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Description of key information

In accordance withREAChRegulation (EC) No 1907/2006 Annex VIII section 8.8.1, a toxicokinetic study is not required as assessment of the toxicokinetic behaviour of the substance has been derived from the relevant available information.

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to thetoxicokineticsof Acid Blue 324. 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 Acid Blue 324 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 for mammalian species.

On the basis ofthese 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
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

Introduction

No ADME studies are available for Acid Blue 324. Therefore, the toxicokinetic assessment of the test substance is predicted based on its physico-chemical properties and available toxicological study data. 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 KOW, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Acid Blue 324 given below is based on the results obtained for, the following toxicological endpoints:

 

  • Acute oral toxicity in rats
  • Acute dermal toxicity in rats (RB19:1)
  • In vivo skin irritation in rabbits
  • In vivo eye irritation in rabbits
  • Skin sensitization in mice
  • Bacterial reverse mutation test
  • In vitro mammalian cell gene mutation test (HPRT-assay)
  • In vitro chromosome aberration assay (RB19)
  • In vivo micronucleus test in rats (RB19)
  • Subacute oral toxicity in rats (RB19:1)
  • Developmental toxicity study (RB19)

 

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

 

Physico-chemical properties

Name:                               Acid Blue 324

EC Number:                     274-675-7

EC Name:                         sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

CAS number:                   70571-81-2

Physical state:                  solid, dark blue powder

Empirical formula:           C22H17N3NaO6S

Molecular weight:            473.434 g/mol              (<500 daltons = good bioavailability)

Water solubility:              20 g/L                                                        (= soluble in water)

Partition coefficient:        log Kow 1.6                 (>-0.4 or <5.6 = good bioavailability)

                                          Log Kow(version 1.68 estimate): -0.46

Surface tension:               ca 56.7 mN/m                                        (<60 = surface active)

Melting point:                  decomposition                                                  (= not volatile)

Vapor pressure:                <1E-5 Pa                                                          (= not volatile)

Atom count (natoms):      32                                                (<70 = good bioavailability)

H-bond acceptor (nON):  9                                                  (<10 =good bioavailability)

H-bond donor (nOHNH): 4                                                    (<5 = good bioavailability)

 

 

Toxicological Profile

In various acute oral toxicity studies with Acid Blue 324 at a limit dose level of 5000 mg/kg body weight to male and female rats neither deaths nor significant adverse effects occurred. The median lethal dose (LD50) after oral administration to rats was determined to be above 5000 mg/kg body weight. Necropsy of the animals at the end of the observation period showed no abnormalities. Single dermal application of 2000 mg/kg body weight onto male and female rats of Reactive Blue 19:1 produced no deaths or symptoms of systemic toxicity. The median lethal dose (LD50) after dermal administration to rats is greater than 2000 mg/kg body weight.

Testing for skin irritating properties of Acid Blue 324 on shaved skin of rabbits did not lead to any signs of systemic toxicity. The test substance was not irritating according to EU guidelines.

The administration of Acid Blue 324 into the conjunctival sac of rabbit eyes did neither result in adverse systemic effects nor in significant irritation of the eye. Consequently, Acid Blue 324 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 Acid Blue 324 was performed in the Local Lymph Node Assay in mice and revealed no sensitizing properties of Acid Blue 324.

To assess the toxicity of Acid Blue 324 after repeated administration, read across with a structurally similar dye, Reactive Blue 19:1 was used. In this study, the test substance was administered orally by gavage to SPF-Wistar rats over a period of 29 days (a total of 28 applications, 7 days a week) at dose levels of 0.62.5.250 or 1000 mg kg body weight. There was no evidence for compound-related toxicity in haematology and clinical chemistry. Urine was red to red-brown discoloured in all animals of the 250 and 1000 mg/kg bw/day test groups, giving evidence of absorption, metabolisation and excretion of the test substance. Evaluation of absolute and relative organ weights showed no compound-related effects. Macroscopic and microscopic examination revealed no test compound-related adverse effects. Summarizing, the 29-day oral administration of Reactive Blue 19:1 at dose levels of 62.5, 250, and 1000 mg/kg body weight/day did not result in any test substance-related toxicity. Consequently, the “no adverse effect level” was considered to be 1000 mg/kg bw/day or above.

Acid Blue 324 was tested for mutagenicity with the bacterial strains TA100, TA1535, TA1537, TA98 and TA102 of Salmonella typhimurium and in an HPRT assay in mammalian cells (V79). The test substance is not mutagenic in these test systems either with or without exogenous metabolic activation at the dose levels investigated. Although, in the presence of metabolic activation, treatment of the bacterial strain with Acid Blue 324 resulted in increases in the number of revertant colonies with the strain TA 98 without showing any dose-dependency; hence not meeting the criteria for a positive result. In addition, the structural analogue Reactive Blue 19 was tested for clastogenicity and aneugenicity in the in vitro chromosome aberration assay (V79 cells) and the in vivo micronucleus test in the mouse. The results indicated that, under the conditions of the present studies, the test substance is not genotoxic in these test systems.

Evaluation and Assessment

Based on all available data, Acid Blue 324 does not exhibit a conspicuous toxicokinetic behaviour. 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 high solubility of the test substance in water and the molecular weight of above 400 g/mol, giving evidence of a poor skin penetration rate.

According to the modified Lipinski rule of five, Acid Blue 324 should have a rather good oral bioavailability, although no staining of urine or internal organs was reported during the acute oral toxicity study. The structural analogue has most likely a slightly lower oral bioavailability, due to its higher molecular weight and the lower log Kow. However, the staining of the urine during the repeat-dose study showed that the test substance was absorbed and metabolised and excreted via the urine. According to the molecular weight, Acid Blue 324 and its structural analogues are 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. Due to the good water solubility and low log Kow, Acid Blue 324 is not bioaccumulative. This is confirmed by the results of the bioaccumulation modelling (BCFBAF v3.01), excluding a significant bioaccumulation potential of Acid Blue 324. Additionally, Acid Blue 324 was also not genotoxic in a mammalian in-vitro cell mutagenicity test. Therefore, a metabolisation towards genotoxic structures by 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 Acid Blue 324. 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 Acid Blue 324 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 for mammalian species.

On the basis of these 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.

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