Reaction mass of lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate and lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-{[4-(vinylsulfonyl)phenyl]diazenyl}naphthalene-2,7-disulfonate

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 FC 05717. 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 FC 05717 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 metabolised 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 FC 05717 given below is based on the results obtained for the following toxicological endpoints of the test substance:

·       Acute oral toxicity in rats

·       Acute dermal toxicity in rats

·       In vivo skin irritation in rabbits

·       In vivo eye irritation in rabbits

·       Skin sensitisation in guinea pigs

·       Bacterial reverse mutation test

·       In-vitro mutagenicity assay in mammalian cells

·       In-vitro UDS test

·       In-vivo micronucleus assay in mammalian cells

·       Subacute oral toxicity in rats

·       One generation study in rats

·       Developmental toxicity study in rats

Allstudieswere carried out according to the principles or in the spirit 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 FC 05717

EC number:                            401-560-2

EC name:                                lithium sodium hydrogen 4-amino-6-(5-(5-chloro-2,6-difluoropyrimidine-4-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-(sulfonatooxy)ethylsulfonyl) phenylazo)naphthalene-2,7-disulfonate

CAS number:                          108624-00-6

CAS name:                              2,7-Naphthalenedisulfonic acid, 4-amino-6-((5-((5-chloro-2,6 -difluoro-4-pyrimidinyl)amino)-2-sulfophenyl)azo)-5-hydroxy- 3-((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)azo)-, lithium sodium salt

Physical state:                        solid, dark blue odourless powder

Empirical formula:                C₂₈H₂₁ClF₂N₈O₁₆S₅.xLi.yNa

Molecular weight:                 959.268 - 1047.195g/mol                     (<500 daltons = good absorption)

Water solubility:                   123.8 g/L                                                  (= soluble in water)

Partition coefficient:             log Kow = -4.99                                       (>-0.4 or <5.6 = good absorption)

Surface tension:                     59.84 mN/m                                             (<60 = surface active)

Vapour pressure:                   decomposition prior to melting            (not volatile)

Atom count (natoms):           60                                                               (<70 =good bioavailability)

H-bond acceptor (nON):      24                                                               (<10 = good bioavailability)

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

Toxicological Profile

The acute oral toxicity testing of Reactive Blue FC 05717 in Wistar rats. Groups of five female and five male fasted rats received a single oral (gavage) dose of 3100 or 5000 mg/kg bw. No mortality occurred at 3100 mg/kg bw. One male (Day 2) and one female (8 h) died at 5000 mg/kg bw. Blue discoloration of stomach mucous and abdominal organs was seen in the animals which died in the course of the study. The animals killed at the end of the observation period showed no macroscopically visible changes. Clinical signs after administration of the test substance included degradation of the general condition and rough fur. Under the study conditions, the oral LD50 was found to be >5000 mg/kg bw in rats.

The acute dermal toxicity of the test substance was tested in male and female Wistar rats, which were exposed to a single dose of the test substance as a dermal application at a nominal concentration of 2000 mg/kg bw. No mortality, clinical signs and effect on body weight were observed in this study. At test end, no macroscopic lesions were found. Under the study conditions, the dermal LD50 was determined to be >2000 mg/kg bw.

No systemic toxicity was observed during testing for skin sensitising and skin or eye irritating properties of Reactive Blue FC 05717 in guinea pigs and rabbits, respectively. Reactive Blue FC 05717 proved not to be irritating to skin or eyes according to the classification criteria of Regulation (EC) No 1272/2008, but was skin sensitising in the GPMT.

The toxicity of the test substance administered once daily over a period of 28 days at dose levels up to 1000 mg/kg body weight was determined in male and female rats. The test substance did not cause any compound related mortality. No treatment-related clinical signs of toxicity were observed. There were no treatment-related adverse effects on the body weight, food consumption, haematological and clinical biochemistry examinations could be observed. All animals at 1000 mg/kg bw/day presented a blue coloration of the visible connective tissue, particularly in the skin area, dark blue kidneys and bluish gastrointestinal contents. This was attributed to the inherent colour of the test substance. Also, in the females of the highest dose, approximately 10% higher liver weight was observed which was interpreted by the authors as a sign of a non-specific adaptation due to increased metabolic activity. Based on the results, the systemic NOAEL of the test substance for the study was considered to be 1000 mg/kg bw/day.

In a modified one generation study in rats, groups of 28 male and 28 (27 in the high-dose group) female Sprague Dawley rats received the test substance orally once daily at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 10 weeks (males) and 4 weeks (females), prior to mating.Daily oral administrations of 1000 mg/kg bw/day was well tolerated in rats within the first 5 weeks of treatment, but thereafter, from Week 6, caused mortality due to dental lesions with subsequent disability of food uptake and starvation (clinical picture of dental fluorosis). This finding was time-dependent, with a threshold dose of 250 mg/kg bw/day for males, and could be related to the fluoride impurity (0.3%) of this batch tested as shown by the analysis of broken teeth. Although there was marked pigment storage of the test substance in several organs, there was no clear functional or histopathological correlation. Impairment of reproduction and fertility at high dose parental animals was primarily the result of severe dental problems. In the presence of severe dental problems at 1000 mg/kg bw/day and threshold dose of 250 mg/kg bw/day for this finding, there was no evidence of selective reproductive toxicity in rats for the test substance. Under the study conditions, the reproductive and developmental NOAELs of the test substance were both considered to be 1000 mg/kg bw/day.

In a study to investigate the effects of the test substance on embryonic and foetal development in mated Sprague-Dawley rats, the test substance was administered orally by gavage once daily from Day 6 - 19 of pregnancy at 0, 62.5, 250 or 1000 mg/kg bw/day. The treated rats were sacrificed on Day 20 of pregnancy. No mortality or substance-related clinical signs of toxicity occurred throughout the study. No substance-related adverse effects were observed at necropsy. There was a slight increase in the number of early or late conceptuses undergoing resorption (post-implantation loss), as well as a slight and not statistically significant decrease in the number of live foetuses at birth in the high dose dams. However, these numbers were within the normal ranges for this strain and, taking into consideration the normal intrauterine development of the conceptuses, considered to be of no toxicological significance. No adverse effects were observed in pup examinations. The no observed adverse effect level (NOAEL) of the test substance was considered to be 1000 mg/kg bw/day. The test substance was not teratogenic in the rat

The test substance was negative in the Ames test in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 at concentrations ofup to and including 10,000 µg/plate. In addition, Reactive Blue FC 05717 was negative in the DNA repair test (UDS) in primary hepatocytes of male rats according to OECD Guideline 482. Reactive Blue FC 05717 did not show any clastogenic or aneugenic effects in an in-vivo Micronucleus Test (MNT) in miceata single oral gavage dose of the test substance at 7500 mg/kg bw.

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce genotoxic effects in bacterial and mammalian test systems. Therefore, Reactive Blue FC 05717 is considered to harvest no genotoxic properties.

Evaluation and Assessment

Based on all available data, Reactive Blue FC 05717 does not exhibit conspicuous toxicokinetic behaviour.

Reactive Blue FC 05717 is a solid at room temperature conditions. The degree of purity of the substance is ca. 53%. The substance decomposes prior to melting; therefore a significant inhalation exposure to vapours is not expected. In view of the low n-octanol/water partition coefficient (log Kow < ‑4.99 at 21°C), systemic bioavailability after dermal exposure is not anticipated.

Reactive Blue FC 05717 has a very low acute toxicity potential. The data of the dermal irritation test indicate low dermal permeability, owing to the fact that neither systemic nor irritating 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 Blue FC 05717 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 Blue FC 05717 is absorbed from the gastrointestinal tract and is systemically available to some extent, as proved by the tissue and kidney staining. The latter being a good indication of the bioelimination of absorbed Reactive Blue FC 05717 or its metabolites. According to the molecular weight, excretion of Reactive Blue FC 05717 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.

Due to its high water solubility and low log Kow, Reactive Blue FC 05717 is not bioaccumulative. This is confirmed by the results of the bioaccumulation modelling, excluding a significant bioaccumulation potential of Reactive Blue FC 05717. Additionally, Reactive Blue FC 05717 was also not genotoxic; therefore, 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 FC 05717. 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 FC 05717 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 metabolised for excretion with little subsequent toxicity. The substance is therefore not considered to be of concern for ADME related effects.