Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011-09-19 to 2012-01-20
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies

Data source

Reference
Reference Type:
publication
Title:
Toxic effects of various modifications of a nanoparticle following inhalation
Author:
Creutzenberg, O.
Year:
2013
Bibliographic source:
Federal Institute for Occupational Safety and Health. Project F 2246

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
2009-09-07
Deviations:
yes
Remarks:
BAL & histopatology conducted in the same lung lobe; highest concentrations caused lung overload; heamatology, clinical chemistry, & food consumption not determined; actual conc. measured once/day only; particle size distribution not determined weekly
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform, surface-treated
Details on test material:
NOTE: the particles were characterised by the supplier. The specific surface (BET method) was also measured by the performing laboratory.

- Name of test material (as cited in study report): TiO2 UV TITAN M262 (European nanomaterials repository code: NM-103) (rutile)
- Primary particle diameter: 20 nm
- Specific surface : 60 m² / g (given by supplier), 56.2 m² / g (determined by the performing laboratory)
- Surface properties: hydrophobic, dimethicone (silicone)
- bulk materials consist of a mixture of various agglomerates sizes including some individual primary particles.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on species / strain selection:
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

*Reference:
- Vu V., Barrett J.C., Roycroft J., Schuman L., Dankovic D. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg. Tox. Pharm. 24, 202-212 (1996)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 270 grams
- Housing: housed in Makrolon® (polycarbonate) cages type III, two rats per cage; bedding material: absorbing softwood ('ssniff BK 8-15')
- Diet (fresh weekly or more often): commercial chow in pellet form (ssniff "V1534")
- Water (fresh weekly or more often): tap water from the Hannover city water supplier
- Acclimation period: approx. 1 week acclimatized to laboratory environment; 3 weeks prior to the expsoure start, all rats were trained to the 6-hour restraint in nose-only tubes.

DETAILS OF FOOD AND WATER QUALITY:
A certificate of water analysis issued by the water supplier (Stadtwerke Hannover) is sent periodically to the laboratory. A certificate of feed analysis is issued by the supplier periodically. The analysis is done at the LUFA-ITL in Kiel, Germany (certified lab; DIN EN ISO/IEC 17025:2000).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Relative humidity: 55 % ± 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 0.62 - <= 1.17 µm
Remarks on MMAD:
- Geometric standard deviation (GSD): 2.64 - 4.27
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Prior to the 28-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.

- Exposure apparatus: aerosol was given to the rats by a flow-past nose-only inhalation exposure system.

- Method of holding animals in test chamber: for exposure to the test item the rats were restrained in acrylic tubes with a flexible stopper. The exposure tubes were arranged around a cylinder capable to take up 16 tubes per platform. The rat nose is located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The airflow to each rat was approximately 1 L/min.

- System of generating particulates/aerosols: the particulate sample aerosols were generated by dry dispersion with pressurized air. Dispersion was achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle developed by laboratory (Koch, 1998)*. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.

- Temperature, relative humidity, air flow: parameters were recorded by 20-minute means. The limits were set at 22 °C ± 2 °C for temperature and 55 % ± 15 % for relative humidity.

- Method of particle size determination: mass median aerodynamic diameter (MMAD) was determined 2-3 times using a cascade impactor (Marple impactor).

TEST ATMOSPHERE
- Brief description of analytical method used: filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to the target concentrations.
- Samples taken from breathing zone: yes

*Reference:
- Koch W. Application of aerosols. In: Pulmonary Research, S. Uhlig, A.E. Taylor (eds.), Birkhäuser, Basel-Boston-Berlin (1998)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see above ("Details on inhalation exposure")
Duration of treatment / exposure:
28 days (20 effective exposure days per rat)
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
3.06 mg/m³ air (analytical)
Remarks:
Standard deviation: 0.18 mg/m³ air
Dose / conc.:
12.13 mg/m³ air (analytical)
Remarks:
Standard deviation: 0.25 mg/m³ air
Dose / conc.:
49.7 mg/m³ air (analytical)
Remarks:
Standard deviation: 8.20 mg/m³ air
No. of animals per sex per dose:
12 male rats/dose/recovery time point (total: 36 rats)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: This dosing scheme was aiming at achieving non-overload, partial overload and complete overload conditions in the low, mid and high dose groups, respectively.
- Post-exposure recovery period: 3, 45 or 94 days
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week in the first 4 weeks of exposure, thereafter once a week

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
- bronchoalveolar lavage was performed in 6 rats per time point and group, i.e. after end of exposure (day 3) and after 45 and 94 days of post-observation.
- method of Henderson et al. (1987)* was used with minor modifications.
- bronchoalveolar lavage (left lobe lungs): two lavages were performed (without lung tissue massage). The 1st lavage fluid was collected and an aliquot was taken for tumor necrosis factor-α (TNF-α) analysis. Then, the 2nd lavage fluid was combined with the 1st lavage fluid and taken for differential cell count (macrophages, neutrophils, eosinophils, lymphocytes) and enzyme/total protein analysis. For differential cell count, two slides were prepared with 200 leucocytes per slide (total of 400 individual leucocytes).
A second series of 3 lavages with lung tissue massage was performed, which were combined and analysed for reactive oxygen intermediates (ROI; dose groups: 5 animals/time point/group).
- leucocyte concentration was determined in the lavage.
- after centrifugation of the lavage fluid, biochemical indicators relevant for diagnosis of lung damage were determined in the supernatant (lactic dehydrogenase (LDH), β-glucuronidase, total protein)
- during ROI analysis, cell count and viability were also measured.

*Reference:
- Henderson, R.F., Mauderly, J.L., Pickerell, J.A., Hahn, R.F., Muhle, H., Rebar, A.H. Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329-342 (1987)
Sacrifice and pathology:
GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
- rats were sacrifced on day 3, 45 and 94 after administration
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights was determined for the following organs: lungs, trachea (lower part), liver, kidneys, adrenals, testes epididymes, thymus, spleen, brain, and heart
- histopathology: the following tissues were prepared for histopathology: adrenals, bone marrow (and/or fresh aspirate), brain (including sections of cerebrum, cerebellum,
and medulla/pons), heart, kidneys, larynx (3 levels , 1 level to include the base of the epiglottis), liver, lung (all lobes at one level, including main bronchi), lymph nodes from the hilar region of the lung, nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the nasal associated lymphoid tissue (NALT), oesophagus, ovaries, seminal vesicles, spinal cord (cervical, mid-thoracic, and lumbar), spleen, stomach, testes, thymus, thyroid, trachea (at least 2 levels including 1 longitudinal section through the carina and 1 transverse section), uterus, and all gross lesions.
The following histopathology was performed in 6 animals per group after end of exposure (day 3) and in the recovery group animals on day 45 and day 94 after exposure:
- full histopathology on the respiratory tract and other organs and tissues, as listed above of all animals in the clean air control group and the 48 mg/m³ dose groups and all animals that died or were killed during the study.
- histopathology of the left lung lobe, including bronchi and the lung-associated lymph nodes (LALN), trachea, larynx, pharynx, the nasal cavities (turbinales) and visceral pleura in all animals of all groups.
During histopathology, lungs were treated with a special stain for the diagnosis of fibrotic changes (Masson trichrome)

TEST ITEM ANALYSIS IN ORGANS
- after sacrifice the right lung lobes were subjected to lyophilisation and subsequent low-temperature ashing and the test items retained in lung tissue were determined using ion-coupled plasma mass spectroscopy (ICP-MS).
- particle retention was also determined in liver and brain

TRANSMISSION ELECTRON MICROSCOPY (TEM) ANALYSIS
At the respective time points to be investigated (recovery day 3, 45 and 94) the right part of the lung including the right cranial, right middle and right caudal lung lobe as well as the accessory lung lobe were fixed for at least 24 hours.
Following fixation of the tissue, the volume of the fixed tissue was determined using the method of Scherle (1970)* in order to detect a possible age- or treatment-dependent affect on the lung volume which would influence the measurement of the particle amount in the transmission electron microscope.
Thereafter, multiple specimens per organ were taken. These samples were postfixed, dehydrated, infiltrated with epoxy resin, embedded, and sectioned (70 nm). Sections were observed with a transmission electron microscope (magnification of 10.000x). The amount and location of the nanoparticles found was noted and assigned to compartments. The compartments in which nanoparticles were detected were defined as follows: intraalveolar macrophage, free within the alveolus, pneumocyte type I, pneumocyte type II, free within the interstitium, interstitial macrophage, interstitial cellular (not otherwise definable), bronchiolar epithelium.

*Reference:
- Scherle, W. A simple method for volumetry of organs in quantitative stereology. Microscopy 26, 57-60 (1970).


Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test. If necessary, further statistical procedures will be applied.
For statistical analysis of the transmission electron microscopy results an analysis of variance was applied.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An statistically significant increase in the absolute and relative organ weights of the lung was observed in the 49.70 ± 8.20 mg/m³ air group 3 days and 94 (but not 45) days post exposure.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopical findings observed upon necropsy at scheduled dates showed dose-dependent effects as follows:
Lungs:
12.13 ± 0.25 mg/m³ air:
- lungs slightly marbled in single animals
49.70 ± 8.20 mg/m³ air:
- lungs slightly to moderately (severely) marbeled/discoloured
- lungs had white/discoloured areas
- changed consistency in the majority.
The findings reflect the dose-dependent retention of test items.

Lung-associated lymph nodes (LALN):
12.13 ± 0.25 mg/m³ air:
- slight enlargement in the majority of rats
49.70 ± 8.20 mg/m³ air:
- moderate to severe enlargement in the majority of rats
- white/discoloured areas
The findings reflect the dose-dependent retention of test item in the lung.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(Raw data attached in section "Attached background material")
low dose: 3.06 ± 0.18 mg/m³ air
mid dose: 12.13 ± 0.25 mg/m³ air
high dose: 49.70 ± 8.20 mg/m³ air

Nasal Cavity:
1. Recovery day 3
An exposure-related effect was the dose-dependent occurrence of very slight (minimal) epithelial hyaline droplets (eosinophilic inclusions), mainly within the respiratory epithelium of the nasal septum and ventral nasal meatus at levels II to III of the nasal cavity sections. While in the clean air control group no animal showed this lesion, this change was observed (multi)focally in the particle exposure groups at incidences of 2/6 in the mid-dose and 3/6 in the high-dose group.particle-laden macrophages were seen to a very slight (minimal) extent in the nose-associated lymphatic tissue (NALT) in 1 of 6 animals of the low-dose and in all animalsof the mid- and high-dose group. In addition, particles were detected in the cytoplasm of epithelial cells above the NALT in 6/6 high-dose animals.
2. Recovery day 45
Very slight multifocal intracytoplasmic hyaline droplets were seen in 1/6 high-dose animals. Furthermore, particle-laden macrophages were seen to a very slight (minimal) extent in the nose-associated lymphatic tissue (NALT) in 1 of 6 animals of the low-dose, in 5/6 animals of the mid-dose and in all animals of the high-dose group. In addition, particles were detected in the cytoplasm of epithelial cells above the NALT only in 1/6 of the mid-dose NM-103 treated animals. Further findings in the nasal cavity included only a moderate chronic active inflammation in one animal which was interpreted as incidental finding.
3. Recovery day 94
Very slight multifocal intracytoplasmic hyaline droplets were still observed with a low incidence in 1/6 low-dose NM-103 treated animals. Furthermore, particle-laden macrophages were seen to a very slight (minimal) extent in the nose-associated lymphatic tissue (NALT) in 2 of 6 animals of the low-dose, in 5/6 animals of the mid-dose and in all animals of the high-dose NM-103 treated. In contrast to recovery day 3 and 45, particles were no longer detected within the epithelium above the NALT.
- further findings in the nasal cavity were only in single animals and included subepithelial mononuclear cells filtration very slight in one animal of the mid dose and one animal of the high dose with a slight chronic active granulomatous inflammation (findings incidental and unrelated to exposure).

Larynx:
1. Recovery day 3
A very slight subepithelial accumulation of particle-laden macrophages were found in one animal in the mid-dose NM-103 treated group.
2. Recovery day 45
A very slight subepithelial accumulation of particle-laden macrophages were found in one animal in the mid-dose and 3 animals in the high-dose NM-103 treated group.
3. Recovery day 94
A very slight subepithelial accumulation of particle-laden macrophages were found in 2 animals in the high-dose NM-103 treated group.

Further findings in the larynx were single events and found in only a few animals randomly distributed in the examined groups including recovery day 3, 45 and 94 as well as the clean air control group and were therefore interpreted as incidental findings. These findings were very slight epithelial hyperplasia, very slight subepithelial granulocytic infiltration, very slight chronic inflammation, very slight granulomatous inflammation, very slight and slight subepithelial/periductal mononuclear cell infiltration, very slight dilatation of the submucosal glands and very slight inspissation of plant fibers. In addition, one animal of the clean air control group showed a moderate granulomatous inflammation.

Trachea:
1. Recovery day 3
No effects were observed.
2. Recovery day 45
A very slight subepithelial accumulation of particle-laden macrophages was seen in one mid-dose and 2 high-dose NM-103 treated.
3. Recovery day 94
In detail, a very slight
subepithelial accumulation of particle-laden macrophages was seen in 2 high-dose NM-103 treated. Further findings included a slight subepithelial mixed inflammatory cell infiltration in one clean air control animal.

Lung:
1. Recovery day 3
Multifocal very slight to moderate alveolar accumulation of particle-laden macrophages was observed dose-dependently in all NM-103 (low-dose: all very slight; middose: 4/6 very slight, 2/6 slight; high-dose: 3/6 slight, 3/6 moderate) exposure groups.(Multi)focal very slight to slight bronchiolo-alveolar hyperplasia was diagnosed dose dependently in 3/6 (all very slight) mid-dose NM-103 and 6/6 (4 very slight, 2 slight) highdose NM-103 treated animals. This adaptive type of hyperplasia describes the presence of bronchiolar epithelium within alveolar ducts and adjacent alveoli. Exposure-related (multi)focal interstitial mononuclear-cell infiltration occurred in 4 mid-dose and 3 high-dose (all very slight) NM-103 treated animals. Another dose-dependent change was the interstitial accumulation of particle-laden macrophages in 3/6 (all very slight) mid-dose and 6/6 (all very slight) high-dose NM-103 animals. Very slight to slight (multi)focal interstitial fibrosis was found in 3 (all very slight) highdose NM-103 animals. In addition, an accumulation of particle-laden macrophages in the bronchus associated lymphoid tissue (BALT) was seen in all treated animals in a dose-dependent way resulting in 6/6 of the NM-103 (low-dose and mid-dose: 6/6 very slight; high dose: 4/6 very slight, 2/6 slight) group. Additionally, particles were seen dose-dependently within the epithelium located above the BALT with 3/6 (all very slight) in the mid-dose and 6/6 (all slight) in the high-dose NM-103 group.
Another dose-dependent finding consisted of intraalveolar granulocytic cell infiltration in 6/6 (allvery slight) mid-dose and 6/6 (1 very slight; 5 slight) high-dose NM-103 treated animals. Furthermore, debris and remnants of degenerated macrophages were diagnosed in this study as alveolar lipoproteinosis. This finding was also dose dependent and was observed in 2/6 (all very slight) mid-dose and 6/6 (3 veryslight, 3 slight) high-dose NM-103 treated animals.
Further pulmonary changes such as slight peribronchiolar and very slight to slight perivascular granulocytic inflammation was also found in the clean air group (finding incidental and unrelated to the particle exposure).
2. Recovery day 45
Similarly to the degree (very slight to moderate) found in the 1 day recovery group, multifocal intraalveolar accumulation of particle-laden macrophages were detected dose-dependently in all NM-103 (low-dose: all very slight; mid-dose: all slight; highdose: 4/6 slight, 2/6 moderate) animals. The bronchiolo-alveolar hyperplasia decreased slightly compared to the 1 day recovery group to a very slight degree in 2/6 high dose NM103.
The multifocal interstitial mononuclear cell infiltration increased slightly in 2/6 (all very slight) low-dose, 6/6 (all very slight) mid-dose and 6/6 (2 very slight, 4 slight) high-dose NM-103 treated animals. This change was accompanied by an interstitial accumulation of particle-laden macrophages in 2/6 (all very slight) low-dose, 6/6 (5 very slight, 1 slight) mid-dose and 6/6 (1 very slight, 5 slight) high-dose group. Very slight interstitial fibrosis was an additional finding with 2/6 mid-dose and 5/6 high-dose in NM-103 treated animals. Similarly, accumulations of particle-laden macrophages in the BALT increased slightly compared to the 1 day recovery groups (low-dose: 6/6 very slight; mid-dose: 4/6 very slight, 2/6 slight; high-dose: 4/6 slight, 2/6 moderate). Additionally, particles were seen dose-dependently within the epithelium located above the BALT with 3/6 (all very slight) in the low-dose, 4/6 (all very slight) in the mid-dose and 6/6 (all slight) in the high-dose group. Alveolar lipoproteinosis was still observed in 6/6 (all slight) high-dose animals. Moreover, intraalveolar granulocytic cell infiltration was still be seen in 6/6 (all very slight) mid-dose and 6/6 (all slight) high-dose treated animals.
One animal (high dose NM103) showed in addition to be described lesions a very slight lymphoid hyperplasia of the BALT probably due to treatment.

3. Recovery day 94
Compared to recovery day 3 and 45 the multifocal intraalveolar accumulation of particle-laden macrophages was similar in a dose-dependent way in all NM-103 (lowdose: all very slight; mid-dose: 1/6 very slight, 5/6 slight; high-dose: 5/6 slight, 1/6 moderate) treatment groups.In addition, the bronchiolo-alveolar hyperplasia stayed at a similar (very slight) degree with an incidence of 1/6 mid-dose and 3/6 high-dose NM-103 treated animals. The multifocal interstitial mononuclear cell infiltration increased partly slightly again to 3/6 (all very slight) low-dose, 6/6 (5/6 very slight, 1/6 slight) mid-dose and 6/6 (all slight) high-dose treatment goup. The change was again accompanied by a slight increase in an interstitial accumulation of particle-laden macrophages showing 4/6 (all very slight) low-dose, 6/6 (4/6 very slight, 2/6 slight) mid-dose and 6/6 (all slight) high-dose NM-103 group. The incidence of the very slight interstitial fibrosis increased slightly compared to recovery day 45 with 3/6 mid-dose and 6/6 high-dose animals. Similarly, accumulations of particle-laden macrophages in the BALT increased
slightly compared to the 45 day recovery groups (low-dose: 5/6 very slight; mid-dose: 4/6 very slight, 2/6 slight; high-dose: 3/6 slight, 3/6 moderate). Particles within the epithelium located above the BALT were still detectable in a dose-dependent way with 1/6 (very slight) in the low-dose, 6/6 (all very slight) in the
mid-dose and 6/6 (1/6 very slight, 5/6 slight) in the high-dose group. The alveolar lipoproteinosis, though decreasing slightly in intensity compared to the prior time points, was still evident in a very slight degree in 6/6 high-dose animals. Furthermore, intraalveolar granulocytic cell infiltration was still be seen in 1/6 (very slight) low-dose, 4/6 (all very slight) mid-dose and 6/6 (4/6 very slight, 2/6 slight) high-dose NM-103 treated animals.
Additional findings at this time point consisted of a very slight osseous metaplasia in a mid-dose animal (incidental finding).

Lung-associated Lymph Nodes (LALN):
1. Recovery day 3
Very slight to moderate dose-dependent accumulation of particle-laden macrophages was observed in 1/6, 2/6 and 4/6 animals of the low-, mid-, and high doe groups, respectively. Furthermore, 2/6 high-dose NM-103 and 1/6 mid-dose NM-104 animals showed an exposure-related very slight lymphoid hyperplasia.
2. Recovery day 45
The degree and incidence of accumulation of particle-laden macrophages increased compared to recovery day 3 still showing a dose-dependent manner ranging from very slight to moderate in 5/6, 6/6 and 6/6 animals of the low-, mid- and high-dose group, respectively.
3. Recovery day 94
The degree of accumulation of particle-laden macrophages further increased compared to recovery day 45 ranging from very slight to severe in 5/6, 6/6 and 6/6 animals of the low-, mid- and high-dose group, respectively. Furthermore, 1/6 high-dose animals exhibited a very slight lymphoid hyperplasia probably exposurerelated. However, one clean air control group animal showed a slight lymphoid hyperplasia of the lymph nodes.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
1) Absolute and relative wet lung weights:
- lung wet weights showed dose-dependent increases (statistically significant in the 12.13 mg/m³ and 49.70 mg/m³ dose groups).
- during the post-exposure observation period a clear recovery effect was observed. After 3 months of recovery the 12.13 mg/m³ dose group had returned to control levels whereas lungs weights in the 49.70 mg/m³ dose group were still statistically significant increased.

2) Lactic dehydrogenase, β-glucuronidase and total protein
12.13 ± 0.25 mg/m³ air:
- after 3 days of recovery: biochemical parameters in the BAL supernatant showed statistically significant increases for lactic dehydrogenase (LDH) and total protein levels.
- after 45 days of recovery: only total protein was still statistically significantly increased.
- after 94 days of recovery: dose group had returned to normal level.

49.70 ± 8.20 mg/m³ air
- after 3 days of recovery: the test item showed statistically significant increases in LDH, β-glucuronidase and total protein as compared to clean air control groups.
- dose group showed signficant increases for LDH, β-glucuronidase and total protein up to 3 months recovery.

3) Differential cell count
3.06 ± 0.18 mg/m³ air
- after 3 days of recovery: a slight inflammation reaching approx. 10 % polymorphonuclear cells was observed. After 45 and 94 days of recovery in clean air the values returned to normalisation.

12.13 ± 0.25 mg/m³ air
- after 3 days of recovery: statistically significant increases in leucocyte concentrations were observed.
- strong and severe polymorphonuclear cells levels were observed. Following the statistically significant increases in the inflammatory parameters, only a partial recovery effect was observed.

49.70 ± 8.20 mg/m³ air
- after 3, 45, and 94 days of recovery: statistically significant increases in leucocyte concentrations were observed.
- strong and severe PMN levels were observed. Following the statistically significant increases in the inflammatory parameters, only a partial recovery effect was observed.

The same tendencies can be observed evaluating the differential cell count on the basis of absolute cell numbers.

RETENTION OF TEST ITEM IN TARGET AND OTHER ORGANS
Retained test item masses predicted by the MPPD model (Ashgarian et al. 1995); National Institute RIVM, 2002; unisex; minute volume : 0.2 mL/min)* were approx. 0.2 - 1.0 - 6.0 mg/lung for the TiO2 low, mid, and high dose groups, respectively.

The following amount of test item retention in the lung was determined for NM-103:
3.06 ± 0.18 mg/m³ air: 0.4 mg/lung
12.13 ± 0.25 mg/m³ air: 1.6 mg/lung
47.70 ± 4.80 mg/m³ air: 7.0 mg/lung

During the recovery period almost no lung clearance was observed in the 47.70 ± 4.80 mg/m³ air group. In the high dose (47.70 ± 4.80 mg/m³ air) the retention half-times resulted in 373 days, i.e. t1/2 = approx. 1 year. In the 12.13 ± 0.25 mg/m³ air and 3.06 ± 0.18 mg/m³ air groups a partial and a physiological lung clearance was found, respectively.
The soluble moiety of the test item in lungs reached up to 5.5% of the total mass in the 3.06 ± 0.18 mg/m³ ai group, however, not more than 2.2% and 0.9% in the 12.13 ± 0.25 mg/m³ air and 47.70 ± 4.80 mg/m³ air dose groups, respectively.
In liver and brain, the detected amounts of TiO2 test items were generally below the limit of detection.

*Reference:
- National Institute for Public Health and the Environment (RIVM) (2002). Multiple Path Particle Dosimetry Model (MPPD v 1.0): A Model for Human and Rat Airway Particle Dosimetry. Bilthoven, The Netherlands. RIVA Report 650010030.
Details on results:
CLINICAL SIGNS:
3.06 ± 0.18 mg/m³ air, 12.13 ± 0.25 mg/m³ air, and 49.70 ± 8.20 mg/m³ air :
- generally, all animals tolerated well the exposure in all concentration groups. No clinical observations outside the normal limits were recorded.

MORTALITY:
3.06 ± 0.18 mg/m³ air, 12.13 ± 0.25 mg/m³ air, and 49.70 ± 8.20 mg/m³ air :
- all animals survived the exposure period of the study
- in the post-exposure observation period, one rat of the control group was sacrificed moribund (reason for death unknown).

BODY WEIGHTS AND WEIGHT CHANGES.
3.06 ± 0.18 mg/m³ air, 12.13 ± 0.25 mg/m³ air, and 49.70 ± 8.20 mg/m³ air :
- statistically significant changes were not observed in the treatment groups as compared to clean air controls.

GROSS PATHOLOGY FINDINGS:
- one control animal (sacrificed during the post-exposure observation period): findings observed were as follows:
epididymis: lardaceous nodule and regional lymph nodes enlarged
thymus: multiple red areas
lungs: changed consistency, solitary white areas

HISTOPATHOLOGY: NON-NEOPLASTIC:
Nasopharynx
Within the nasopharynx no pathohistological lesions were detected in all animals investigated including all animals of recovery day 3, 45 and 94.

BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
3) Tumour necrosis factor (TNF-α)
3.06 ± 0.18 mg/m³ air, 12.13 ± 0.25 mg/m³ air, and 49.70 ± 8.20 mg/m³ air:
- particles did not significantly stimulate or decrease the production of TNF-α in BAL of exposed animals compared to BAL of clean air exposed animals. None or only small changes in treated samples have been observed compared to reference samples. Most of the samples were measured close to or below the lower limit of detection. In single samples measurable amounts of the respective cytokine were detected, but the result did not show any correlation to a certain exposure scenario.

4) Analysis of reactive oxygen intermediates (ROI)
3.06 ± 0.18 mg/m³ air, 12.13 ± 0.25 mg/m³ air, and 49.70 ± 8.20 mg/m³ air:
- statistically significantly increased ROI secretions were not observed.

TEM ANALYSIS (raw data attached in section "Attached background material"):
The initial conducted measurement of the lung volumens at the different time points investigated (recovery day 3, 45 and 94) revealed a statistical significant (p < 0.01) influence of the recovery day on the lung volume.

Low-dose group:
In general in the low-dose group, particles were only detected predominantly in intraalveolar macrophages, to a lesser extent in pneumocytes type I and rarely free in the alveoli. The overall amount of particle detected was low. However, the amount of particles found in intraalveolar macrophages decreased statistically significant (p<0.001) over the time period investigated in the treatment group. The amount of particles found in pneumocytes I was to low to do a proper statistical analysis. Furthermore, particles free in the alveoli represented single events at this time point.

Mid-dose group:
In general in the mid-dose group, particles were detected predominantly in intraalveolar macrophages, to a lesser extent in pneumocytes type I and rarely freein the alveoli. In addition, as single events <1% particles were found in the interstitium free, in macrophages or cellular. The amount of particles found in intraalveolar macrophages decreased statistically significant (p=0.004) over the time period investigated in the treatment group. Furthermore, statistical analysis of the amount of particles detected in pneumocyte type I revealed a statistical significant decrease over the time (p<0.001).

High-dose group:
In general in the high-dose group, particles were detected predominantly in intraalveolar macrophages, to a lesser extent free within the alveoli, in macrophages in the interstitium, in pneumocytes type I, and rarely intracellular in the interstitium. In addition, as single events <0.1% particles were found free in the interstitium. In contrast to the low- and mid-dose group, the amount of particles found in intraalveolar macrophages did not decrease significantly over the time period investigated (recovery day 3 to 94). In addition, the amount of particles found free in the alveoli decreased significantly of the time period. The statistical analysis revealed no influence of the time point investigated on the amount of particles within the interstitium in macrophages, or intracellular.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
3.06 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: BAL
Key result
Dose descriptor:
NOAEC
Effect level:
12.13 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: observed at lung overload conditions, in exceedance of the maximum tolerated dose (MTD)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Discussion of Kinetic Data Referring to the Paper of Pauluhn (2011)

Pauluhn (2011)* has presented an approach how to predict NOAEC values for granular poorly soluble particles following inhalation. In terms of the volume-based cumulative dose in lungs, target volume loads in rat studies should be in the range of approx. 1 μL/lung (no adverse effects expected) to maximum 10 μL/lung, lest a lung clearance collapse with clearance half-times > 1 year should occur.

The lung overload threshold in rats is defined as 4.2 μL particulate matter/kg bw. Up to this value adverse effects due to particle load will not be observed for low soluble dusts without special surface reactivity (“inert dusts”). Using this value of 4.2 μL/kg bw, fractional doses/day on volume basis (unit: μL/day) can be derived, varying depending on study duration, to attain the steady state (for a 4-wk study: 17.5; for a 3-mth study: 40). The NOAEC (unit: mg/m³) can be calculated using the following equation:

NOAECpred. (mg/m³) = 1 μL x ρ (mg/μL) / 17.5 (AF) x 0.29 m³ (MV) x PMresp

pred. = predicted;

AF = accumulation factor;

MV = daily respiratory volume;

PMresp = deposition fraction (%according to MPPD model)

Calculation of NOAECpred. for the 4-week inhalation test in this study:

NOAECpred. (mg/m³) = 1 μL x ρ (mg/μL) / 17.5 (AF) x 0.29 m³ (MV) x PMresp = 1 x 1.7 / 17.5 x 0.29 x 0.07

NOAECpred. = 4.8 mg/m³

The predicted NOAEC is approx. 5 mg/m³ and is well reflected by the experimental NOAEC detected at 3 mg/m³ for the test item in this study.

*Reference:

- Pauluhn J. Poorly soluble particulates: Searching for a unifying denominator of nanoparticles and fine particles for DNEL estimation. Toxicology 279, 176-188 (2011).

Applicant's summary and conclusion

Conclusions:
The NOAEC for male rats based on findings in BAL: 3.06 ± 0.18 mg/m³ (analytical)
Key events: Data of polymorphonuclear cells (PMN) as inflammation indicator showed a slight inflammation in the low dose group reaching approx. 10% PMN. After 45 and 94 days of recovery in clean air NM-103 values of treated animals returned to normalisation. In the mid and high dose groups an increase in inflammatory effect was observed. The same tendencies can be observed evaluating the differential cell count on the basis of absolute cell numbers.

The NOAEC for male rats based on histopathological findings observed at lung overload conditions: 12.13 ± 0.25 mg/m³ (analytical)
Key events: The distribution of the intraalveolar particle-laden macrophages changed over the time period slightly from an initially more disseminated distribution to a more multifocal distribution with a higher concentration of these cells in the vicinity of the alveolar ducts. The interstitial accumulation of particle- laden macrophages was accompanied in a dose-dependent manner by a very slight interstitial fibrosis and an interstitial mononuclear cell infiltration which increased slightly during the time period investigated. In addition, a very slight bronchiolo- alveolar hyperplasia was observed in the mid- and high-dose group. This type of hyperplasia is considered to be non-preneoplastic and to represent an “attempt of the lung” to facilitate a more efficient removal of inhaled materials. Within the alveoli a lipoproteinosis and an infiltration of granulocytes were noticed in a dose dependent way

The design of repeated inhalation studies precludes such effects to occur based on adequately designed pilot studies, which are dealt with in the respective TG. Insoluble materials deposited in the alveolar region of the lung may accumulate over time with resultant impairment of particle clearance and particle-mediated inflammatory response. Hence, the lung dose accumulated over time may be decisive for the outcome of the test. Hence, mechanistic considerations need to be applied whether effects are related to an MTC, which is often the case for non-accumulating agents, or a maximum tolerated dose (MTD) which is usually the case for accumulating agents.
The objective of these inhalation study series was to reveal target organs and sensitive non-lethal endpoints characterizing toxicity, including an analysis of the entire concentration-response/effect relationship. The lowest concentration represents the no-observed-adverse-effect concentration (NOAEC), the mid concentration shows signs of slight exceedance of the maximum tolerated dose (MTD) and at the highest concentration and clear signs of the exceedance of the maximum tolerated dose were obvious.

The guideline OECD 451 for the conduct of carcinogenicity studies, in conjunction with the relevant OECD guidance document 116, highlights on various occasions that inhalation concentrations overwhelming physiological mechanisms are in exceedance of the MTD:
The guidance explicitly states that “inhalation of doses that overwhelm pulmonary clearance may lead to tissue responses that are specific to the species being tested” (section 94, p.54).
“The robustness of a carcinogenicity or chronic toxicity study, in particular the former, is dependent on a demonstration that the dose levels selected in the study are adequate to show an effect or effects of the test substance, without producing either false negative results (because the doses selected were too low) or false positive results (because metabolic/homeostatic mechanisms are overwhelmed, etc.), which may be problematic in assessing risk in humans” (section 101, page 55).

In the selection of the maximum concentration, it should be considered that “disturbances of physiology or homeostasis that would compromise the validity of the study should be considered in the dose-selection process. Examples include hypotension, inhibition of blood clotting, overwhelming normal pulmonary clearance mechanisms, immune system effects, and in some cases hormonal imbalance” (p.63).
“For substances likely to accumulate in the lung over time due to poor solubility or other properties, the degree of lung-overload and delay in clearance needs to be estimated based on adequately designed pre-studies; ideally a 90-day study with post-exposure periods long enough to encompass at least one elimination half-time. The use of concentrations exceeding an elimination half-time of approximately 1 year due to lung-overload at the end of study is discouraged” (section 135, p.71).
The study summarised and discussed above clearly shows that the mid and high dose were in slight and clear exceedance of the maximum tolerated concentration. This finding is in good accordance with the predicted concentration of 4.8 mg/m³ using Pauluhn's (2011) general approach for poorly soluble particulates, as described by the authors.
Based on this lack of guideline compliance as described above, the study is considered as not reliable.