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Neurotoxicity

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Description of key information

- rat, 28 d subacute repeated dose study, oral: NOAEL neurotoxicity >= 1000 mg/kg bw/d (GLP, OECD guideline 407; 1991)

- Neurotoxicity in mice, s.c.: NOAEL = 2 mg/kg bw / day (non-GLP, NIH conform; Xiao et al., 2000)

- Neurotoxicity in rats, stereotactic injection into the ventral mesocephalon: LOAEL = 400 ng per animal; robust rotations toward the lesioned side, tissue destruction, specific deleterious effects on dopaminergic neurons (non-GLP, according to NIH guidelines, Jackson-Lewis & Liberatore, 2000)

- Neurotoxicity in vitro (E18 mesencephalic cells) and in vivo (rats): LOAEL = 4 µl per animal; massive DA cell loss (non-GLP, non-guideline, Masalha et al., 1997)

Key value for chemical safety assessment

Additional information

There are reliable studies available to assess the potential of the substance for neurotoxicity. Wilkinson (2000) discussed the studies of Masalha et al. (1997), Xiao et al. (2000), Jackson-Lewis & Liberatore (2000) and an GLP-compliant 28d study as follows: "Masalha et al. (1997) reported that the test substance exhibited "profound and specific cytotoxicity for dopaminergic neurons" in in vitro cultures of E18 mesencephalic cells obtained from fetal substantia nigra primordia. Addition to the cultures of 0.3 µl of the test substance (1 mg/ml DMSO) for three consecutive days reportedly resulted in a greater than 80% loss of dopaminergic cells as measured by a tyrosine hydroxylase (TH)-immunohistochemical technique. In contrast, non-dopaminergic cells were said to be unaffected. The researchers further claimed that stereotaxic injection in vivo in adult rats caused massive (>90%) dopaminergic cell loss but little damage to non-dopaminergic neurons. It was suggested that the test substance had a close structural resemblance to 1-Methyl-4-phenyl-pyridin (MPP+) the presumed toxic metabolite from (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) MPTP and that this was probably the reason for its PD action.

 

Xiao et al. (2000) investigated the effects of two (16h apart) subcutaneous injections of the test substance (2, 20 or 200 mg/kg) in C57BI/6 mice, the most sensitive strain to MPTP-induced PD. They measured the number of TH-immunoreactive neurons in midbrain sections (containing the substantia nigra) and also measured the concentrations of dopamine and its metabolites dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) in striatal neurons.

The treated mice showed no mortality, no reduction in body weight and no dopaminergic neurotoxicity and there was no reduction in TH-immunoreactive neurons in brain sections taken one week after treatment. While there was no effect on striatal levels of dopamine or its metabolites at 2 mg/kg test substance, the levels of dopamine (but not dopamine metabolites) were reduced by 31 and 38%, respectively, at total cumulative doses of 40 and 400 mg/kg (i.e., two doses of 20 and 200 mg/kg, respectively).

Xiao et al. (2000) concluded that there was no evidence of test substance-induced dopaminergic toxicity under the conditions used in the study and that the observed decrease in dopamine in striatal neurons following treatment with high doses of the test substance may be due to "minor inhibition of dopamine-synthesizing enzymes" or to differences in the metabolic turnover of dopamine. The main difference from the results of Masalha et al. (1997) is clearly the fact that this study did not demonstrate any effect of the test substance in reducing dopaminergic neurons in the substantia nigra portion of the mid-brain.

 

Jackson-Lewis & Liberatore (2000) provide important new data that relate more directly to the work of Masalha et al. (1997). Indeed, Jackson- Lewis used the same technique, direct stereotaxic injection of the test substance into the left ventral mesencephalon (containing the substantia nigra) of adult rats and subsequently examined the morphology, dopaminergic response and levels of dopamine and its metabolites in appropriate sections of the brain. The responses observed with the test substance were compared with those of 6-hydroxydopamine (6-OHDA), a known dopaminergic neurotoxin.

Following injection of 6-OHDA, the rats showed a strong spontaneous rotation to the side of the injection for about 48h. This rotational effect was stimulated by amphetamine (which stimulates dopamine release) and was reversed by apomorphine (that binds to the post-synaptic dopamine receptor) resulting in the 6-OHDA-treated animals rotating away from the side of the lesion. Following exposure to the test substance, the rats rotated towards the side of the lesion with either amphetamine or apomorphine, a fact that indicates severe general nonspecific tissue damage on the side of the injection. This provides strong evidence that, contrary to the conclusions of Masalha et al. (1997), the damage caused by the test substance is not specific for dopaminergic neurons.

The non-specificity of the test substance lesion was clearly established by the dramatic cell loss in the substantia nigra on the side of the lesion and the fact that the cells lost were not limited to those that were TH-immunoreactive as had been suggested by Masalha et al. (1997). Glutamic acid dehydrogenase (GAD)- responsive neurons were also destroyed and, near the actual injection site, there was almost complete tissue destruction as indicated by release of glial fibrillary acid protein (GFAP). This was quite distinct from the effect of 6-OHDA that specifically affected only the dopaminergic neurons.

Dopamine levels in the striatum of test substance-treated rats were decreased to an extent similar to that observed following treatment with 6-OHDA. With 6-OHDA, however, the reduction of dopamine was about the same as the reduction in its metabolites. As noted previously by Xiao et al. (2000), in the case of the test substance, the ratio of the levels of dopamine metabolites (DOPAC and HVA) to dopamine was increased. Jackson-Lewis (2000) suggests that this indicates that dopamine turnover is greater in Tinuvin 123-treated animals with more of the total dopamine-derived material being in the form of the metabolites.

Jackson-Lewis concludes that, under the experimental conditions used, the test substance caused a marked cytotoxicity at the site of injection with no evidence of specific dopaminergic toxicity as suggested by Masalha ef al. (1997). She further concludes that, in her view, the test substance cannot be considered to be parkinsonian toxin.

 

A 28-day subacute rat oral toxicity study on the test item is available. The study was conducted in full compliance with the Principles of Good Laboratory Practice (GLP) and followed OECD and U.S. EPA guidelines. The test substance was administered by gavage to groups of 20/dose/sex albino rats at 0,10, 100 and 1,000 mg/kg/day for 28 consecutive days. After 28 days, 10 animals/group were sacrificed and the other 10 were allowed to recover for two weeks prior to sacrifice. No mortality occurred and no relevant clinical signs or symptoms were observed during either treatment or recovery periods; bodyweight and food intake were not different from controls. Neurological observations (including a Functional Observation) revealed no treatment-related effects and there were no neuropathological changes in the central or peripheral nervous systems. Based on an observation of extramedullary hematopoietic activity in the liver of males at 1,000 mg/kg/day, a NOEL of 100 mg/kg/day was assigned.

The neuropathological evaluation included light microscopic examination of only the controls and the high dose animals since no treatment-related changes were observed. CNS tissues examined included the forebrain, midbrain, center of cerebrum, cerebellum and pons, and medulla oblongata. The report clearly states that:

All sections [of areas of the central and peripheral nervous systems examined] presented normally distributed and well-preserved nerve cells and nerve fibers as well as the glia cells and other supporting tissues. The examination provided no evidence of treatment-related damage of the nervous system.

Following publication of the Masalha et al. (1997) report, a reexamination of the slides from this study was undertaken to confirm that there was no damage to the brain in the area of the substantia nigra. In some brains, sections were recut to improve the visibility of the substantia nigra. The results confirmed those of the earlier study that there were no neuropathological differences between control and high-dose animals and no signs of any type of brain damage.

The study designed to investigate potential neurological effects in view of the publications issued is the 28-day oral toxicity study in rats conducted by Ciba-Geigy. This study, initially reported in 1991, clearly showed that, at a high daily dose of 1000 mg/kg/day, Tinuvin 123 caused no damage to the nervous system. The recent reevaluation of the study confirmed that there was no damage to the substantia nigra area containing dopaminergic neurons and no sign of any other damage to the brain.

In addition, state-of-the-art neurological investigations were performed in the scope of the 90 -day oral toxicity study in rats.Histochemically stained sections of the brain for glutamic acid dehydrogenase (GAD) tyrosine hydroxylase (TH) and glial fibrillary acid protein (GFAP) showed no change in the pattern or distribution of positive staining when compared to controls."

Based on the scientific evidence currently available, there is no scientific evidence to support the view that the test substance should be considered a neurologically potent parkinsonian agent.

Justification for classification or non-classification

Based on the available in vivo and in vitro studies, there is no indication of a neurotoxic property of the test substance. Thus, no classification is warranted according to 67/548/EEC and EC/1272/2008, respectively.