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Key value for chemical safety assessment

Additional information

Toxicokinetic parameters such as uptake, distribution, metabolism and excretion form the essential toxicological profile of a substance. An approximate indication of the toxicokinetic pattern can be gained from the physico-chemical properties (solubility in solvents, log POW, hydrolytic stability) and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Thiazol Blau given below is based on the results obtained for, the following toxicological endpoints:

-          Acute oral toxicity in rats

-          Acute dermal toxicity in rats

-          In vivo skin irritation in rabbits

-          In vivo eye irritation in rabbits

-          Local lymphnode assay in mice

-          Subacute (28-day) oral toxicity in rats

-          Bacterial reverse mutation test

-          In vivo Micronucleus Test in rats

All studies were carried out according to the principles of Good Laboratory Practice and met the requirements of the OECD and EU-Guideline for the Testing of Chemicals.

Physico-chemical properties

Physical state:                     solid, microgranular powder

Empirical formula:               C27H32ClN5O7S

Molecular weight:              606 g/mol                                                      (>500 daltons= bad absorption)

Water solubility:                 < 26.6 µg/L                                                   (= insoluble in water)

Partition coefficient:           log Po/w = 6.4                                               (<-0.4 or >5.6= bad absorption)

Surface tension:                  NA                                                                (>60 = no activity)

Vapor pressure:                  2.1E-10 hPa                                                  (= not volatile)

Atom count:                        41                                                                  (>70 = bad bioavailability)

H-bond acceptor (nON):   12                                                                   (>10= bad bioavailability)

H-bond donor (nOHNH):  1                                                                     (>5 = bad bioavailability)

Toxicological Profile

After single oral administration of Thiazol Blau at a dose level of 2000 mg/kg body weight to female rats neither deaths nor significant adverse symptoms occurred. Similarly, single dermal application of 2000 mg/kg body weight onto male and female rats produced no deaths or symptoms of systemic toxicity. Test item related blue staining of the skin was observed up to and including Day 10. The median lethal dose (LD50) of Thiazol Blau after oral and dermal administration to rats is greater than 2000 mg/kg body weight.

Thiazol Blau is not irritating to skin or eyes according to the classification criteria of Directive 2001/59/EC or Regulation (EC) No 1272/2008.

Testing for sensitizing properties of Thiazol Blau was performed in the Local Lymphnode Assay (LLNA) in female mice. No evidence of skin sensitizing properties was found.

To assess the toxicity of Thiazol Blau after repeated administration, male and female rats received the test substance at dose levels of 62.5, 250, or 1000 mg/kg body weight per day for a period of 28 days by oral gavage. 14-day recovery groups (controls and high dose animals) were included in the study.

There was no mortality. Black discoloration of the faeces was noted at all dose levels tested from Day 2 (1000 mg/kg bw/day dose level, males and females), Day 3 (250 mg/kg bw/day dose level, males and females) or Day 5 onwards (62.5 mg/kg bw/day dose level, males and females). Black discolored faeces were still visible 3 days after the last dose administration in the 1000 mg/kg bw/day recovery animals, thereafter no test item-related effects were noted until completion of the 14-day recovery period. In addition, blue urine was noted during the treatment period at 250 and 1000 mg/kg bw/day from Day 5 or Day 4 onwards, respectively, and up to and/or including Day 28. These changes were ascribed to elimination of Thiazol Blau or its metabolites through faeces and/or urine (cage side observations) and an expected staining effect. In the absence of any clinical pathology alterations or pathology findings, they were not considered to be adverse effects. The behaviour and general condition of the animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative of neurotoxicity were observed. Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats. There were no toxicologically significant changes in body weight, body weight gain or animal food consumption between the control and test item treated groups. Minor variations, on occasion attaining statistical significance, were noted in the clinical pathology parameters (haematology, coagulation, or clinical chemistry) in both main and recovery animals. However, no dose or gender-response was observed, and/or the results were within the historical range. These changes were not considered toxicologically significant. There were no macroscopic or microscopic findings, or changes in the absolute or relative organ weights that could be ascribed to Thiazol Blau-administration. No induction of micronuclei in bone marrow erythrocytes was observed, thus, there was no evidence of any genotoxic activity of the test item. Under the conditions of this study, the no observed adverse effect level (NOAEL) for Thiazol Blau is considered to be 1000 mg/kg bw/day. 

Thiazol Blau was tested negative for bacterial mutagenicity. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2 uvrA) in the presence and absence of a metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction prepared from rat and hamster liver.

Thiazol Blau has been assessed for its clastogenic and aneugenic potential in an in vivo Micronucleus Assay in the rat. This assessment was included in the 28-day repeat dose study. No induction of micronuclei in bone marrow erythrocytes was observed, thus, there was no evidence of any genotoxic activity of the test item.

Evaluation and Assessment

Based on all available data, Thiazol Blau does not exhibit a conspicuous toxicokinetic behavior. The data of the acute dermal toxicity, dermal irritation test and skin sensitization testing indicate low dermal permeability, owing to the fact that neither systemic nor irritating or sensitizing effects were observed. This is in accordance with the physico-chemical data of the test substance.

In the subacute oral toxicity study, Thiazol Blau was dissolved in an organic solvent (PEG400); hence, the low water solubility should not play a major role in the bioavailability in this study. This is confirmed by the blue discoloration of the urine during the treatment period. According to its molecular weight, partition coefficient and number of H-bond acceptors, Thiazol Blau should have a low absorption potential. According to the molecular weight, excretion of Thiazol Blau is most likely predominantly eliminated via intestine, as substances with a molecular weight above 300 g/mol are preferentially excreted via the feces in rats. However, it is most likely that the test compound is at least partly eliminated via kidneys/urine, too, as shown by the discoloration of the urine.

Taking the results of the bioaccumulation modeling into account, a significant bioaccumulation potential can most probably be excluded. This is confirmed by the fact, that with cessation of the treatment no further staining of the urine was observed. Additionally, Thiazol Blau was also not genotoxic in an in-vitro cell mutagenicity test and an in-vivo MNT test. Therefore, a metabolisation towards genotoxic structures can most probably be excluded.

Summary

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of Thiazol Blau. The data indicate that there is little or no dermal absorption. No signs of a significant systemic toxicity associated with absorption potential have been observed. Bioaccumulation of Thiazol Blau can most probably be excluded due to the available data. Based on the results of genotoxicity assays, a metabolisation towards genotoxic metabolites can also be excluded.

On the basis of the results, it is anticipated that the substance does not undergo significant metabolic activity; rather it is metabolized for excretion with little subsequent toxicity. The substance is therefore not considered to be of concern for ADME related effects.