2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium 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 or inhalation uptake of Basic Blue 41. Also, there is wide tissue distribution of Basic Blue 41 after oral exposure, but bioaccumulation is 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 41 is non-mutagenic in mammalian species. Therefore, biotransformation of Basic Blue 41 towards genotoxic metabolites appears to be unlikely in mammals. On the other hand, a combined repeated dose/reproductive screening study demonstrated that Basic Blue 41 chloride targets the heart and triggers inflammation and cardiomyopathy in both male and female exposed rats. The lack of blue colouring observed in the heart after necropsy also presents the possibility that metabolites of Basic Blue 41 might be involved in the adverse effects observed in the heart.

Considering its molecular weight, it is predicted that Basic Blue 41 is excreted predominantly via faeces.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

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 K_ow), 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 41 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 inhalation toxicity in rats

•In vivo skin irritation in rabbits

•In vivo eye irritation in rabbits

• Skin sensitisation in guinea pigs

• Skin sensitisation in human volunteers

• Bacterial reverse mutation test

•In vitro genotoxicity/mutagenicity assays in mammalian cells

• Combined repeated dose toxicity/reproductive-developmental screening study in rats

It should be mentioned that for a number of these toxicological endpoints, Basic Blue 41 chloride (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 41 methyl sulfate

CAS number:                                   12270-13-2

CAS name:                                         Benzothiazolium, 2-[2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]diazenyl]-6-methoxy-3-methyl-, methyl sulfate (1:1)

Physical state:                                 Blue liquid (as solely produced dissolved in acetic acid)

Empirical formula:                         C₁₉H₂₃N₄O₂S.CH₃O₄S

Molecular weight:                         482.57 g/mol                                   (< 500 daltons = good absorption)

Water solubility:                             46 g/L @ 20 °C                                 (using the chloride analogue as read-across)

Partition coefficient:                     log K_ow= -1.6 @ 20 °C                   (using the chloride analogue as read-across)

Surface tension:                             52.7 mN/m                                       (using the chloride analogue as read-across)

Melting point:                                 90 °C                                                   (using the chloride analogue as read-across)

Vapour pressure:                           1.3E-6 Pa @ 20 °C                          (using the chloride analogue as read-across)

Atom count (chromophore):    26                                              (< 70 = good bioavailability)

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

H-bond donor:                         1                                                             (< 5 = good bioavailability)

  

Name:                                                Basic Blue 41 Chloride

CAS number:                                   26850-47-5

CAS name:                                         Benzothiazolium, 2-[2-[4-[ethyl(2-hydroxyethyl)amino]pheyl]diazenyl]-6-methoxy-3methyl, chloride (1:1)

Physical state:                                 Dark olive green solid

Empirical formula:                         C₁₉H₂₃N₄O₂S.Cl

Molecular weight:                         406.93 g/mol                                   (< 500 daltons = good absorption)

Water solubility:                             46 g/L @ 20 °C                                 (soluble in water)

Partition coefficient:                     log K_ow= -1.6 @ 20 °C                   (< -0.4 or > 5.6 = poor absorption)

Surface tension:                             52.7 mN/m                                       (< 60 = surface active)                  

Melting point:                                 90 °C                                                   (= not volatile)

Vapour pressure:                           1.3E-6 Pa @ 20 °C                          (= not volatile)

 

Toxicological profile

Extrapolation of combined oral LD₅₀ from two acute oral toxicity rat studies of mixtures containing up to 20% of Basic Blue 41 methyl sulfate resulted in a calculated LD₅₀ ranging from 412 to 675 mg/kg bw. However, acute oral toxicity test using Basic Blue 41 chloride resulted in LD₅₀ of 3700 mg/kg bw in male rats and 4300 mg/kg bw in female rats with a preparation of unknown dye content. No data regarding Basic Blue 41 methyl sulfate is available for acute toxicity via the inhalation route. However, as Basic Blue 41 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. Nevertheless, there is available acute inhalation toxicity data using Basic Blue 41 chloride, which can be used in a read-across approach. Based on the results from an in vivo acute inhalation study using Basic Blue 41 chloride, the LC₅₀ value in rats (both sexes) is considered to be greater than 1.0408 mg/L.

For skin and eye irritation/corrosion, in vivo studies conducted using Basic Blue 41 (methyl sulfate or chloride form) were evaluated in a weight-of-evidence approach. Basic Blue 41 chloride was tested negative for acute dermal irritating properties in in vivo rabbit studies conducted accordingly or equivalently to OECD test guideline 404, showing that the chromophore itself does not elicit skin-irritating effects. However, in vivo rabbit studies conducted with mixtures containing up to 20% of Basic Blue 41 methyl sulfate showed different effects on the skin. One study testing Basic Blue 41 methyl sulfate in a mixture formulation containing up to 15.3% acetic acid showed irritating effects on skin, which were not fully reversible after 8 days. On the other hand, another study testing a mixture containing Basic Blue 41 methyl sulfate without acetic acid was considered as non-irritating. Two in vivo rabbit studies conducted accordingly or equivalently to OECD test guideline 405 are available for Basic Blue 41 chloride to assess the eye irritating potential. Both studies demonstrated the eye-irritating potential of Basic Blue 41 chloride. Similarly, two in vivo eye irritation rabbit studies were conducted with mixtures containing up to 20% of Basic Blue 41 methyl sulfate and reported severe eye-irritating effects.

The potential of the Basic Blue 41 chloride (90% purity) to cause skin sensitisation was assessed using the modified Buehler test (OECD 406). Based on the results obtained from this test, Basic Blue 41 chloride was considered to be a non-sensitiser. Furthermore, the skin sensitisation potential of Basic Blue 41 chloride was determined in two human repeated insult patch tests with 200 volunteers each. Both studies had negative outcome showing no visible skin changes in any subjects except one volunteer having a severe reaction. However, this reaction was attributable to one of the other tested composite materials and not to Basic Blue 41 chloride.

Genotoxicity of Basic Blue 41 was assessed in an in vitro testing battery under GLP (OECD 471, 473 and 476). Basic Blue 41 chloride was not genotoxic in an in vitro chromosome aberration test (OECD 473) but was tested positive in a bacterial reverse mutation assay (OECD 471). In an in vitro mammalian mutagenicity test (OECD 476) Basic Blue 41 methyl sulfate was tested negative. 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 41 is considered to be non-mutagenic.

Basic Blue 41 chloride (90.6% purity) was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) at dose levels of 0, 40, 80 and 160 mg/kg bw/day in 10 animals per sex/dose. Due to the toxicity observed in the high dose group, the high dose level was reduced to 120 mg/kg bw/day from day 17 onwards. Males were treated for a total of 28 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post-partum, for at least 41 days. During the study, a total of 13 high dose animals died or were sacrificed for humane reasons during the treatment period. Five mid-dose group animals died during the treatment period. Generally, clinical signs observed were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. In addition, clinical signs such as hair loss, piloerection, cannibalisation by cage mates were generally observed in a limited number of animals. No toxicological significant effects on body weight were noted throughout the study in males. Before pairing, body weight of treated females was comparable to the control. At the end of the post coitum period, the body weight of females of the high dose group was only slightly (but with statistical significance) lower than that of controls. At post-partum period, body weight of females in the mid and high dose levels were lower than that in the control group with statistical significance observed in the mid-dose group. In the recovery groups (2 weeks after end of exposure) no effect on body weight were seen. Food consumption in treated males and females, before pairing, was comparable to the control group. On day 7 post coitum, food consumption was slightly but statistically significant lower than the control in females of the mid-and high dose groups. In the recovery groups, no differences from controls were noted in food consumption. A prolonged prothrombin time was recorded in mid and high dose male animals. In the recovery groups this effect was complete reversible. Decreases of urea, creatinine, protein, albumin, globulin and calcium were observed in treated males. However, due to the absence of a dose-relationship, these findings cannot be definitively attributed to the treatment. A statistically significant decrease was observed in terminal body weight of mid-dose females when compared to controls. A significant increase in absolute and relative heart weight was noted in females of the mid-dose group. Treatment-related changes were seen in the heart of all high dose males and one surviving female, consisting in moderate to marked cardiomyopathy, which was also observed with minimal to moderate degree in males and mild to marked degree in females of the mid-dose group, while mononuclear cell infiltration associated or not with single necrosis of cardiomyocytes was also reported in low dose males and females. Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL for general toxicity cannot be determined. Thus, based on the findings from the study, the LOAEL is considered to be 40 mg/kg bw/day for systemic toxicity.

Evaluation and assessment

Results of acute and repeated toxicity studies have demonstrated that there is good oral bioavailability of Basic Blue 41 chloride upon exposure. On the other hand, based on the chemical structure and molecular weight, it is predicted that Basic Blue 41 has limited dermal bioavailability. Basic Blue 41 chloride has a low volatility (1.3E-6 Pa at 20°C) and therefore, no significant uptake of the substance via inhalation is expected. This might be further substantiated by an acute inhalation study of Basic Blue 41 chloride (up to 1.04 mg/L), which reported no treatment-related clinical signs or mortality. In addition, Basic Blue 41 is marketed as an aqueous solution containing significant concentrations of free acetic acid as stabiliser, and thus, inhalation to Basic Blue is not considered as a relevant route of human exposure.

Basic Blue 41 shows potential of wide tissue distribution as a repeated oral dose toxicity study in rats reported blue colouring of various organs throughout the body. According to the low log K_ow and the results of the repeated oral dose toxicity study, which show lack of significant colouring of organs after a 2-week recovery period, Basic Blue 41 chloride does not show potential for bioaccumulation.

Basic Blue 41 chloride was not genotoxic in an in vitro chromosome aberration test (OECD 473) but was tested positive in a bacterial reverse mutation assay (OECD 471). In an in vitro mammalian mutagenicity test (OECD 476) the Basic Blue 41 methyl sulfate was tested negative. From the overall evaluation of the available data using a weight-of-evidence approach Basic Blue 41 is considered as non-mutagenic. It can be subsequently deduced that biotransformation of Basic Blue 41 to genotoxic metabolites is unlikely to occur in mammalian species. The repeated oral dose toxicity study observed a dose-dependent increase in inflammation and cardiomyopathy in Basic Blue 41 chloride-exposed male and female rats, providing evidence that Basic Blue 41 chloride targets the heart. The lack of blue colouring observed in the heart after necropsy presents the possibility that metabolites of Basic Blue 41 chloride might be involved in the adverse effects observed in the heart.

There is no available data on the excretion of Basic Blue 41. Nevertheless, according to the molecular weight, Basic Blue 41 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.

Summary

The results of the toxicity testing battery give no concern to anticipate unusual characteristics with regard to toxicokinetics of Basic Blue 41. Physicochemical properties of this substance provide indication that there is limited absorption of Basic Blue 41 upon dermal or inhalation exposure. Acute and repeated oral toxicity studies using Basic Blue 41 chloride indicate that there is good oral bioavailability and wide tissue distribution. Bioaccumulation of Basic Blue 41 can most likely be ruled out due to the low log K_ow as well as the results from the repeated dose oral toxicity study, which showed only transient staining of organs after exposure to Basic Blue 41 chloride. Based on the results of genotoxicity assays, biotransformation of Basic Blue 41 to genotoxic metabolites appears unlikely in mammalian species. There is evidence to suggest that Basic Blue 41 or its metabolites target the heart, triggering inflammation and cardiomyopathy. There is no available data on the excretion of Basic Blue 41, but based on its molecular weight, Basic Blue 41 is most likely eliminated via faeces. Basic Blue 41 is therefore not considered to be of concern for ADME-related effects.

The information on the chloride variant is used to read-across to Basic Blue 41 methyl sulfate.