Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
calculation (if not (Q)SAR)
Remarks:
Migrated phrase: estimated by calculation
Adequacy of study:
key study
Reliability:
other: A written assessment based on the toxicological profile of the substance.
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A written assessment of toxicokinetic behaviour is considered appropriate for the substance. The substance displays only minor toxicological effects in any of the studies proposed, and is deemed to be be not harmful for health effects. As such, it is deemed inappropriate in terms of animal welfare to conduct a toxicokinetic assessment when no harmful effects are predicted based on known toxicology. A written assessment has therefore been prepared to address this endpoint.

Data source

Materials and methods

Objective of study:
other: Assessment of toxicokinetic behaviour
Principles of method if other than guideline:
A written assessment based on the toxicological profile of the substance.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Not applicable

Test animals

Species:
other: Not applicable
Details on test animals and environmental conditions:
Not applicable

Administration / exposure

Details on exposure:
Not applicable
Duration and frequency of treatment / exposure:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
Not applicable
No. of animals per sex per dose:
Not applicable
Positive control:
Not applicable
Details on study design:
Not applicable
Details on dosing and sampling:
Not applicable
Statistics:
Not applicable

Results and discussion

Preliminary studies:
Not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable

Metabolite characterisation studies

Details on metabolites:
Not applicable

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: no bioaccumulation potential based on review of study results
Executive summary:

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 KOW, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Reactive Blue 19 given below is based on the results obtained for, the following toxicological endpoints:

 

·        Acute oral toxicity in rats

·        Acute dermal toxicity in rats (parabase form)

·        In vivo skin irritation in rabbits

·        In vivo eye irritation in rabbits

·        Skin sensitization

·        Bacterial reverse mutation test

·       In vitro mammalian cell gene mutation test (HPRT-assay)

·       In vitro chromosome aberration assay

·        In vivo micronucleus test in rats

·        Subacute oral toxicity in rats (meta- and parabase form)

·        Teratogenicity study

Allstudieswere 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:                                       Reactive Blue 19(metabase form)

CAS number:                         2580-78-1; 122392-55-6; 12640-88-9; 90598-99-5; 95145-50-9

CAS name:                              2-Anthracenesulfonic acid, 1-amino-9,10-dihydro-9,10-dioxo-4-((3-((2-(sulfooxy)ethyl)sulfonyl)phenyl)amino)-, sodium salt (1:2)

 

Name:                                       Reactive Blue 19:1 (parabase form)

CAS number:                         12225-52-4; 16102-99-1

CAS name:                              2-Anthracenesulfonic acid, 1-amino-9,10-dihydro-9,10-dioxo-4-((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)amino)-, sodium salt (1:2)

 

Physical state:                         solid, blue powder

Empirical formula:              C22H16N2Na2O11S3

Molecular weight:                626.5 g/mol                                                         (>500 daltons= bad absorption)

Water solubility:                   > 140 g/L                                                              (= soluble in water)

Partition coefficient:            log Kow ca -7                                                      (<-0.4 or >5.6 =bad absorption)

Surface tension:                     50.9 mN/m (meta); 56.2 mN/m (para)    (<60 = surface activ)

Melting point:                        > 500°C                                                                 (= not volatile)

Vapor pressure:                     2.4E-5 Pa                                                              (= not volatile)

Atom count (natoms):       38                                                                            (<70 =good bioavailability)

H-bond acceptor (nON):  13                                                                            (>10 =bad bioavailability)

H-bond donor (nOHNH):                                                                                3  (<5 =good bioavailability)

Toxicological Profile

After single oral administration of Reactive Blue 19 at dose levels of 6300, 8000, 10000, 12500 mg/kg body weight to female rats neither deaths nor significant adverse symptoms occurred at 6300 mg/kg bw. The median lethal dose (LD50) of Reactive Blue 19 after oral administration to rats was calculated to be 10135 mg/kg body weight; died animals showed ataxia, prone position, and/or lateral position. The visible skin was bluish discolored in all treated animals. Similarly, single dermal application of 2000 mg/kg body weight onto male and female rats of the parabase dye 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 Reactive Blue 19 on shaved and abraded 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 Reactive Blue 19 into the conjunctival sac of rabbit eyes did neither result in adverse systemical effects nor in significant irritation of the eye. Consequently, Reactive Blue 19 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 Reactive Blue 19 was performed in the Magnusson&Kligman test in female guinea pigs revealed no sensitizing properties of Reactive Blue 19.

To assess the toxicity of Reactive Blue 19 after repeated administration, 10 male and 10 female mixed-race healthy albino rats were dosed orally 14-times in a 21-day study. The test substance, Remazolbrillantblau R (Reactive Blue 19) was administered in aqueous solution 14-times within 21 days by gavage at a dose level of 500 mg/kg bw. All animals were weighed weekly and in 5 animals/sex, the urine and blood was analysed at start and end of the study. There was a 5-day recovery period at the end of the study prior to necropsy. Body weight gains were slightly retarded in the 500 mg/kg bw group when compared to control animals. There were no changes in behavior in substance-treated animals. There were no haematological changes. Urinalysis showed a slightly increased protein excretion. The dye was excreted via feces and urine. At necropsy, no macroscopic changes were observed. Histopathologically, substance-related changes in the kidneys were observed, however these changes were assessed not to be adverse effects. The No Observed Effect Level is 500 mg/kg bw/day.

Reaktiv-Blau F-64 357 FW (the parabase form of Reactive Blue 19) 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 hematology and clinical chemistry. Urine was red to red-brown discolored in all animals of the 250 and 1000 mg/kg test groups. 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 Reaktiv-Blau F-64 357 FW 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 toxic effect level" was considered to be 1000 mg/kg bw/day or above.

Reactive Blue 19 was tested for mutagenicity with the bacterial strains TA100, TA1535, TA1537, TA1538, TA98 of Salmonella typhimurium and Escherichia coli WP2uvrA 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. In addition, 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, Reactive Blue 19 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 extremely high solubility of the test substance in water and the low log Kow and with a molecular weight above 400 g/mol, giving evidence of a poor skin penetration rate.

The molecular weight, number of H-bond acceptors and the low log Kow indicate a bad gastroenteral absorption of the test substance; whereas according to the atom count and H-bond donors, Reactive Blue 19 should be absorbed from the gastrointestinal tract to some extent. This assumption is confirmed by the blue or red to red-brown urine staining, which was a good indication of the bioelimination of absorbed test substance. According to the molecular weight, excretion of Reactive Blue 19 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, the test results showed that the test compound is at least partly eliminated via kidneys/urine, too. Due to its high water solubility and low log Kow, Reactive Blue 19 is not bioaccumulative. This is confirmed by the results of the bioaccumulation modeling (BCFBAF v3.01), excluding a significant bioaccumulation potential of Reactive Blue 19. Additionally, Reactive Blue 19 was also not genotoxic in a mammalian in-vitro cell mutagenicity test and an in-vivo MNT 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 Reactive Blue 19. 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 19 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 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.