7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

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 Orange 72/78. 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 Orange 72/78 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 Orange 72/78 given below is based on the results obtained for, the following toxicological endpoints:

 

·        Acute oral toxicity in rats

·        In vivo skin irritation in rabbits

·        In vivo eye irritation in rabbits

·        Bacterial reverse mutation test

·        In-vitro mutagenicity assay in mammalian cells

·        Subacute (14-day) oral toxicity in rats

Allstudies were 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 Orange 72/78

CAS number:                         85536-87-4

CAS name:                            2-Naphthalenesulfonic acid, 7-(acetylamino)-4-hydroxy-3-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, sodium salt

Physical state:                        solid, orange odourless powder

Empirical formula:               C₂₀H₁₉N₃O₁₁S₃.xNa / C₂₀H_(19-X)N₃Na_XO₁₁S₃

Molecular weight:                 573.564 to 617.527 g/mol                    (<500 daltons = good absorption)

Water solubility:                    248 g/L                                                         (= soluble in water)

Partition coefficient:             log Kow = -2.61                                       (>-0.4 or <5.6 = good absorption)

Surface tension:                      67.25 mN/m                                              (<60 = surface active)

Vapor pressure:                     decomposition prior melting             (not volatile)

Atom count (natoms):          37                                                                   (<70 =good bioavailability)

H-bond acceptor (nON):   14                                                                   (<10 = good bioavailability)

H-bond donor (nOHNH): 2                                                                     (<5 =good bioavailability)

Toxicological Profile

The acute oral toxicity testing of Reactive Orange 72/78 in the female Wistar rat was carried out at dose levels of 6300, 8000, 10000, and 12000 mg/kg body weight. Lethality occurred up to day 3 of the study. The animals showed prone position and disorders of balance. The test substance was excreted with feces and urine. Development of body weight was not impaired. Necropsy of the deceased animals revealed orange discolouration of organs and connective tissue. Based on the results obtained in this study the median lethal dose value (LD50) of Reactive Orange 72/78 was calculated by probit analysis for the female Wistar rat to be 8377 mg/kg body weight.

During testing for skin irritating properties of Reactive Orange 72/78 in rabbits, the skin of the animals was discoloured orange 30 - 60 minutes after removal of the patches up to the end of the study. Testing of Reactive Orange 72/78 for primary dermal irritation in the rabbit showed that the substance is not irritating to skin. The administration of Reactive Orange 72/78 into the conjunctival sac of rabbit eyes did not result in significant irritation of the conjunctiva. Consequently, Reactive Orange 72/78 is not irritating to skin or eyes according to the classification criteria of Directive 2001/59/EC or Regulation (EC) No 1272/2008.

Reactive Orange 72/78 was administered over a period of 21 days to 10 mixed race albino rats per sex at a dose level of 500 mg/kg body weight per day orally by gavage (total 14 applications, 5 days a week), followed by a 3-day post-observation time. There were no adverse effects observed in clinical signs, body weight development, urinalysis, haematology, macro- and microscopic evaluation. The test item was excreted via faeces and urine. The No Observed Effect Level is the highest administered dose, 500 mg/kg bw/day.

Reactive Orange 72/78 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA1538 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate.

The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study. In the standard Ames test, the test compound did not show a relevant increase in the number of revertants in any of the five bacterial strains either in the absence or presence of a metabolic rat liver activation system. In the Prival modification, a slight increase in the number of revertant colonies with the tester strain TA 98 (2.1 -fold) was observed in the presence of the hamster liver S9-mix at 2500 µg/plate only. This effect was only marginal, was not seen in the highest concentration and not dose dependant and despite it could also be seen in the repeat experiment was hence considered incidental. Reactive Orange 72/78 did not induce a significant increase in the number of revertant colonies in any other strains.

Hence, Reactive Orange 72/78 is not mutagenic in the standard plate test (Ames Test) and is also considered not mutagenic in the preincubation method according to Prival. The potential of Reactive Orange 72/78 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration of the pre-test on toxicity and both main experiments with metabolic activation (9100 µg/mL equal to approximately 5000 µg/mL of the pure substance) was chosen with respect to the current OECD Guideline 476 and the purity of the test item. The dose range of the experimental parts of the main experiments without metabolic activation was limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Reactive Orange 72/78 is considered to be non-mutagenic in this HPRT assay.

Evaluation and Assessment

Based on all available data, Reactive Orange 72/78 does not exhibit conspicuous toxicokinetic behaviour.

Reactive Orange 72/78 is an orange powdered solid at room temperature conditions. The degree of purity of the substance is ca. 55%. The substance decomposed 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 = ‑2.67 at 20°C), systemic bioavailability after dermal exposure is not anticipated.

Reactive Orange 72/78 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 Orange 72/78 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 Orange 72/78 is absorbed from the gastrointestinal tract and is systemically available to some extent, as proved by the urine staining, which was a good indication of the bioelimination of absorbed Reactive Orange 72/78 or its metabolites. According to the molecular weight, excretion of Reactive Orange 72/78 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 Orange 72/78 is not bioaccumulative. This is confirmed by the results of the bioaccumulation modelling, excluding a significant bioaccumulation potential of Reactive Orange 72/78. Additionally, Reactive Orange 72/78 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 Orange 72/78. 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 Orange 72/78 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.