Pentasodium 5-[4-chloro-6-[4-(2-sulfatoethylsulfonyl)phenylamino]-1,3,5-triazin-2-ylamino]-4-hydroxy-3-(1,5-disulfonatonaphthalen-2-ylazo)naphthalene-2,7-disulfonate

Endpoint summary

Description of key information

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regard to the toxicokinetics of Reactive Red 239

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 Kow, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Reactive Red 239 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

·        Bacterial reverse mutation test

·        In vivo micronucleus test

·        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:                                       Reactive Red 239/Reactive Red F-52 167 FW

CAS number:                         89157-03-9

CAS name:                              1,5-Naphthalenedisulfonic acid, 2-[2-[8-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl] sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3,6-disulfo-2-naphthalenyl]diazenyl]-, sodium salt (1:5)

Physical state:                         solid, red odourless powder

Empirical formula:              C₃₁H₁₉ClN₇Na₅O₁₉S₆

Molecular weight:                1136.3g/mol                               (>500 daltons =bad absorption)

Water solubility:                   299 g/L                                           (= soluble in water)

Partition coefficient:            log Kow< -2                               (<-0.4 or >5.6 =bad absorption)

Surface tension:                     55.6 mN/m                                   (<60 = surface active)

Vapor pressure:                     NA                                                  (not volatile)

Atom count (natoms):       64                                                     (<70 =good bioavailability)

H-bond acceptor (nON): 26                                                     (>10 =bad bioavailability)

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

Toxicological Profile

After single oral administration of Reactive Red F-52 167 FW at a dose level of 5000 mg/kg body weight to male and female rats no deaths or clinical signs other than unspecific signs of toxicity occurred. Similarly, single dermal application of 2000 mg/kg body weight onto male and female rats produced no deaths or signs of systemic toxicity. The median lethal dose (LD₅₀) of Reactive Red F-52 167 FW after oral and dermal administration to rats is greater than 5000 and 2000 mg/kg body weight, respectively.

Testing for skin irritating properties of Reactive Red F-52 167 FW in rabbits led to a minimal erythema up to 48 hours after test item administration. The administration of Reactive Red F-52 167 FW into the conjunctival sac of rabbit eyes did not result in significant irritation of the conjunctiva. Consequently, Reactive Red F-52 167 FW 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 Red F-52 167 FW was performed in the guinea pig maximisation Assay. No evidence of skin sensitizing properties was found.

Reactive Red F-52 167 FW was administered over a period of 30 days to SPF-Wistar rats at a dose of 1000 mg/kg body weight per day orally by gavage (total 29 applications, 7 days a week). In all experimental groups, the behavior and general health were checked twice daily, on weekends and holidays once daily. Body weight gain and food consumption were determined twice a week; water consumption weekly. Haematological, clinical chemistry and urinalysis tests were performed at the end of the study.

At necropsy, the animals were examined macroscopically for organ changes; the major organs were weighed and relative organ weights were calculated. The most important organs of these animals were collected and histological specimens were prepared and examined for microscopic changes. Body weights, haematology and clinical chemistry parameters, albumin and globulin values, urinalysis (pH, density) and the absolute and relative organ weights were statistically tested for differences compared to the control groups.

The 29-day administration of 1000 mg/kg bw Reactive Red F-52 167 FW resulted in no compound-related impairment of general health status or body weight gain. Feed and water consumption were also unaffected by the test substance.

The haematological investigations showed no evidence of compound-related toxicity. The evaluation of the clinical-chemical parameters showed a slight increase in serum urea levels in females; however, the values were still within the normal range. During analysis of the relative and absolute organ weights in both sexes, a statistically significant increase in absolute and relative kidney weights were noted. In male animals also the relative lung weights were increased.

The urinalysis was unremarkable and revealed no evidence of toxic effects to the animals. During evaluation of sediments, epithelial cells was observed sporadically in the 1000 mg / kg bw. dose group.

At necropsy, Reactive Red F-52 167 FW-treated animals showed a red discoloration of the connective tissue and of the kidneys.Microscopically, resorption vacuoles were found in the proximal convoluted tubules of the renal cortex; this effect was much more pronounced in females. These changes suggest a re-absorption of the substance from the primary urine and a lysosomal deposition in the tubular cells. Overall, it can be concluded that the 29-day administration of Reactive Red F-52 167 FW resulted in a deposition of the substance in the kidneys; whereupon no evidence of impaired renal function were seen in clinical chemistry.

Consequently, the 'no toxic effect level' was below 1000 mg/kg bw/day in this study.

In a second study, Reactive Red F-52 167 FW was administered over a period of 29 days, SPF-Wistar rats at dose levels of 0, 40, 200 and 1000 mg/kg body weight per day orally by gavage (total 28 applications, 7 days a week). In all experimental groups, the behaviour and general health were checked twice daily, on weekends and holidays once daily. Body weight gain and food consumption were determined twice a week; water consumption weekly. Haematological, clinical chemistry and urinalysis tests were performed at the end of the study.

At necropsy, the animals were examined macroscopically for organ changes; the major organs were weighed and relative organ weights were calculated. The most important organs of these animals were collected and histological specimens were prepared and examined for microscopic changes. Body weights, haematology and clinical chemistry parameters, albumin and globulin values, urinalysis (pH, density) and the absolute and relative organ weights were statistically tested for differences compared to the control groups.

The 28-day administration of 1000 mg/kg bw Reactive Red F-52 167 FW resulted in no compound-related impairment of general health status or body weight gain. Feed and water consumption were also unaffected by the test substance.

The haematological investigations showed no evidence of compound-related toxicity.
The evaluation of the clinical-chemical parameters resulted in lower creatinine values in females of the 1000 mg/kg bw-group. All other changes were within the normal range of the used rat strain and are therefore not related to the administration of the test substance administration. In the animals of the 200 and 1000 mg/kg bw-groups, the urine was brown-yellow to red discoloured. Otherwise, the urinalysis was normal.

In male animals at 1000mg/kg bw, the adrenal weights were increased. In females at the 200 and 1000 mg/kg bw, increases in relative liver weights and at 1000 mg/kg bw increases in relative kidney weights were observed.

The macroscopic examinations revealed reddish discoloration of various tissues and organs. The affected organs were the kidneys of the 40 mg/kg bw-group females, 200 mg/kg bw-group males, and 1000 mg/kg bw-group rats, as well as the connective and stomach tissue at 1000 mg/kg bw. In addition, in males the testes and colon were stained red.

Microscopically, dose-dependent increase in deposits of the substance in the proximal tubules of the kidneys were found in the top two dose groups (200 and 1000 mg/kg bw), which is interpreted to be a sign of reabsorption of the dye resulting in intra-cellular lysosomal storage. No other substance-related changes were observed.

In summary, in this 29-day subacute toxicity test (28 applications in 29 days)the administration of Reactive Red F-52 167 FW resulted in a dose-dependent incorporation of the substance in the proximal tubule of the kidneys without causing changes in renal morphology in animals of the 200 and 1000 mg/kg bw groups. Since theclinicalchemistry parametersrevealedalsono evidenceof impairedrenal function in the 1000 mg/kg bw-group,a toxiceffectisunlikelyin thisdosegroup.

Consequently, the 'no observed effect level' (NOEL) in this study is 200 mg/kg bw/day. However, clear signs of toxic effects were also not seen in the 1000 mg/kg bw group, which is defined as 'no observed adverse effect level' (NOAEL).

 

Reactive Red F-52 167 FW was tested for bacterial mutagenicity in two independent tests. The first experiment was carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(TA98, TA100, TA1535, TA1537 and TA1538), 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 (S9-mix). The second study was carried out in theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 andEscherichia coli(WP2 uvrA) in the presence and absence of rat and hamster S9-mix. The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S9-mix in either of these tests.

Reactive Red F-52 167 FW has been assessed for its clastogenic and aneugenic potential in an in vivo Micronucleus Assay in the mouse at a dose level of 6250 mg/kg bw. Due to the colour of the test compound urine, faeces and skin were red coloured, which proved sufficient exposure to Reactive Red F-52 167 FW and/or its metabolites. 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, Reactive Red F-52 167 FW does not exhibit a conspicuous toxicokinetic behavior.

Reactive Red F-52 167 FW is a dark red powdered solid at room temperature conditions. The degree of purity of the substance is 45 - 75%. The melting point of the substance is >320°C therefore a significant inhalation exposure to vapours is not expected. In view of the low n-octanol/water partition coefficient (log Kow <– 2 at 20°C), systemic bioavailability after dermal exposure is not anticipated.

Reactive Red F-52 167 FW has a very low acute toxicity potential. 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 good 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.

According to its atom count and H-bond donors, Reactive Red F-52 167 FW should be absorbed from the gastrointestinal tract to some extent, whereas the molecular weight, log Kow and number of H-bond acceptors indicate a low absorption of the test substance. Taking the results of the subacute oral toxicity study into account, Reactive Red F-52 167 FW is absorbed from the gastrointestinal tract and is systemically available to some extent, as proved by the red urine staining, which was a good indication of the bioelimination of absorbed Reactive Red F-52 167 FW or its metabolites. The discoloration of urine and/or skin was related to the tinctorial properties of the test substance (dye-stuff), but does not represent a toxicologically relevant finding. According to the molecular weight, excretion of Reactive Red F-52 167 FW 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 Red F-52 167 FW is not bioaccumulative. This is confirmed by the results of the bioaccumulation modelling, excluding a significant bioaccumulation potential of Reactive Red F-52 167 FW. Additionally, Reactive Red F-52 167 FW was also not genotoxic in an 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 Red F-52 167 FW. 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 Red F-52 167 FW 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.