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Hematurrea

Three studies were performed to investigate for hematurea in the rat (Cyanamid, 1975). In the first study, doses of 6000 and 18000 ppm were fed for 20, 28 or 35 days. Hematurea, proteinurea and crystallurea were observed only in treated animals and not in control animals. As a follow-up study, a purified sample and the original test substance were applied in the feed at concentrations of 2000, 6000 and 18000 ppm to each four male rats for 21 days. This time, occult blood, hematurea, proteinurea and crystallurea were observed both in control group animals and in treatment group animals. The incidence increase was increased at 18000 ppm and appeared to be in the range of the control group at the other dose groups.  

Endocrine activity

The potential of Benzophenone and its derivatives, including Octabenzone (BP12), for endocrine disruption was assessed in a series of in vitro and in silico studies. Based on this reliable dataset BP12 - in contrast to e.g. BP1 or BP2 - possesses no potency for endocrine disruption, i.e. no estrogenic, androgenic or antiandrogenic activity.

Additional information

Haematurea

Whereas no effects on kidney or urine parameters were observed in the subacute gavage study in rats, repeated-dose feeding studies gave indication of adverse effects on kidney as indicated from organ weight changes and histopathology findings at doses of 2000 ppm and above (Ciba, 1968) or 18000 ppm (Cytec, 1965). Feeding of 25000 ppm for 30 days resulted in hematurea and severe kidney damage (Cytec, 1965). Urinalysis was not included in any of the feeding studies.

The results from the three studies on hematurea correlate well with the findings of the available feeding studies (American Cyanamid Company, 1975). There is a clear increase in the incidence of hematurea, proteinurea, crystallurea and occult blood after feeding of 18000 ppm for 21 days whereas the incidence at 2000 ppm is within the range of the control animals.

 

Effects on the Endocrine System

Specific comparative investigations on putative endocrine disrupting properties of Benzophenones/Octabenzone (BP12) including in silico, in vitro and combined assessments are available in the open literature. In these studies, without exception, BP12 was shown to display no estrogenic, androgenic or anti-androgenic activity (Kawamura et al., 2003, 2005; Kristensen et al., 2011; Miller et al., 2001; Nashev et al., 2010; Ogawa et al., 2006; Rabinowitz et al., 2009; Takatori et al., 2003). This lack of endocrine disrupting potency was also noted when a mammalian metabolic activation system (S9 mix) was added to an in vitro test system (Ogawa et al., 2006; Takatori et al., 2003), indicating that metabolites of BP12 similarly do not exert and do not inhibit Estrogen receptor mediated effects. Of note, the max. BP12 concentrations applied in the in vitro assays exceeded the effect concentrations of the respective receptor agonists (E2, DHT) generally by several orders of magnitude. Thus, in contrast to some other Benzophenone derivatives known to display endocrine disrupting properties, like e.g. BP1 and BP2, no reproduction (fertility) hazard was identified for BP12 when considering the available substantial weight of evidence.

 

Several studies assessed the putative endocrine disrupting properties of Benzophenones in a comparative approach by means of the yeast two-hybrid assay and/or in silico methods:

 

In a study by Kawamura et al. (2003) UV stabilizers used in food contact plastics, including Benzophenones, were tested for their estrogenic activity by the yeast two-hybrid assay. Among 11 kinds of UV stabilizers, BP3 and BP8 displayed estrogenic activity, while BP12 and the Salicylate-, Benzoate- and Benzotriazole-derivatives tested had no activity. Therefore, Benzophenone and 19 hydroxylated Benzophenone-derivatives were further studied. Of these, 15 chemicals showed estrogenic activity. The strongest activity was determined for BP1, 4 -Hydroxy-4'-chlorobenzophenone, 4 -Hydroxybenzophenone and 2,3,4 -Trihydroxybenzophenone, no activity was determined for BP12. The following structure activity relationships of Benzophenones were obtained. The activity of Benzophenones with a hydroxyl group at the 3 or 4 -position was positive and rather strong. The effects of the hydroxyl group in the Phenol moiety were in order of 4- > 3- >> 2 -position. A hydroxyl group added at the 2 -position of the 4 -hydroxylated Benzene ring enhanced the activity.

 

In a study by the same group (Kawamura et al., 2005) estrogenic and anti-androgenic activities of Benzophenone and 19 hydroxylated Benzophenone derivatives were measured in reporter gene assays using transfected human Estrogen and Androgen receptors in Chinese hamster ovary cells. Eighteen Benzophenones had estrogenic activity and seventeen also had anti-androgenic activity. In both assays, 2,4,4'-Trihydroxybenzophenone and BP2 showed the strongest activity which was comparable to the know endocrine disrupting compounds Bisphenol A or DDE. The structure-activity relationships of the estrogenic activity in this mammalian reporter gene assay were mostly similar to the yeast two-hybrid assay previously reported (Kawamura et al., 2003). Benzophenones hydroxylated at the 3 or 4 -position showed the estrogenic activity, while the others displayed negative or weakly positive activities. Moreover, a hydroxyl group added at the 2 -position of the 4 -hydroxylated Benzophenone enhanced activity, but reduced activity at the 3 -position. In contrast, different results were obtained when a hydroxyl group was added to another Benzene ring. The added hydroxyl group enhanced the activity in this reporter gene assay, but reduced it in the yeast two-hybrid assay. Results from the reporter gene assay corresponded with the in vivo uterotrophic assay. On the other hand, a hydroxyl group at the 2-position generally enhanced the anti-androgenic activity, though the effect of other hydroxyl groups was less clear. Meanwhile, these Benzophenones had no or very weak androgen agonistic activities. The target chemical BP12 displayed no estrogenic, androgenic or anti-androgenic activity at >1 x 10E-4 M under the conditions of this study.

 

A study by Kristensen et al. (2011) revealed that many putative endocrine disruptors inhibit Prostaglandin synthesis (most probably via Cycolooxygenase inhibition) in a mouse Sertoli cell line, in human primary mast cells and in ex vivo rat testis. This reduction was correlated with a reduced Testosterone production. Under the conditions of this study, the half maximal inhibitory concentrations (IC50 values) for Prostaglandin secretion from mouse Sertoli cells was highest (i.e. reflected lowest inhibitory potency) for BP12 as compared with the Benzophenone-derivatives BP4, BP7 and BP3.

 

When assessed by an in vitro yeast assay, a substantial number of phenolic additives (including Benzophenones) incorporated in a group of different products displayed slight estrogenic activity (Miller et al., 2001). The strongest activity was chiefly displayed by those compounds in which a phenolic -OH group was in a para position to some other substituents and the molecular mass fell within the range 200 -250 g/mol. BP12 with a molecular mass of 326.4 g/mol and a phenolic -OH group only in ortho position displayed no estrogenic activity under the conditions of this study.

 

For a virtual screening using chemical-feature-based pharmacophores (Nashev et al., 2010), a 3D-structural library of chemicals with proven and suspected endocrine disrupting effects was constructed. The presented approach was used to facilitate the identification of chemicals acting on a given target of interest as well as the identification of the target(s) responsible for an observed adverse effect. A novel mechanism for the previously reported endocrine effects of UV filters was proposed and it was demonstrated that some of these chemicals interfere with Androgen metabolism by inhibiting 17β-HSD enzymes. BP1 was identified as potent inhibitor of 17β-HSD3 -dependent Testosterone formation. In addition, BP1 acted as a direct Androgen receptor (AR) antagonist, suggesting that BP1 may cause synergistic effects in vivo by inhibiting Testosterone synthesis and blocking AR activation. Overall, the potency of the various Benzophenones for exerting the described inhibitory effects on Testosterone synthesis and AR activation showed very high variability ranging from marked effects (BP1) to no effects (BP12).

 

In a study by Ogawa et al. (2006), chemicals related to food contact plastics and rubbers (including Benzophenones), and their metabolites were tested for estrogenic activities using the yeast two-hybrid assay. Among the 150 chemicals tested, 10 chemicals including BP3 and BP8, their metabolites and the metabolites of 6 other chemicals with inactive parent compound displayed estrogenic activities. All of them contained a Phenol group in their chemical structure or formed one easily by hydrolysis or metabolism. However, most of the chemicals related to food contact plastics and rubbers, including BP12, and their metabolites did not show any estrogenicity under the conditions of this assay.

 

The interaction between a series of environmentally relevant chemicals (including BP6 and BP12) and models of the rat Estrogen receptors (ER) was computed and the results compared to an experimental data set of their relative binding affinities (Rabinowitz et al., 2009). The experimental data set consisted of 281 chemicals, selected from the U.S. EPA’s Toxic Substances Control Act (TSCA) inventory, that were initially screened using the rat uterine cytosolic ER-competitive binding assay. Secondary analysis, using Lineweaver-Burk plots and slope replots, was applied to confirm that only 15 of these test chemicals were true competitive inhibitors of ER binding with experimental inhibition constants (Ki) less than 100μM. Two different rapid computational docking methods were applied. Each provided a score that was a surrogate for the strength of the interaction between each ligand-receptor pair. Using the score that indicated the strongest interaction for each pair, without consideration of the geometry of binding between the toxicant and the target, all of the active molecules were discovered in the first 16 % of the chemicals. When a filter was applied on the basis of the geometry of a simplified pharmacophore for binding to the ER, the results were improved, and all of the active molecules were discovered in the first 8 % of the chemicals. In order to obtain no false negatives in the model that included the pharmacophore filter, only 8 molecules were false positives. These results indicated that molecular docking algorithms that were designed to find the chemicals that act most strongly at a receptor can efficiently separate weakly active chemicals from a library of primarily inactive chemicals. BP6 and BP12 were identified as primarily inactive chemicals in this computational molecular modeling study using the toxicant-target approach.

 

Finally, in a study by Takatori et al. (2003) the estrogenic activities of metabolites induced by means of a mammalian metabolic activation system (S9 mix) of Benzophenone, Benzophenone derivatives (BP1, BP3, BP8, BP12 and 2,3,4 -Trihydroxybenzophenone) and Benzhydrol were examined with a yeast two-hybrid screening system. After the chemicals were incubated with S9 mix prior to their incubation with the yeast strain, the S9 mix containing metabolites was tested for estrogenic activity by the yeast two-hybrid assay. With inactive S9 mix, BP1 and 2,3,4 -Trihydroxybenzophenone showed estrogenic activity; with active S9 mix, MXC, Benzophenone, BP3 and BP8 exhibited estrogenic activity. No estrogenic activity was shown for BP12 with inactive or active metabolic activation system under the conditions of this study.