Retention of Test Items in Target and other Organs

In Table 4.8 the mean data of retained masses in lungs are summarized. Individual data are given in Appendix V.

For the chemical analysis only the 4 right lobes of each rat were available as the left lobe was needed for BAL analysis. Although separation into 2 lung halves for two endpoints is a compromise, good experience is existing at Fraunhofer ITEM regard-ing the quality of experimental results. As a homogeneous deposition in the entire lung can be expected it is a reliable practice to calculate the whole lung retention data based on the analysis in only one half of lungs.

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.

Assumption for model calculations:

Deposition fraction : approx. 0.07 ; Agglomerate density : ρAgg = approx. 1.7 g/cm³;MMAD: approx. 1 µm

In the low dose groups 0.4, 0.4 and 0.5 mg/lung, in the mid dose groups 1.6, 1.7 and 1.8 mg/lung, and in the high dose groups 7.0, 3.8 and 5.9 mg/lung were determined for NM-103, NM-104 and NM-105, respectively. For validation of the analytical method see Table 4.8. The retained masses correspond quite well to the theoretical data derived from the model and show a high similarity in the groups of the same dose, with one exception: An underproportional lower mass was detected in the NM-104 high dose group; this can be explained by a higher MMAD value in this group (1.6 µm, see Table 3.4), i.e. a stronger tendency to build agglomerates in the aerosol as compared to NM-103 and NM-105 (MMADs: approx. 0.6 µm); consequently, the deposition rate has been lower.

During the recovery period almost no lung clearance was observed in the high dose groups. In contrary, in the mid and low dose groups a partial and a physiological lung clearance was found, respectively. This reflects well the different grade of clearance retardation due to the various lung loads.

The soluble moiety of the test items in lungs - as measured by separating the par-ticulate and dissolved parts by filtration (0.2 µm pore diameter) - reached up to 5.5%

of the total mass in the low dose groups, however, not more than 2.2% and 0.9% in the mid and high dose groups, respectively. This results suggests that solubility of the test item is limited by a given maximum under the conditions of the lung ambi-ence.

In liver, the detected amounts of TiO2 test items were generally below the limit of de-tection. However, in some individual rats considerable masses of particulate test items were detectable:

 approx. 14 µg/liver in 2 rats of the NM-104 mid dose group and even 206 µg/liver in the NM-104 high dose group at day 94

 approx. 16 µg/liver at day 45 and 67 µg/liver at day 94, each in 1 rat of the NM-105 high dose group.

In brain, the detected amounts of TiO2 test items were generally below the limit of detection.

Tab. 4.8 Overview table on retention results

PARTICULATE Mean ASD RSD Mean ASD RSD Mean ASD RSD (µg/lung) (%) (µg/lung) (%) (µg/lung) (%)

Day 3 Day 45 Day 94

Clean Air Control < 5 < 5 < 5

NM-103, low 358 80 22.3 179 44 24.4 122 30 24.3 NM-103, mid 1625 248 15.3 1530 199 13.0 1107 166 15.0 NM-103, high 7081 918 13.0 7664 987 12.9 6028 510 8.5

NM-104, low 436 41 9.5 370 147 39.8 209 43 20.6 NM-104, mid 1698 189 11.1 1674 145 8.6 1344 92 6.8 NM-104, high 3782 507 13.4 3928 479 12.2 3113 211 6.8 NM-105, low 477 140 29.4 255 93 36.3 121 27 22.5 NM-105, mid 1819 180 9.9 1806 356 19.7 1345 141 10.5 NM-105, high 5879 594 10.1 6679 622 9.3 5212 844 16.2 SOLUBLE Mean ASD RSD Mean ASD RSD Mean ASD RSD

(µg/lung) (%) (µg/lung) (%) (µg/lung) (%)

Day 3 Day 45 Day 94

Clean Air Control < 2.5 < 2.5 < 2.5

NM-103, low 8.5 2.3 27.6 5.8 2.1 36.7 4.8 0.6 11.8 NM-103, mid 9.6 6.3 66.2 10.7 2.6 24.6 13.2 5.7 42.9 NM-103, high 40.0 16.6 41.5 37.0 8.1 21.8 16.9 3.2 19.0 NM-104, low 12.1 5.2 43.3 7.2 1.3 17.8 11.4 3.2 27.9 NM-104, mid 11.2 5.2 46.7 17.4 4.7 26.8 9.8 3.8 38.9 NM-104, high 34.6 5.1 14.8 16.1 3.7 22.9 17.9 8.5 47.8 NM-105, low 21.8 9.3 42.6 7.9 3.6 46.0 4.3 1.4 31.8 NM-105, mid 17.4 10.6 60.7 39.9 18.6 46.6 18.1 2.6 14.3 NM-105, high 54.0 6.5 12.0 51.8 12.2 23.6 44.4 4.4 9.8 ASD Absolute standard deviation

RSD Relative standard deviation Processing of organ samples

Samples were cut into smaller pieces and oxidized using low-temperature plasma ashing. The remaining ash was put into 25 ml water (Milli-Q) and shaken until homogeneity. After a further 30-min period the particle sus-pension was filtrated using a 0.2 µm Whatman-Nuclepore filter (pore filter); then the filter was rinsed with 25 ml additional water. Recoveries: Plasma ashing - Ionic Ti: 103-105%; Filtration - Ionic Ti: 96%; Chemical analysis - QC standards: 101%; NBS SRM 349: 102%

Fig. 4.10 Retained masses in lungs (µg/lung)

Tab. 4.9 Juxtaposition of particulate vs. soluble moieties of the test items (lungs, in µg/lung)

Day 3 Day 45 Day 94

P S T S P S T S P S T S

(%) (%) (%)

Control < 5 < 2.5 < 5 < 2.5 < 5 < 2.5

NM-103,

low 358 8.5 366 2.3 179 5.8 185 3.1 122 4.8 135 3.5 NM-103,

mid 1625 9.6 1635 0.6 1530 10.7 1541 0.7 1107 13.2 1120 1.2 NM-103,

high 7081 40.0 7121 0.6 7664 37.0 7701 0.5 6028 16.9 6045 0.3

NM-104,

low 436 12.1 448 2.7 370 7.2 377 1.9 209 11.4 220 5.5 NM-104,

mid 1698 11.2 1710 0.7 1674 17.4 1691 1.0 1344 9.8 0.7 NM-104,

high 3782 34.6 3817 0.9 3928 16.1 3944 0.4 2860 17.9 2878 0.6

NM-105,

low 477 21.8 499 4.4 255 7.9 262 3.0 121 4.3 125 3.5 NM-105,

mid 1819 17.4 1836 0.9 1806 39.9 1846 2.2 1345 18.1 1363 1.3 NM-105,

high 5879 54.0 5933 0.9 6679 51.8 6731 0.8 5527 44.4 5531 0.8

P Particulate moiety

S Soluble moiety

T Total: Particulate + soluble moiety S (%) Soluble moiety in percent of total mass

Fig. 4.11 Kinetic data shown for NM-103, NM-104 and NM-105 (lung burdens given with linear y axis)

Fig. 4.12 Kinetic data shown for NM-103, NM-104 and NM-105 (lung burden given with logarithmical y axis)

Figures 4.11 and 4.12 illustrate the kinetic data of the 3 time-points day 3, day 45 and day 94 after end of exposure presenting the lung burdens (unit: µg/lung) on lin-ear and logarithmical scale vs. study duration, each for 103, 104 and NM-105. From the calculated retention half-times a non-overload, a slight overload and a moderate overload can be deduced for the low, mid and high dose groups.

Tab. 4.10 Retention half-times during a 90-day recovery period Half-times (days) NM-103 NM-104 NM-105

Low dose 59 85 46

Mid dose 162 267 204

High dose 373 315 485

In the high doses the retention half-times resulted in 373, 315 and 485 days, i.e. val-ues close to approx. 1 year for NM-103 and NM-104. For NM-105 a higher t1/2 = 1.3 years was calculated. The t1/2 data are within a range recommended by Pauluhn (2011) as upper limit. Beyond the 1 year threshold of lung retention half-time toxico-logical findings may be detected that are not longer relevant for occupational expo-sure scenarios.