-related influence was noted in the proportion of the examined lymphocyte subtypes to each other between the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

URINALYSIS:
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Increased values (statistically significant or not) were noted for the female animals of all treatment groups for the pH value and the urine volume. However, as no dose-response relationship was noted, no changes were noted for the male animals and all values were in the range of historical control data, the differences that were noted for the female animals were considered to be spontaneous.

SEXUAL HORMONES:
MALES: No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Increased estrone levels in comparison to the control group were noted at the intermediate and the high dose level, statistically significant at the high dose level. Any increased estrone levels were considered to be spontaneous and within circadian variation.
FEMALES: No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

THYROID HORMONE LEVELS:
MALES: No test item-related differences for the examined thyroid hormone levels (T3, T4 and TSH) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).
FEMALES: No test item-related differences for the examined thyroid hormone levels were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A statistically significantly decreased T3 level in comparison to the control group was noted at the low dose level. However, this slight decrease was considered to be spontaneous as no dose-response relationship was noted. Furthermore, all mean values and individual values were in the range of the circadian cycle.
Markedly increased values were noted for the TSH concentrations at the low, the intermediate and the high dose group, although they were not statistically significant. The lack of statistically significance was due to a high variability of the individual values. Only a few values were above the upper range of the individual values of the circadian cycle. However, as no statistical significance was noted, the few increased values were considered to be spontaneous.

SEXUAL MATURATION:
MALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of balanoprepuital gland cleavage and the body weight at the time point of balanoprepuital gland cleavage.
FEMALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.
No test item-related influence was noted on the time point for the appearance of cornified cells in the vaginal smear and no test item-related influence was noted on the period between the day of vaginal opening and the day of the appearance of cornified cells in the vaginal smear for the females of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

ESTROUS CYCLE- 2-WEEK PERIOD:
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

SPERMIOGRAM:
Sperm number: No test item-related difference was noted between the rats of the control group and the rats treated with 100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.
Sperm motility: No test item-related differences were noted between the rats of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.
Sperm morphology: The examination of spermatozoa from the epididymal cauda revealed no increased number of spermatozoa with a malformation in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) in comparison to the control group. In detail, the examination of 4000 spermatozoa (200 per animal) from each test group revealed no malformation.

BODY WEIGHT AT NECROPSY:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the body weight at autopsy.

GROSS NECROPSY:
No observations were noted in the control group and no test item-related observations were noted in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).
The following observations were considered to be spontaneous:
Group 2 (males): Lungs: reddened (no. 410)
Group 2 (females): Stomach: scattered haemorrhagic foci, approx. 0.5 – 10 mm in diameter (no. 421)
Group 2 (females): Kidney, left: few light brownish foci, approx. 3.0 - 8.0 mm in diameter (no. 431)

ESTROUS CYCLE AT NECROPSY:
The examination at necropsy revealed an increased number of females in the high dose group that were in an estrous stage and a decreased number of females that were in a diestrous stage in comparison to the control group and the low and the intermediate dose group. However, this difference markedly decreased when comparing the results from the histopathological examination. In detail, during the histopathological examination 11 females with a diestrous stage were noted in the control group in comparison to 8 in the high dose group. Seven females of the control group were in an estrous or a proestrous stage in comparison to 11 in the high dose group. Hence, the difference that was noted between the control group and the high dose group during the examination at necropsy could be considered as irrelevant, as the more accurate histopathological examination revealed only small differences.

ORGAN WEIGHTS:
MALES: No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day), with the following exceptions: Only slightly but statistically significantly reduced absolute organ weights were noted for the right kidney and the right testes at the intermediate and the high dose level. However, as the differences were only slight and statistically significantly reduced values were not noted for the absolute organ weights of the left kidney and the left testes, these changes were considered to be spontaneous and not of toxicological relevance.
FEMALES: No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Decreased absolute and relative organ weights (statistically significant or not) in comparison to control were noted for the mesenteric lymph node of all treatment groups. However, the histopathological examination of the mesenteric lymph node of the high dose females revealed no test item-related changes. Furthermore, no dose response-relationship was noted (the percentage of organ weight decrease in comparison to control was equal in all treatment groups). Hence, the observed decrease in the absolute and relative organ weights was considered to be spontaneous.

BONE MARROW:
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day).

HISTOPATHOLOGY:
Selected organs form all male and female animals of the control group and the high dose group (1000 mg Titanium Dioxide E171-E/kg b.w./day) (20 animals per sex and group) were examined microscopically. These examinations did not reveal any test item-related changes.
Histopathological examination performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases)and histopathology of the interstitial testicular structure) of all males of groups 1 and 4 (20 males per group), did not reveal any test itemrelated effects.
The examination of one ovary each from 20 females of the control group and 20 females of the high dose group revealed no test item-related differences in the number of primordial and small growing follicles and the number of corpora lutea.


COHORT 2A

All examinations were performed with 10 animals/sex/dose.

MORTALITY:
No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals.

CLINICAL SIGNS:
No changes in behaviour, the external appearance and the consistency of the faeces were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT AND BODY WEIGHT GAIN:
MALES: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).
FEMALES: No differences in body weight and body weight gain were noted between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A slightly decreased body weight (statistically not significant) was noted for the female animals of the low dose group (8.0% below the control on test day 57). However, as no differences were noted between the control group and the intermediate and the high dose group, the decreased values that were noted at the low dose level were considered to be spontaneous.

FOOD CONSUMPTION:
MALES: No test item-related difference in food consumption was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A statistically significantly increased food consumption was noted at the intermediate and the high dose level between test days 8 to 15 (23.3 % or 22.5 % above the control; p ≤ 0.01). As the observation was only temporary, it was considered to be spontaneous. An additional statistically significant difference in the form of a temporary decreased food consumption was noted at the low dose level between test days 43 to 50 (6.7 % below the control, p ≤ 0.05). This observation was also considered to bespontaneous, due to its small and temporary nature.
FEMALES: No test item-related difference in food consumption was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Statistically significantly increased values were noted between test days 8 and 15 at the low and the intermediate dose level (24.6 % or 14.8 % above the control; p ≤ 0.05). A statistically significantly decreased value was noted between test days 22 to 29 at the high dose level (27.5 % below the control; p ≤ 0.01). As both observations were only temporary, they were considered to be spontaneous.

TEST ITEM INTAKE:
The actual mean test item intake during the whole study was in the range of the nominal values for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

SEXUAL HORMONES:
No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals. Differences (statistically not significant) between the mean values of the test groups and the control group were noted for the estrone and testosterone concentrations of the male animals and the estradiol concentration of the female animals. However, all individual concentrations of estradiol of the female animals were within the circadian variation. In case of the testosterone concentration of the male animals, one or a few individual values per group were slightly below the circadian variation, whereas all other values were within the circadian variation (the circadian variation of estrone was not examined). As the differences in the estrone, the testosterone and the estradiol concentrations were not statistically significant and completely (estradiol concentration) or nearly completely (testosterone concentration) within the circadian variation, they were considered to be spontaneous.

SEXUAL MATURATION:
MALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of balanopreputial gland cleavage and the body weight at the time point of balanopreputial gland cleavage.
FEMALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.

BODY WEIGHT AT AUTOPSY:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the body weight at autopsy.

GROSS NECROPSY:
No observations were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BRAIN WEIGHTS:
No test item-related differences for the absolute and relative brain weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals.

NEUROHISTOPATHOLOGY:
The neurohistopathological examination of different brain regions from all male and female animals of the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day) did not reveal any test item-related effects when compared to the male and female animals of the control group (n = 10 animals per sex and group). The results of the quantitative evaluation (comparison of the relative size of different brain regions) of groups 1 and 4 was still ongoing at the time of preparation of this robust study summary, but will be given in an update.

SCREENING FOR NEURODEVELOPMENTAL EFFECTS

AUDITORY STARTLE RESPONSE:
No test item-related difference was noted for the reaction of the animals in the auditory startle response test between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day), when exposed to a short noise stimulus on postnatal days 23, 24 or 25. The reaction was noted as yes / no answer.
At the beginning all animals react with a short startle to nearly every sound stimulus. During the course of the experiment, the animals adapt to the stimuli. At the end of the experiment the animals showed no reaction to the stimulus in more than half of the cases. This course of the experiment was noted for in the control group and in the treatment groups.

OBSERVATIONAL SCREENING:
No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faces were noted for the male and female animals of all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. A statistically significantly increased hind-leg splay was noted for the female animals at the low and the high dose level. As no dose-response relationship was seen and no changes were noted for the male animals, the increased values at the low and the high dose level were considered to be spontaneous.

FUNCTIONAL TESTS:
GRIP STRENGTH
No test item-related influence on the fore- and hindlimb grip strength was noted for the male and female animals of all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A statistically significantly increased forelimb grip strength was noted for the male and the female animals of the intermediate dose group. However, as the values of the high dose group were again in the range of the control group (no dose-response relationship) the increased values at the intermediate dose level were considered to be spontaneous.

SPONTANEOUS MOTILITY:
No test item-related influence on the spontaneous motility was noted for the male and female animals of all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).


COHORT 2B

All examinations were performed with 10 animals/sex/dose.

BODY WEIGHT:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the body weight of the male and female animals of Cohort 2B on their lactation day 21. Slight and statistically not significantly decreased body weights were noted for the male animals of each treatment group (between 10.4% below the control in the low dose group and 7.5% below the control in the high dose group). For the female animals a decreased body weight was noted for the animals of the intermediate dose group (7.6% below the control, statistically not significant). However, regarding the other cohorts, slightly reduced body weights in comparison to the control group were noted for the male and female animals of the intermediate and the high dose group in most cases on test day 1 of the F1 Generation study. In all these cases, the difference between the treatment groups and the control group decreased or even completely disappeared during the course of the study. Therefore, the differences in body weight that were noted on test day 1 for the different cohorts were considered to be spontaneous. Thus, it can be assumed that also the differences in body weight that were noted for the animals of cohort 2B on lactation day 21 (which was only a few days before test day 1 of the other F1 cohorts) would have been disappeared during the course of the study.

SEXUAL HORMONES:
No test item-related differences for the examined sexual hormone levels (estradiol, estrone and testosterone) were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals. A decreased testosterone concentration was noted for the animals of the intermediate dose group. However, as no dose-response relationship was noted, the observation was considered to be spontaneous and not test item-related. Furthermore, nearly all individual values were within the circadian variation, only a few individual values form group 3 and one value from group 2 were slightly below the lower range of the circadian cycle.

BODY WEIGHT AT AUTOPSY:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the body weight at autopsy. Slight but statistically significantly decreased body weights at autopsy were noted for the male and the female animals of the treatment groups, statistically significant for the female animals of the intermediate dose group. However, the differences corresponded to the differences that were noted for the last live body weight from lactation day 21, which was considered to be spontaneous (see above). Hence, the differences in the body weight at autopsy could also be considered as spontaneous.

GROSS NECROPSY:
No observations were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BRAIN WEIGHTS:
No test item-related differences for the absolute and relative brain weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) for the male and female animals. Slightly increased relative brain weights were noted for the male and female animals, statistically significant for the female animals of the intermediate dose group. However, no changes were noted for the absolute brain weights. Hence, the observed differences in the relative brain weight were only due to the differences that were noted for the body weight at autopsy and no dose response could be observed. Therefore, the observed differences in the relative brain weight were of no toxicological relevance.

NEUROHISTOPATHOLOGY:
The neurohistopathological examination of different brain regions from all male and female animals of the high dose group (1000 mg Titanium dioxide E171-E/kg b.w./day) did not reveal any test item-related effects when compared to the male and female animals of the control group (n = 10 animals per sex and group).


COHORT 3

All examinations were performed with 10 animals/sex/dose.

MORTALITY:
No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

CLINICAL SIGNS:
No changes in behaviour, the external appearance and the consistency of the faeces were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

BODY WEIGHT AND BODY WEIGHT GAIN:
MALES: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) between test day 1 and test day 29 (test day 29 was the last day of body weight measurement for the complete groups). However, a slightly reduced body weight (statistically not significant) was noted for the male animals of the intermediate and the high dose group (at maximum 10.3 % or 11.3 % below the control on test day 1). During the further course of the study the difference between the intermediate and the control group slightly decreased (7.6 % below the control on test day 29). In contrast to the intermediate dose group, the difference between the high dose group and the control group even nearly completely disappeared during the course of the study (1.4 % below control on test day 29). As the difference at the high dose level nearly completely disappeared, the reduced body weights that were noted at the beginning of the study for the intermediate and the high dose level were considered to be spontaneous and not test item-related.
FEMALES: No differences in body weight and body weight gain were noted between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). As for the male animals, reduced body weights in comparison to the control were noted for the animals of the intermediate (10.3 % below the control) and the high dose group (16.3 % below the control; p ≤ 0.01) on test day 1. As the differences in comparison to the control group nearly completely disappeared during the course of the study (1.5 % or 0.3 % below the control on test day 29), the differences that were noted at start of the study, were considered to be spontaneous and not test item-related.

FOOD CONSUMPTION:
MALES: No test item-related differences in food consumption were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A slightly but statistically significantly (p ≤ 0.01) increased food consumption was noted at the high dose level for test week 3 (test days 15 to 22) and for test week 4 (test days 22 to 29) (at maximum 12.4 % above the control in test week 3). This slight increase was considered to be spontaneous.
FEMALES: No test item-related differences in food consumption were noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). A statistically significantly increased food consumption (17.1 % above the control; p ≤ 0.01) was noted at the high dose level on test day 1. This temporary observation was considered to be spontaneous.

TEST ITEM INTAKE:
The actual mean test item intake during the whole study was in the range of the nominal values for all treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).

SEXUAL MATURATION:
MALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of balanoprepuital gland cleavage and the body weight at the time point of balanoprepuital gland cleavage.
FEMALES: No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.

GROS NECROPSY:
No test item-related observations were noted for the male and female animals of the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day). The following singular observations were considered to be spontaneous:
- Dilatation of the renal pelvis (male no. 865; control)
- Small spleen (male no. 904; high dose group)

ESTROUS CYCLE AT NECROPSY:
No test item-realted changes in estrous cycle at necropsy were noted of the control and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day).


SCREENING FOR DEVELOPMENTAL IMMUNOTOXIC EFFECTS

LYMPHOCYTE TYPING IN SPLEEN:
No differences in the relative size of the lymphocyte subpopulations were noted between the control group and the animals treated orally with 100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day, after immunization of the animals of the control group and the treatment groups with KLH. However, when compared to animals of cohort 1A, the KLH injected animals of cohort 3 revealed a shift towards a higher percentage of B cells in the lymphocyte population of the spleen. This shift in the lymphocyte subpopulation indicates the successful activation of the immune system by injection of KLH with increased B-cell proliferation to produce the antigen-specific antibodies. As this shift towards the B cell population was equally noted for all animals of cohort 3 (animals of the control group and the treatment groups) this can be taken as a reference that the immune response was not adversely affected by the test item.

T-CELL DEPENDENT KLH RESPONSE (KLH ASSAY):
No test item-related difference was noted for the anti-KLH IgM serum levels between the male and the female animals of the control group and the test-item treated groups (100, 300 or 1000 mg Titanium dioxide E171-E/kg b.w./day) after treatment of the animals from group 1 to 4 with KLH.

It was decided that the KLH immunisation was not successful and thus no final conclusion on possible effects of Titanium dioxide E171-E on the developing immune system can be provided. Thus, it was decided to repeat this part of the study. In early 2021 parental animals will be dosed for 10 weeks with 0, 100, 300 and 1000 mg/kg bw/day, then paired and the F1 generation will be used to investigate possible developmental immunotoxic effects. Final results will be provided with the next dossier update.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects were observed

2

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects were observed

3

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2B)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects were observed

4

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 3)

Basis for effect level:

other: No final conclusion of effects of TiO2 E171-E available. Will be provided with the next dossier update.

Remarks on result:

other: No final conclusion of effects of TiO2 E171-E available. Will be provided with the next dossier update.

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

ANOGENITAL DISTANCE:
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (100, 300 or 1000 mg Titanium dioxide E171/kg b.w./day). A slight but statistically significantly decreased relative ano-genital distance was noted at the low dose level for the male pups. However, the slight decrease was considered to be spontaneous.

For further results for the F2 generation please refer to the field "Details on results (P1)".

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2 (cohort 1B)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects were observed

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

F0 Generation
General toxicity: NOAEL above 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet.
Reproductive toxicity: NOAEL above 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet.

F1 Generation
Reproductive and developmental toxicity (Cohort 1A and 1B): NOAEL above 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet.
Developmental neurotoxicity (Cohort 2A and 2B): NOAEL above 1000 mg Titanium dioxide E171-E/kg b.w./day via the diet.
Developmental immunotoxicity (Cohort 3): needs to be determined (early 2021)

Executive summary:

The extended one generation reproduction (EOGRT) study was commissioned by the Titanium Dioxide Manufactures Association (TDMA) in response to a request by the European Food Safety Authority (EFSA) as set forth in their Scientific Opinion of June 2016 (EFSA, 2016) on the re-evaluation of titanium dioxide (E171) as a food additive.

During the fully guideline compliant extended one generation reproductive toxicity study (OECD 443) 20 male and 20 female rats were dosed with 0, 100, 300 and 1000 mg/kg bw/day of food-grade Titanium Dioxide E171-E via the diet for 10 weeks before mating. After mating, all males were analysed for general toxicity. After weaning of F1 animals, all parental females were sacrificed and analysed for reproductive and gerneral toxicity and all F1 pups were allocated to Cohort 1A, 1B, 2A, 2B and 3 for the assessment of reproductive/developmental endpoints (cohort 1A and 1B), potential impact on the developing nervous system (Cohort 2A adult rats, Cohort 2B weaning rats) and potential impact on the developing immune system (Cohort 3). Cohort 1B animals were maintained on treatment until postnatal day 90 and bred to obtain the F2 generation. The F2 generation was analysed for developmental toxicity on postnatal day 7.

In addition to that, 10 male and 10 female F0 satellite animals per group were paired and sacrificed after weaning and used for evaluation of aberrant crypt foci. Further F0 satellite animals were used for hormone analysis of beta-estradiol, Testosterone, T3, T4 and TSH (group 1 and 4 only). This analysis was performed twice, pre-dose and at the end of the pre-mating period (after 10 weeks) throughout a 24-hour cycle. Blood sampling was performed every 2 hours during the 24-hour period of the cycle.

Further, blood samples were taken at sacrifice from F0, Cohort 1A, 1B, 2A and 2B animals for the determination of sexual hormones (estradiol, testosterone and estrone) and whole blood and urine samples taken at sacrifice from F0, Cohort 1A, 1B and F2 pups were used for titanium level analysis.

No test item-related influence was noted on the general toxicity and the reproductive performance of the parental animals of the F0 Generation as well as on the pre- and postnatal development of the F1 pups. No test item-related changes were noted during the histopathological examination (including a detailed examination of the testis and the epididymides) and the examination of the intestines for aberrant crypt foci (ACF). There were no treatment-related effects on hormone levels (estradiol, estrone and testosterone, plus T3, T4 and TSH) in any of the treatment groups of each cohort compared to controls. During their post-weaning development, the animals of the F1 Generation showed no signs of general toxicity. No test item-related influence was noted on the development of the reproductive system (levels of sexual hormones, time points of sexual maturations, number and length of estrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries). Also no test item-related influence was noted on the reproductive performance of the F1 males and females (fertility index, gestation index, pre-coital time and gestation length) and on the pre- and postnatal development of the F2 pups until sacrifice on lactation day 4 (number of resorptions, stillborns, live born pups and the viability index after birth until lactation day 4-7). No test item-related influence was noted on the neurological function of the young adult male and female animals of cohort 2A. The neurohistopathological examination of the brains from the high dosed adult animals of cohort 2A and from the high dosed recently weaned animals of cohort 2B did not reveal any test item-related effects when compared to their control group. The examination of the lymphpcyte subpopulations in the spleen and the anti KLH IgM serum levels was not successful, thus complete cohort 3 will be repeated in early 2021. Final results on cohort 3 will be provided with the next dossier update. The analysis of titanium in whole blood and urine is also still ongoing and will be reported with the next dossier update.