5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Endpoint summary

Description of key information

Based on physicochemical properties and data from toxicity tests, there appears to be a good oral bioavailability but limited dermal absorption of Basic Blue 159. The lack of staining in the inner organs during repeat-dose studies leads to the conclusion that the dye is rapidly metabolised into non-coloured structures leading to adaptive changes in kidneys and liver. Bioaccumulation of the test item is hence unlikely.

Results from the higher tier mammalian cell assay for mutagenicity and the overall evaluation of the available data using a weight-of-evidence approach indicate that Basic Blue 159 is non-mutagenic in mammalian species. Therefore, biotransformation of Basic Blue 159 towards genotoxic metabolites appears to be unlikely in mammals.

Considering its molecular weight, it is predicted that unmetabolised Basic Blue 159 is excreted predominantly via faeces. Whereas the metabolites are likely excreted via the kidneys.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

20

Absorption rate - inhalation (%):

100

Additional information

Introduction

Toxicokinetic parameters such as absorption, distribution, metabolism and excretion can be determined by evaluating the physicochemical properties as well as the toxicological profile of the test substance. In particular, the toxicokinetic of the test substance may be predicted using the physicochemical properties such as molecular weight, the substance’s volatility, the solubility in solvents (e.g. log Kow), etc. Furthermore, the results of standard toxicity testing of a substance may provide additional information about the substance’s bioavailability, distribution and metabolism.

 

With this mentioned, the toxicokinetics assessment of Basic Blue 159 is based on the physicochemical properties of the substance as well as 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
• Skin sensitisation in guinea pigs
• Bacterial reverse mutation test
• In vitro mutagenicity assays in mammalian cells
• In vivo micronucleaus test in mice
• Repeated dose toxicity study in rats
• Teratogenicity study in rats

       

It should be mentioned that for a number of these toxicological endpoints, Basic Blue 159 trichlorozincate (a chemical analogue of the methyl sulfate form) was used as the test material. Majority of the mentioned studies below were carried out accordingly or equivalently to the principles of Good Laboratory Practice and/or met the requirements of the OECD and EU-Guideline for the Testing of Chemicals.

Substance identity and physicochemical properties

Name:                               Basic Blue 159 methyl sulfate

EC number:                      281-589-3

EC name:                          5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

CAS number:                   83969-12-4

CAS name:                       1,3,4-Thiadiazolium, 5-(bis(1-methylethyl)amino)-2-[2-[4-(dimethylamino)phenyl]diazenyl]-3-methyl-, methyl sulfate (1:1)

Physical state:                  dark blue liquid

Empirical formula:          Salt:C₁₈H₃₀N₆O₄S₂
Chromophore:C₁₇H₂₇N₆S

Molecular weight:            Salt:458.6g/mol
Chromophore: 347.5 g/mol                  (< 500 daltons = good absorption)

Water solubility:              25.22 g/L @ 20 °C(using the ZnCl₃analogue as read-across)

Partition coefficient:        log Kow= -0.4 @ 20 °C    (using the ZnCl₃analogue as read-across)

Melting point:                  > 195 °C (decomposition)(using the ZnCl₃analogue as read-across)

Atom count (natoms):      24                                                      (< 70 = good absorption)

H-bond acceptor (nON):  6                                                        (< 10 = good absorption)

H-bond donor (nOHNH): 0                                                          (< 5 = good absorption)

 

Name:                               Basic Blue 159 trichlorozincate

EC number:                      298-265-2

EC name:                          5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium trichlorozincate(1-)

CAS number:                   93783-70-1

CAS name:                       1,3,4-Thiadiazolium, 5-[bis(1-methylethyl)amino)-2-[2-[4-(dimethylamino) phenyl]diazenyl]-3-methyl-, trichlorozincate(1-) (1:1)

Physical state:                  blue solid

Empirical formula:          Salt:C₁₇H₂₇Cl₃N₆SZn
Chromophore:C₁₇H₂₇N₆S

Molecular weight:            Salt: 519.2 g/mol
Chromophore: 347.5 g/mol                  (< 500 daltons = good absorption)

Water solubility:              25.22 g/L @ 20 °C                                       (soluble in water)

Partition coefficient:        log Kow= -0.4 @ 20 °C   (> -0.4 or < 5.6 = good absorption)

Melting point:                  > 195 °C (decomposition)                               (= not volatile)

Atom count (natoms):      24                                                      (< 70 = good absorption)

H-bond acceptor (nON):  6                                                        (< 10 = good absorption)

H-bond donor (nOHNH): 0                                                          (< 5 = good absorption)

Toxicological profile

Extrapolation of oral LD50 from an acute oral toxicity rat study containing 57.4% of Basic Blue 159 methyl sulfate resulted in a LD50 of 216 mg/kg bw (124 mg/kg bw for 100% active ingredient). Acute oral toxicity test using Basic Blue 159 trichlorozincate resulted in LD50 of 395 mg/kg bw in male rats and 275 mg/kg bw in female rats based on test material (LD50 (male) = 178 mg/kg bw; LD50 (female) = 124 mg/kg bw for 100% active ingredient). No data regarding Basic Blue 159 is available for acute toxicity via the inhalation route. However, as Basic Blue 159 methyl sulfate is marketed as an aqueous solution containing significant concentrations of free acetic acid used as stabiliser, this route of exposure is not relevant for humans and animal testing is not considered to be in accordance with animal welfare regulations. An acute dermal toxicity study in rats withBasic Blue 159 trichlorozincate in male and female rats, led to an LD50 of above 2000 mg/kg bw, leading to the assumption that Basic Blue 159 is practically not absorbed through the skin.

For skin and eye irritation/corrosion, in vivo studies conducted using Basic Blue 159 (methyl sulfate ortrichlorozincateform) were evaluated in a weight-of-evidence approach. Basic Blue 159 trichlorozincate was tested negative for acute dermal irritating properties in in vivo rabbit studies conducted according to OECD test guideline 404, showing that the chromophore itself does not elicit skin-irritating effects. Also, an in vivo rabbit study conducted with Basic Blue 159 methyl sulfate containing 57.4% dye and 26% acetic acid (pH 0.5) showed no effects on the intact rabbit skin. In in-vivo rabbit studies conducted according to OECD test guideline 405 with Basic Blue 159 (methyl sulfate ortrichlorozincateform), both salt forms demonstrated severe eye-irritating potential of Basic Blue 159. The potential of the Basic Blue 159 (94% purity - 68.4 % Basic Blue 159 ZnCl3 (CAS 93783-70-1), 25.59 % Basic Blue 159 chloride (CAS 81921-84-8)) to cause skin sensitisation was assessed using the Guinea Pig Maximisation Test according to Magnusson & Kligman (OECD 406). Based on the results obtained from this test, Basic Blue 159 was considered to be a sensitiser. No systemic effects were noted in these local toxicity studies.

Genotoxicity of Basic Blue 159 was assessed with the methyl sulfate in a bacteria reverse mutation assay (OECD 471) and an in-vivo mouse micronucleus test (OECD 474) and with the trichlorozincate in an in vitro mammalian mutagenicity test (HPRT) (OECD 476) under GLP. Basic Blue 159 was not genotoxic in the in-vitro mammalian mutagenicity test and the in-vivo mouse micronucleus test, but was tested positive in a bacterial reverse mutation assay (OECD 471). Based on the result of the higher tier mammalian cell assays and the overall evaluation of the available data in a weight-of-evidence approach Basic Blue 159 is considered to be non-mutagenic.

Basic Blue 159 trichlorozincate (86.6% purity) was tested in an oral 28-day repeat-dose toxicity study in rats at dose levels of 0, 10, 20 and 40 mg/kg bw/day in 10 animals per sex/dose. No alterations of toxicological relevance were observed in this study. Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls. No remarkable changes in terminal body weight and in abosolute/relative organs weight were noted in animals of the recovery phase. Histopathologically, in male animals of Group 4, moderate to marked hyaline droplets accumulation in kidneys and multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed, which showed complete reversibility at the end of the recovery period. These findings were considered to be adaptive changes due to and increased metabolisation of the dye and showed a complete reversibility at the end of the recovery period. Hence, the dose level of 40 mg/kg body weight/day is considered to be the No Observed Adverse Effect Level (NOAEL) in this study. The fact that no test substance specific discolouration of organs/tissues were noted during macro- and microscopic evaluation is an indicator of a fast metabolism of the dye into non-coloured metabolites.

Basic Blue 159 trichlorozincate (85.3% purity) was tested in a preliminary teratogenicity study at dose levels of 5, 20, and 80 mg/kg bw/day in 6 females per dose group and a main teratogenicity study at dose levels of 1.5, 5, and 15 mg/kg bw/day in 21 females per dose group. No teratogenic effects were seen in the preliminary and main study at dose levels up to 80 mg/kg bw/day. Maternal effects were seen at 80 mg/kg bw/day (deaths and unspecific toxic effects) leading to reduced foetal and placental weight in the offspring of the one surviving dam. As a consequence, the NOAEL for teratogenic effects could be considered to be 80 mg/kg bw/day and the NOAEL for maternal effects lies between 20 and 80 mg/kg bw/day. Again, no test item specific discolouration of organs/tissues were observed during necropsy.

Evaluation and assessment

Results of acute and repeated toxicity studies have demonstrated that there is good oral absorption of Basic Blue 159 upon exposure. On the other hand, based on the chemical structure and molecular weight, it is predicted that Basic Blue 159 has limited dermal absorption which is confirmed in the acute dermal and local toxicity studies. Basic Blue 159 has a low volatility and therefore, no significant uptake of the substance via inhalation is expected. In addition, Basic Blue 159 methyl sulfate is marketed as an aqueous solution containing significant concentrations of free acetic acid as stabiliser, and thus, inhalation to Basic Blue 159 is not considered as a relevant route of human exposure.

Basic Blue 159 is most likely rapidly metabolised in a first-past effect, as a repeated oral dose toxicity studies in rats did not report any blue colouring of inner organs or tissues. This assumption is confirmed by the adaptive organ changes observed in kidneys and liver in the oral 28-day repeat-dose study. According to the low log Kow, the lack of colouring of organs and reversibility of adaptive organ changes, Basic Blue 159 or its metabolites do not show potential for bioaccumulation.

Basic Blue 159 methyl sulfate was not genotoxic in an in vivo mouse micronucleus test but was tested positive in a bacterial reverse mutation assay. In an in vitro mammalian mutagenicity test Basic Blue 159 trichlorozincate was tested negative. From the overall evaluation of the available data using a weight-of-evidence approach Basic Blue 159 is considered as non-mutagenic. It can be subsequently deduced that biotransformation of Basic Blue 159 to genotoxic metabolites is unlikely to occur in mammalian species.

There is no available data on the excretion of Basic Blue 159. Nevertheless, according to the molecular weight, Basic Blue 159 is most likely eliminated via faeces – as it has been shown that substances with molecular weight above 300 g/mol are preferentially excreted via faeces in rats – whereas its metabolites are most likely excreted via urine.

Summary

The results of the toxicity testing battery give no concern to anticipate unusual characteristics with regard to toxicokinetics of Basic Blue 159. Physicochemical properties of this substance provide indication that there is limited absorption of Basic Blue 159 upon dermal exposure. Acute and repeated oral toxicity studies using Basic Blue 159 indicate that there is good oral absorption and rapid metabolisation. Bioaccumulation of Basic Blue 159 can most likely be ruled out due to the low log Kow as well as the results from the repeated dose oral toxicity study, which showed no staining of organs after exposure to Basic Blue 159. Based on the results of genotoxicity assays, biotransformation of Basic Blue 159 to genotoxic metabolites appears unlikely in mammalian species. There is no available data on the excretion of Basic Blue 159, but based on its molecular weight, Basic Blue 159 is most likely eliminated via faeces, whereas its metabolites are most likely excreted via urine. Basic Blue 159 is therefore not considered to be of concern for ADME-related effects.