Reddish Blue

Endpoint summary

Description of key information

Toxicokinetic Assessment

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 Reddish Blue given below is based on 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 sensitization (Local lymphnode assay in mice)

·        Bacterial reverse mutation test

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

·        In vivo micronucleus test in rats

·        Subacute (28-day) oral toxicity 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:                                       Reddish Blue

Chemical name:                    2-oxopropyl N-{5-(acetylamino)-4-[(2,4-dinitrophenyl)diazenyl]-2-methoxyphenyl}-N-benzyl-beta-alaninate

Physical state:                         solid, powder

Empirical formula:              C₂₈H₂₈N₆O₉

Molecular weight:                592.57g/mol                               (>500 daltons = bad absorption)

Water solubility:                   <50 µg/L                                       (= insoluble in water)

Partition coefficient:            log Kow = 4.8                              (<-0.4 or >5.6 = bad absorption)

Vapor pressure:                     1.11E-8Pa                                    (= not volatile)

Atom count (natoms):       43                                                     (>70 = bad bioavailability)

H-bond acceptor (nON):  15                                                     (>10 = bad bioavailability)

H-bond donor (nOHNH):                                                         1          (>5 = bad bioavailability)

Toxicological Profile

After single oral administration of Reddish Blue at a dose level of 2000 mg/kg body weight to female rats neither deaths nor significant adverse symptoms occurred. Similarly, single dermal application of 2000 mg/kg body weight onto male and female rats produced no deaths or symptoms of systemic toxicity. Test item related bluish purple staining of the treated area was observed from Day 1 up to Day 12 in all animals. The median lethal dose (LD₅₀) of Reddish Blue after oral and dermal administration to rats is greater than 2000 mg/kg body weight.

Testing for skin irritating properties of Reddish Blue in rabbits led to minimal erythema and edema (score 1) up to 48 hours after test item administration.

The administration of Reddish Blue into the conjunctival sac of rabbit eyes did not result in significant irritation of the conjunctiva. Based on the system of evaluation defined by the EEC, the following group mean scores after 24, 48 and 72 hours were calculated: conjunctiva redness: 0.44, chemosis: 0.0, discharge: 0.0; cornea opacity: 0.0, iris: 0.0. Consequently, Reddish Blue 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 Reddish Blue was performed in the Local Lymphnode Assay (LLNA) in female mice. Dose levels tested were 2.5%, 5%, and 10% in DMF. No evidence of skin sensitizing properties was found.

To assess the toxicity of Reddish Blue after repeated administration, male and female rats received the test substance at dose levels of 62.5, 250, or 1000 mg/kg body weight per day for a period of 28 days by oral gavage. 14-day recovery groups (controls and high dose animals) were included in the study. Reddish Blue administered daily by oral gavage for 28 days in Wistar rats did not result in mortality, clinical signs, changes in body weight, food consumption, coagulation parameters, clinical chemistry parameters or oestrus cycle at dose levels of 62.5, 250, or 1000 mg/kg bw/day during either the treatment or 14-day recovery/observation periods. Haematological changes considered related to treatment and suggesting a regenerative anaemia were observed in the 1000 mg/kg bw/day animals and possibly at the 250 mg/kg bw/day at the end of the treatment period although the findings were not present following the 14 day recovery period. Black discoloration of the faeces was noted at all the dose levels tested from Day 1 (1000 and 250 mg/kg bw/day dose level, males and females) or Day 2 onwards (62.5 mg/kg bw/day dose level, males and females). The discoloration of the faeces persisted in the 1000 mg/kg bw/day recovery animals for 2 days after the last dose administration (on Days 28 and 29); thereafter no test item-related effects were noted until completion of the 14-day recovery period. In addition, grey urine was noted during the treatment period at 250 and 1000 mg/kg bw/day between Days 1 and 28. These changes were ascribed to elimination of Reddish Blue or its metabolites through faeces and/or urine (cage side observations) and an expected staining effect. Macroscopic changes including small testes and/or epididymides and spleen enlargement were observed at 1000 mg/kg bw/day and correlated with the organ weight and microscopic changes. The microscopic changes in the testes consisted in bilateral diffuse mild tubular atrophy/degeneration in the testes and bilateral mild to moderate reduction of the sperm content accompanied with ductal cell debris and apoptotic bodies in the epididymides. These changes were also noted in animals following the recovery period, however with a slightly reduced severity in recovery animals, indicating a possible tendency to recover after cessation of the treatment. In the spleen, a slight increase in the severity of extramedullary haematopoiesis was noted at 1000 and 250 mg/kg bw/day and was considered correlated with the spleen response to the slight regenerative anaemia in these animals. Recovery animals showed no such effects.

Reddish Blue was tested for bacterial mutagenicity. The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(WP2 uvrA) in the presence and absence of a metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction prepared from rat liver. Biologically relevant increases in the number of revertant colonies compared to the solvent control plates were observed in all Salmonella strains with and without metabolic activation. No mutagenic activity of the test item was observed inEscherichia colitester strain.

The potential of Reddish Blue to induce gene mutations in mammalian cells was tested at the HPRT locus in Chinese hamster ovary (CHO KI) cells in vitro using concentrations up to 333 µg/mL. The test substance proved to be non-mutagenic in this test system in the presence and in the absence of a metabolic activation system (S9-mix).

Reddish Blue has been assessed for its clastogenic and aneugenic potential in an in vivo Micronucleus Assay in the rat. This assessment was included in the 28-day repeat dose study. No induction of micronuclei in bone marrow erythrocytes was observed, thus, there was no evidence of any genotoxic activity of the test item.

Evaluation and Assessment

Based on all available data, Reddish Blue 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 low solubility of the test substance in water and with the molecular weight and number of H-bond acceptors, giving evidence of a poor systemic bioavailability.

In the subacute oral toxicity study, Reddish Blue was dissolved in an organic solvent (PEG400); hence, the low water solubility should not play a major role for the bioavailability in this study. This assumption is confirmed by the urine staining, which was a good indication of the bioelimination of absorbed Reddish Blue. According to its log Kow, atom count and H-bond donors, Reddish Blue should be absorbed from the gastrointestinal tract to some extent, whereas the molecular weight and number of H-bond acceptors indicate a low absorption of the test substance. According to the molecular weight, excretion of Reddish Blue 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. The fact that elimination of the test item ceased with the last day of treatment, is a good indicator that the test item has no bioaccumulative properties. This is confirmed by the results of the bioaccumulation modeling, excluding a significant bioaccumulation potential of Reddish Blue. Additionally, Reddish Blue 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 Reddish Blue. 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 Reddish Blue 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.