Phenotyping of leukocytes from whole blood, Peyer’s patches and spleen was performed by flow
cytometry using antibodies directed against rat CD45 to identify rat leukocytes, CD103, MHCII, and
CD11b/c to quantify dendritic cells, CD4 to quantify Thelper cells, CD25 to quantify activated Thelper cells,
and FoxP3 to identify Tregulatory (Treg) cells. In the 100-day study, tissue segments of small intestine, liver,
spleen, lungs, and large intestine were fixed in formalin, and testes were fixed in modified Davidson’s
solution. Tissues from the 100-day study were fixed and processed for histopathology. Hematoxylin and
Eosin-stained sections of distal colon were evaluated for goblet cells, including the number per colonic
gland, length of colonic gland, and number per unit length of glands. In addition, segments of proximal,
mid, and distal colon were evaluated for aberrant crypt foci (ACF).
Total body weight did not change with E171 administration in either the 7- or 100-day study. Food and
water consumption were similar between groups. An increase in spleen cellularity at 5,000 ppm was
observed in the 7-day but not in the 100-day study. No significant differences were observed in any
E171 treatment groups in the 7-day or 100-day studies in the percentage of dendritic cells, CD4+,
activated CD4+ or Treg cells in Peyer’s patches, spleen, or peripheral blood, compared to control diet-fed
animals. This was also true in the rats pretreated with DMH in the 100-day study. The profile of cytokine
production was determined in plasma, sections of jejunum, and colon with no significant changes in the
cytokine profile observed in plasma or in tissues analyzed, except for IL-17A in colon (DMH+400 ppm
E171) and IL-12p70 in plasma (DMH+40 ppm E171). In addition, two rats exhibited exceptionally
elevated plasma IL-6 levels in the DMH+40 ppm E171 treatment group. In the 7-day study, T cells from
Peyer’s patches, peripheral blood, and spleen were activated using anti-CD3/anti-CD28 and cytokine
secretion was also evaluated. No significant changes in cytokine secretion were observed in any of the
E171 treatment groups compared to control.
In the 100-day study, no treatment related changes were visualized by histopathology in the small
intestine, liver, spleen, testis, or lungs. In the large intestine, one rat in the DMH+control diet group had
2 separate invasive adenocarcinomas. Further, a single rat in each of the DMH+40 ppm E171 and
DMH+400 ppm E171 dose groups had adenomas of the large intestine; however, no other animals
exhibited histologic changes in the large intestine, whether pretreated with DMH or vehicle. E171
treatment produced no significant changes in ACF or in numbers of crypts per ACF. As expected, there
were significant differences in the number of ACF in the DMH pretreated rats compared to vehicle only,
but the number of ACF were not significantly influenced by E171 treatment. Furthermore, there were
no differences between groups (even DMH vs. vehicle) in the number of goblet cells per colonic gland,
the length of the glands or in the number of goblet cells per unit length of gland.
In summary, with the exception of an increase in spleen cellularity at the 5,000 ppm E171 dose in the 7-
day study and an increase in IL-17A in colon (DMH+400 ppm E171) and IL-12p70 in plasma (DMH+40
ppm E171) in the 100-day study, we did not observe any statistically significant changes associated with
E171 administration in any other immune parameters in the small intestine, spleen, or blood, whether
administered by itself or after pretreatment with DMH. Furthermore, we did not observe
histopathologic changes related to E171 treatment in the tissues examined, including tumor formation,
ACF, goblet cells, or histologic abnormalities. Our findings support the original observations of the 2-year
bioassay (National Toxicology Program) in rats showing no evidence of a carcinogenic effect of TiO2 in
the intestine or other tissues when administered in the diet at levels of 2.5 or 5% (25,000 and 50,000
ppm). Thus, we conclude that there is minimal to no effect of E171 when administered in the diet on the
immune system and no effect on the intestinal epithelium with respect to proliferation and cancer.