Cuprate(4-), [2-[[[[2-hydroxy-3-sulfo-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]phenylmethyl]azo]-4-sulfobenzoato(6-)]-, sodium

Endpoint summary

Description of key information

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of Reactive Blue 220. 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 Reactive Blue 220 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.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Introduction

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 taking into account the molecular weight, the number of atoms (hydrogen bond donors and acceptors), the solubility in solvents, log K_OW, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Reactive Blue 220 given below is based on the results obtained for, the following toxicological endpoints:

 

·        Acute oral toxicity in rats

·        In vivo skin irritation in rabbits

·        In vivo eye irritation in rabbits

·        Skin sensitization

·        Bacterial reverse mutation test

·       In vitro mammalian cellchromosome aberration test

·        In vivo micronucleus test in mice

·        Developmental toxicity study in rats

Allstudieswere 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

Name:                                       Reactive Blue 220

CAS number:                         90341-71-2

CAS name:                              Cuprate(4-), (2-(2-(2-(2-(hydroxy-kappaO) -3-sulfo-5-(2-(sulfooxy) ethyl) sulfonyl) phenyl) diazenyl-kappaN2) phenylmethyl) diazenyl-kappaN1) -4-sulfobenzoato(6-) -kappaO) -, sodium

Physical state:                         solid, blue powder

Empirical formula:              C₂₂H₁₄CuN₄Na₄O₁₅S₄

Molecular weight:                858.115g/mol                             (>500 daltons= bad absorption)

Water solubility:                   > 492 g/L                                       (= soluble in water)

Partition coefficient:            log Kow < -6                                (<-0.4 or >5.6 =bad absorption)

Surface tension:                     62.0 mN/m                                   (>60 = no activity)

Vapor pressure:                     NA                                                  (= not volatile)

Atom count (natoms):       46                                                     (<70 =good bioavailability)

H-bond acceptor (nON):  19                                                     (>10 =bad bioavailability)

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

Toxicological Profile

After single oral administration of Reactive Blue 220 at a dose level of 5000 mg/kg body weight to female rats neither deaths nor significant adverse symptoms occurred. The median lethal dose (LD₅₀) of Reactive Blue 220 after oral administration to rats is greater than 5000 mg/kg body weight.

Reactive Blue 220 caused neither skin nor eye irritation in in-vivo skin or eye irritation studied in rabbits. No signs of systemic toxicity were noted in these studies.

Testing for sensitising properties of Reactive Blue 220 and for its degradation product Reactive Blue 220-OH was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN. Intradermal induction was performed using 5 % of the respective test substance in isotonic saline. Dermal induction and challenge treatment were carried out with 25 % the respective test substance in isotonic saline.The treated animals showed no clinical signs of intoxication throughout the study. Six of ten animals of the treatment group showed a positive skin response after the challenge procedure with Reactive Blue 220, butnone of the animals of the treatment group showed a positive skin response after the challenge procedure withReactive Blue 220-OH.Based on the results of this studyReactive Blue 220may cause sensitisation by skin contact; however,Reactive Blue 220-OHshowed no evidence for sensitizing properties.

In a limit test, Reactive Blue 220, dissolved in distilled water, was administered orally by stomach tube in a single daily dose of 1000 mg/kg body weight to a group of 20 pregnant female Wistar rats from the 7^th- 16^thday of pregnancy. A simultaneous control group of the same size received the vehicle without test compound. On the 21^stday of pregnancy, the dams were killed and delivered by caesarean section. The foetuses were then examined morphologically for developmental disorders. The studies showed that the repeated oral administration of Reactive Blue 220, at a dose of 1000 mg/kg body weight in the sensitive phase of organogenesis for the conceptuses did not lead to any impairment of the general physical condition of the dams or impaired intrauterine development of the conceptuses. The dams of the compound group and the control dams showed no impairment to behaviour and general condition and survived until the scheduled study end. 17 dams in the compound group excreted black-discoloured faeces, mainly in the period from day 8 - 19 of pregnancy. In the other three dams, the faeces were blue in colour from day 8 until 17 or 18 of pregnancy. Furthermore, 17 dams also voided red-discoloured urine in the period from day 8 - 16 of pregnancy. The administration of Reactive Blue 220 had no influence on the feed consumption of the dams. The dams consumed comparable amounts of feed to those of the controls both during and after the treatment period. The body weight gain of the dams treated with Reactive Blue 220 was likewise unaffected. The dams treated with the test compound gained in weight to the same extent as the controls. The morphological examination of the foetuses with regard to stage of development, outwardly detectable anomalies as well as anomalies of the internal organs and the skeleton showed no indication of an embryotoxic or teratogenic effect of the compound. On the basis of the results of this limit test, the "no observed adverse effect level" for Reactive Blue 220 in rats following oral administration lies at 1000 mg/kg body weight with regard to maternal and embryo-foetal toxicity and teratogenicity. No teratogenic effect was observed.

Reactive Blue 220 wastestedfor mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2 uvrA in the absence and in the presence of a metabolizing systemin the standard plate test. In a second test, the test substance was tested inTA 100, TA 1535, TA 1537, TA 1538 and TA 98 strains of Salmonella typhimuriuminthe standard plate test (Ames Test) and in a modified pre incubation test (Prival Test) with rat and hamster S9, respectively.A dose range of different doses from 4 µg/plate to 5000 µg/plate was used in both studies. In these tests,Reactive Blue 220 was not mutagenic with or without rat or hamster S9-mix.

Reactive Blue 220 was examined for clastogenic activity in two studies with V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix). The first study was conducted at dose levels of 200, 600 and 2000 µg/ml, which was the limit of solubility. In this test, Reactive Blue 220 showed slight clastogenic effects. The second Chromosome Aberration Assay was performed at concentrations of 500, 2500 and 5000 µg/ml. The highest concentration produced a slight decrease of the mitotic index without S9-mix at 7 and 18 h. Up to the highest investigated dose the test compound induced no significant increase in the number of chromosome aberrations. Therefore Reactive Blue 220 is considered to be not clastogenic in this chromosome aberration assay. In a further genotoxicity test, Reactive Blue 220 was tested in an in vivo micronucleus test in mice at the limit does of 5000 mg per kg bodyweight. All animals survived after application of 5000 mg per kg bodyweight. The following signs of toxicity were observed: faeces blue coloured and diarrhoea. 24 hours after application all animals were free of clinical signs of toxicity. The dissection of the animals revealed no test substance related findings.Under the conditions of the present study,Reactive Blue 220is not mutagenic in the micronucleus test. This result confirms the negative outcome of the second Chromosome Aberration Assay in vitro performed with the test substance.

It can therefore be concluded that Reactive Blue 220 is neither genotoxic, not does it form genotoxic metabolites in vitro or in vivo.

Evaluation and Assessment

Based on all available data, Reactive Blue 220 does not exhibit conspicuous toxicokinetic behaviour. The data of the dermal irritation test and skin sensitization testing indicate low dermal permeability, owing to the fact no systemic toxicity was observed in these tests. This is in accordance with the extremely low partition coefficient and the good water solubility of the test substance in water and with the molecular weight and number of H-bond acceptors, giving evidence of a poor dermal or oral systemic bioavailability.

In the oral developmental toxicity study with Reactive Blue 220 reddish discoloured urine was observed. This together with the atom count and H-bond donors, indicate that Reactive Blue 220 was absorbed from the gastrointestinal tract to some extent, whereas the molecular weight, the log Kow and number of H-bond acceptors indicate a low absorption of the test substance. According to the molecular weight, excretion of Reactive Blue 220 is most likely predominantly eliminated via intestine, as substances with a molecular weight above 300 g/mol are preferentially excreted via the faeces in rats. However, the test results showed that the test compound is at least partly eliminated via kidneys/urine, too. The good water solubility and low log Kow is a good indicator that the test item has no bioaccumulative properties. This is confirmed by the results of the bioaccumulation modelling (EpiSuite), excluding a significant bioaccumulation potential of Reactive Blue 220 . Additionally, Reactive Blue 220 was also not genotoxic in in-vitro genotoxicity tests and an in-vivo MNT test. Therefore, a metabolisation towards genotoxic structures by bacterial or mammalian species 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 Reactive Blue 220. 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 Reactive Blue 220 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.