reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: evaluation based on present data

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: assessment based on the physico-chemical and toxicological profile of the substance

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

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Objective of study:

toxicokinetics

Principles of method if other than guideline:

A written assessment based on the toxicological profile of the substance.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

EC Number:

429-070-4

EC Name:

reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

Cas Number:

214362-06-8

Molecular formula:

C26H(25-y-x)KyN5NaxO16S5 / C26H(25-y-x)KyN5NaxO19S6 / C26H(25-y-x)KyN5NaxO19S6 / C26H(25-y-x)KyN5NaxO22S7

IUPAC Name:

octapotassium octasodium 7-amino-4-hydroxy-3,8-bis(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-3,8-bis(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-3-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-8-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-8-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-3-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Radiolabelling:

no

Test animals

Species:

other: rat, rabbit, guinea pig, bacterial cells, mammalian cells,

Details on test animals or test system and environmental conditions:

Not applicable

Administration / exposure

Route of administration:

other: oral (gavage), dermal, ocular

Vehicle:

water

Details on exposure:

Not applicable

Duration and frequency of treatment / exposure:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

oral and dermal acute toxicity

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose repeat-dose

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

mid dose repeat-dose

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

low dose repeat-dpose

No. of animals per sex per dose / concentration:

Not applicable

Positive control reference chemical:

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

Main ADME results

Main ADME results 1

Type:

absorption

Results:

low oral absorption, very low dermal absorption

Main ADME results 2

Type:

distribution

Results:

distribution in blood serum, no permeation of blood-brain-barrier

Main ADME results 3

Type:

metabolism

Results:

mainly reduction of azo bond by azo-reductase, reaction (Michael addition) with GSH

Main ADME results 4

Type:

excretion

Results:

excretion mainly via faeces, partially over kidneys and urine

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:

No bioaccumulation potential based on review of study results

Executive summary:

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 Scarlet F07-0522 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 vivo eye irritation


• Skin sensitization


• Bacterial reverse mutation test


• In vitro mutagenicity test in mammalian cells


• In vivo micronucleus test


• Subacute repeat dose toxicity study


• Reproduction/developmental toxicity screening test

All studies were 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.

The test substance is a solid with a molecular weight range of 903 – 1136 g/mol. It is characterised by a high water solubility of 277 g/L and a low partition coefficient (log Kow < -4.5).

Table 2. Physicochemical properties

Endpoint	Reactive Scarlet F07-0522
Physical state	Solid
Melting point [°C]	> 300
Boiling point [°C]	> 400 735 calculated (Meissner et al.) Adapted Stein and Brown Method^X C1: 1323.59 C2 + C3: 1418.91 C4: 1514.24
Vapour pressure [Pa]	1.22E-23 at 25°C calculated using the Modified Watson Correlation Modified Grain Method^X C1: 1.45E-35 C2 + C3: 1.25E-38 C4: 1.01E-41
Partition coefficient – log Kow	< -4.5 at 20°C; spectrophotometry^†(all coloured components)
Water solubility [g/L]	277 at 20°C; spectrophotometry (all coloured components)
Properties of ionic molecules*	C1	C2	C3	C4
miLogP	-2.09	-3.36	-3.36	-4.49
TPSA	354.06	411.25	411.25	468.45
Atom count (natoms)	52	56	56	60
Molecular weight [g/mol]	820.82	899.87	899.87	978.93
H-bond acceptor (nON)	21	24	24	27
H-bond donor (nOHNH)	3	3	3	3
No of violations	2	2	2	2
No of rotatable bonds (nrotb)	15	16	16	17
volume	586.53	623.23	623.23	659.94
Bioactivity score*	C1	C2	C3	C4
GPCR ligand	-0.88	-1.51	-1.51	-2.34
Ion channel modulator	-1.89	-2.71	-2.71	-3.33
Kinase inhibitor	-1.29	-2.16	-2.16	-3.02
Nuclear receptor ligand	-1.59	-2.48	-2.48	-3.25
Protease inhibitor	-0.41	-0.93	-0.93	-1.61
Enzyme inhibitor	-1.10	-1.83	-1.83	-2.68

X   EpiSuite 4.1; † based on maximum solubilities in n-octanol and water

*    Molinspiration Cheminformatics (https://www.molinspiration.com/cgi-bin/properties)

According to the modified “Lipinski Rule of Five”, the following criteria define the bioavailability of chemical substances:

Partition coefficient:                 log Kow > -0.4 or < 5.6 good bioavailability

Molecular weight:                    < 500 daltons                good bioavailability

Atom count (natoms):               < 70                              good bioavailability

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

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

ADME prediction

Reactive Scarlet F07-0522 is a micro granulated solid at room temperature conditions. The melting point of the substance is > 300°C therefore a significant inhalation exposure to vapours is not expected. In view of the low n‑octanol/water partition coefficient, systemic bioavailability after oral exposure is very low, after dermal exposure it is not anticipated.

Evaluating the two main constituents using SwissADME (http://www.swissadme.ch) of the Swiss Institute of Bioinformatics produced the following results.

Table 3. Prediction of bioavailability using SwissADME

Prediction	Reactive Scarlet F07-0522
	C1	C2	C3	C4
Physico-chemical Properties
Formula	C26H22KN5Na2O16S5	C26H21KN5Na3O19S6	C26H21KN5Na3O19S6	C26H20K2N5Na3O22S7
Molecular weight [g/mol]	905.88	1007.92	1007.92	1126.08
No. heavy atoms	55	60	60	65
No. arom. heavy atoms	22	22	22	22
Fraction Csp3	0.15	0.15	0.15	0.15
Num. rotatable bonds	15	16	16	17
Num. H-bond acceptors	20	23	23	26
Num. H-bond donors	2	2	2	2
Molar Refractivity	174.96	182.97	182.97	190.97
TPSA [Ų]	395.93	461.51	461.51	527.09
Lipophilicity
Log Po/w (iLOGP)	-37.78	-59.8	-65.86	-79.8
Log Po/w (XLOGP3)	0.78	-0.48	-0.48	-1.73
Log Po/w (WLOGP)	7.78	7.76	7.76	7.75
Log Po/w (MLOGP)	0.69	0.34	0.34	0.02
Log Po/w (SILICOS-IT)	-1.06	-2.6	-2.6	-4.13
Consensus Log Po/w	-5.92	-10.95	-12.17	-15.58
Water Solubility
Log S (ESOL)	-5.25	-5	-5	-4.86
Solubility [mg/mL]	5.05e-03	1.00e-02	1.00e-02	1.55e-02
Class [soluble]	Moderately	Moderately	Moderately	Moderately
Log S (Ali)	-8.68	-8.74	-8.74	-8.82
Solubility [mg/mL]	1.91e-06	1.81e-06	1.81e-06	1.69e-06
Class [soluble]	Poorly	Poorly	Poorly	Poorly
Log S (SILICOS-IT)	-6.73	-5.95	-5.95	-5.16
Solubility [mg/mL]	1.69e-04	1.14e-03	1.14e-03	7.88e-03
Class [soluble]	Poorly	Moderately	Moderately	Moderately
Pharmacokinetics
GI absorption	Low	Low	Low	Low
BBB permeant	No	No	No	No
P-gp substrate	Yes	Yes	Yes	Yes
CYP1A2 inhibitor	No	No	No	No
CYP2C19 inhibitor	No	No	No	No
CYP2C9 inhibitor	No	No	No	No
CYP2D6 inhibitor	No	No	No	No
CYP3A4 inhibitor	No	No	No	No
Log Kp (skin perm) [cm/s]	-11.27	-12.79	-12.79	-14.40
Druglikeness
Lipinski	No	No	No	No
violations:	MW>500, NorO>10	MW>500, NorO>10	MW>500, NorO>10	MW>500, NorO>10
Ghose	No	No	No	No
violations:	MW>480 WLOGP>5.6 MR>130 #atoms>70	MW>480 WLOGP>5.6 MR>130 #atoms>70	MW>480 WLOGP>5.6 MR>130 #atoms>70	MW>480 WLOGP>5.6 MR>130 #atoms>70
Veber	No	No	No	No
violations:	Rotors>10, TPSA>140	Rotors>10, TPSA>140	Rotors>10, TPSA>140	Rotors>10, TPSA>140
Egan	No	No	No	No
violations:	WLOGP>5.88 TPSA>131.6	WLOGP>5.88 TPSA>131.6	WLOGP>5.88 TPSA>131.6	WLOGP>5.88 TPSA>131.6
Muegge	No	No	No	No
violations:	MW>600 TPSA>150 Rotors>15 H-acc>10	MW>600 TPSA>150 Rotors>15 H-acc>10	MW>600 TPSA>150 Rotors>15 H-acc>10	MW>600 TPSA>150 Rotors>15 H-acc>10
Bioavailability Score	0.17	0.17	0.17	0.17

Profiling of the four constituents of Reactive Scarlet F07-0522 shows that the substance has a low oral and a very low dermal bioavailability. Bioaccumulation of the substance or its metabolites in not anticipated.

By far the most predominant metabolic pathway for water-soluble azo dyes is cleavage of the azo linkage by azoreductase of the liver and extrahepatic tissue or by intestinal microflora in the body. Reactive Scarlet F07-0522 can be considered to be metabolised by azo-reductase from intestinal bacteria to the common parabase endpoint identified in metabolism studies with Reactive Black 5. Following the same route of metabolisation as Reactive Black 5 is considered to be appropriate for the substance Reactive Scarlet F07-0522 leading to 3,7,8‑triamino-4-hydroxynaphthalene-2-sulfonic acid, 2‑amino-5-[2-(sulfooxy)ethanesulfonyl]benzene-1-sulfonic acid, and [2-(4-aminobenzenesulfonyl)ethoxy]sulfonic acid as metabolites.

Evaluation of Toxicokinetics – Absorption, distribution, metabolism and excretion

Absorption

According to ECHA Guidance R.7c (ECHA (2017c)), the smaller the molecule the more easily it may be taken up via oral route. Generally, oral absorption is favoured for molecular weights below 500 g/mol. As the test substance consists of large molecules (> 800 g/mol), they will therefore be taken up at a very low rate following the oral route. This assumption is supported by the results of the acute oral toxicity study in rats, where no deaths and no clinical signs were noted at the limit dose of 2000 mg dye/kg bw. Red discolouration of faeces was noted up to two days after test substance administration. Similarly, discoloured faeces were observed in the repeat-dose study at the intermediate (200 mg/kg bw/day) and high (1000 mg/kg bw/day) dose groups from day 7 and 4, respectively, until day 3 of the recovery phase. In urine, a dose-dependent discoloration from yellow (control and low dose) through deep-yellow (intermediate dose) to red-brown (high dose) was observed, indicating that the test substance is absorbed to some amount and partly excreted via urine. This discolouration was no longer observed at the end of the 2-week recovery phase. No discolouration of internal organs or tissues were reported during necropsy, resulting in the assumption that the substance is not deposited in any organs or tissues and eliminating any concern for bioaccumulation of the test substance.

For dermal absorption, molecular weights below 100 g/mol favours dermal uptake, molecules with a weight above 500 g/mol may be too large to be absorbed (ECHA 2017c). With molecular weights of the test substance above 800 g/mol, the molecules are too large to allow absorption through skin. The high water solubility of the substances, would favour the dermal absorption rates under normal circumstances, as the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. However, the poor lipophilicity (log Kow < -1) will hinder penetration through the lipid rich environment of the stratum corneum and therefore dermal absorption is limited.

As the test substance has a low volatility (melting point above 300°C), they are not available for inhalation as a vapour. Should particles of the solid form of the substances be inhaled, absorption through the respiratory tract epithelium is not expected due to the molecule sizes.

Distribution

In general, the smaller the molecule, the wider the distribution. Based on their molecular weight distribution and the hydrophilicity it is assumed, that if absorbed, the substances were distributed within the aqueous compartment of the organism. Furthermore, an accumulation within adipose tissue can be excluded based on the low log Kow of all constituents of the test substance. No discolouration of inner tissues was noted during necropsy of the acute and repeat-dose toxicity studies.

Metabolism

The constituents of the substance are not expected to be bioavailable following oral, inhalative or dermal exposure at a relevant level as a result of their properties. Based on the results of in-vitro genotoxicity studies, it can be assumed that the substances are not enzymatically activated (toxified) during metabolism as the metabolic activated test substances showed no higher toxicity compared to the test substances without metabolic activation. Available data indicate that the substances are susceptible to hydrolysis. It is likely that during passage of the GI-tract the azo bond is reduced via azo-reductase. However, as shown in radio-labelled studies with Reactive Black 5, only the parabase ester structure is likely to be absorbed to some extent, and no adverse effects are reported for this structure.

Excretion

According to the physico-chemical properties of the substances, molecular weight and hydrophilic characteristics the main route of excretion is expected to be via faeces, as substances that are excreted in the urine tend to be water-soluble and of low molecular weight (below 300 g/mol in the rat). As seen from the dose-dependent discolouration of urine at the end of the treatment phase in the repeat-dose toxicity study, a very low amount of the substance and/or its metabolites are also excreted via the kidneys.

Conclusion

The results of of ADME prediction and basic toxicity testing give no reason to anticipate unusual characteristics with regard to the toxicokinetics of Reactive Scarlet F07-0522. The data indicate that there is little or no dermal absorption. No signs of a systemic toxicity associated with absorption through skin have been observed. Based on exposure model from AG Textilien des Bundesinstituts fur Risikobewertung (BfR), the dermal penetration rate for dyes through the skin was found to be less than 2%.

Based on physico-chemical data and the results of oral toxicity studies, Reactive Scarlet F07-0522has a limited oral bioavailability. The substance is considered to have low volatility as evident from the vapour pressure measurement and the calculated melting point of > 300°C, so the potential for the generation of inhalable forms is low. The molecular weight is higher than 500 g/mol and the chromophore is negative charged. This together with the high water solubility and low partition coefficient value, indicate the substance is not able to cross the mucous layer of the respiratory tract. Due to the high water solubility, vapours if generated/inhaled, will be trapped in the mucus of the respiratory tract, thereby further limiting the absorption. Hence, the main route of exposure of the substance, if inhaled, will be due to swallowing of particles deposited in the nose/mouth. Therefore, the bioavailability for inhalation is considered the same as for oral intake.

Bioaccumulation of Reactive Scarlet F07-0522 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.