Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. Hypothesis for the analogue approach
The target substance Direct Black 155-Na salt, CAS No. 68877-33-8, Disodium 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate (Direct Black RBB) is defined as a mono-constituent substance.

The available toxicological data on this substance are insufficient to fulfil the data requirements for a REACH Annex VIII dossier.

In order to prevent unnecessary animal testing, the occurring data gaps on toxicity studies might be filled by applying read-across from the similar substance (source) Direct Black 155-NaKLi salt, CAS No. 2196165-14-5, Sodium, potassium, lithium 4-amino-3-[{4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl]-5-hydroxy-6-[phenyldiazenyl]naphthalene-2,7-disulfonate (Direct Black RBK) which is also defined as a mono-constituent substance.

Both substances, target and source, have the same molecular structure. The only difference between the source structure Direct Black 155-NaKLi salt (CAS No. 2196165-14-5) and target Direct Black 155-Na salt (CAS No. 68877-33-8) is the counter ion.

Both substances are synthetized using the same raw materials and following the same manufacturing process. They are identical in relation to the anionic chemical structure.

CAS No. 2196165-14-5 is the result of the precipitation at the final stage of the reaction with potassium chloride and neutralization with lithium hydroxide and sodium hydroxide, whilst in CAS No. 68877-33-8 the precipitating agent is sodium chloride and the neutralization agent is sodium hydroxide.

The read-across is based on the hypothesis that the source and target substances have similar toxicological and environmental fate properties because both molecules have the following similarities:


a) Identical raw materials and manufacturing process.
b) Similar impurities, in comparable amounts.
c) Structural similarity: sulphonated molecules, aromatic rings, azo bonds.
Both dyes have identical anionic structure, the same polyaromatic structures polysulphonated, linked with azo bonds.
d) Both have the same ionic functional groups (sulphonic, amino, phenol).
The substances in a solid state are salts and in water solution at neutral pH are the same polyanions solvated with water.
e) Both have affinity to the same type of substrates/molecules.
The substances are able to be adsorbed on the same type of substance, e.g. polysaccharides (cellulose), polyphenols (lignine) and proteins.
f) Both may release by reductive cleavage the same degradation products belonging to the same family (sulphonamines, diamines), of identical size and identical physicochemical properties
g) Both substances have similar physicochemical properties.


In summary, it is considered that both substances have the same mode of action with regard to the following endpoints:

• Biodegradation
• Genetic toxicity
• Skin Sensitisation

2. Source and target chemicals (Purity / Impurities)
Read-across is possible provided that there is no impact of impurities on the toxicological properties of the target and source chemicals. Both substances have similar composition, same degree of purity and impurities are comparable structurally and in content.
The composition and impurities of the target and source substances are shown in Table 1
(see attached document in Section 13).

3. Analogue approach justification
As per available data, both substances, source and target, have similar structure, physicochemical properties, metabolism, mechanistic considerations and biological activity (predicted and empirical).
Therefore, read-across is an appropriate approach for the toxicity data gap endpoints to be filled.
3.1 Structural Similarity

Both substances, target and source, are considered structurally similar. Both are polysulphonates and consequently are polyanions. They are also polyaromatic substances and contain azo bonds. As a result of common starting materials used during their synthesis, both substances contain aromatic ring structures that contain sulfonated salt functional groups. The alkali metal salts are expected to dissociate in aqueous media and as a result the solubility of these compounds is increased.

3.2 Physicochemical Property Similarity

The physico-chemical properties of both substances are shown in Table 2.

Due to similar chemical structure, the source and target substances are similar with respect to relevant physicochemical properties. As the members of the sulfonated azo compounds group, both substances are solids (at room temperature) with low values of log Kow at expected pH in the small intestine.
In general, sulfonated azo compounds are expected to be ionized at physiological pH and over the pH ranges within the GI tract. Due to similar properties of volatility, solubility and reactivity among others for both substances, source and target, a similar bioavailability is expected.
3.3 Metabolic Similarity

The potential for metabolic reduction of the azo bond to yield aromatic amines is typically the determining factor in the genotoxic mode of action for azo type substances (Brown and De Vito 1993).
The similarity hypothesis of the analogue approach is based on the consideration that after oral intake, both azo direct dyes are metabolically reduced through the action of azoreductase of microflora in the intestine to release the related aromatic amines. The ability of the azo bond to be reduced for a particular substance is influenced by its solubility (Golka et al. 2004).
Nevertheless, some characteristics of the substance may influence the susceptible of cleavage, for example it has been noted that sulfonation of azo dyes may inhibit the release of aromatic amines (Ollgaard at al. 1998).

The source and target chemicals are structurally very close molecules and the expected metabolites via breakdown of the azo linkage are the same:
- Benzene-1,2-4-triyltriamine, EC 210-443-2, CAS 615-71-4
- P-phenylendiamine, EC 203-404-7, CAS 106-50-3
- 3,4,6-Triamino-5-hydroxynaphthalene-2,7-disulfonica cid, CAS 69762-07-8

In conclusion, the potential for both substances to undergo metabolic azo reductions to aromatic amine metabolites is regarded as similar.

3.4 Mechanistic Similarity
Certain azo dyes are mutagenic after reductive cleavage of the azo linkage to their aromatic amine metabolites. The azo linkage is the most labile portion of an azo molecule and the potential for azo compounds to become mutagens is often determined by their ability to undergo enzymatic breakdown in mammalian organisms or micro-organisms. (Brown and DeVito 1993).




Cleavage of aromatic azo bond can yield aromatic amine metabolites that can potentially bind to DNA
leading to gene mutations.

Biodegradation

As seen in Table 1, there is a clear chemical similarity between the source substance Direct Black 155-NaKLi salt and the target substance Direct Black 155-Na salt. Similarity on physicochemical properties is shown in Table 2.
The target and source substances are soluble salts that will be rapidly dissociated in water to the anionic component (identical for both substances) and the corresponding cations.
The degradation pathways, speed and degradation products are expected to be nearly identical, as the functional groups and reactivity are the same and the physico-chemical properties are very similar.
Consequently, the behaviour in regards of biodegradation is considered to be very similar for both substances and the read-across is regarded as feasible for this endpoint.

Sensitisation

The source substance and the target one have identical chemical anionic structure but vary in the cationic part.
The physicochemical properties (water solubility, molecular weight, particle size, log Know, pKa) are also similar. Therefore, a similar toxicokinetic behaviour is expected. The substances are expected to be dissociated shortly after absorption to the same anionic component and the cationic free ions (Na+, Li+ and K+ for the source substance and Na+ for the target). The absorption, distribution, degradation products and excretion will be comparable.
As a result, the sensitisation potential is assumed to be very similar, or equal concerning the anionic part of the molecule.
However, information on potential skin-sensitizing effect caused by the cationic part has to be assessed.
Potassium is the chief cation in the intracellular fluid of muscle and other cells. Potassium ion is a strong electrolyte that plays a significant role in the regulation of fluid volume and maintenance of the water-electrolyte balance. Potassium is the major cation (positive ion) inside animal cells, while sodium is the major cation outside animal cells. The concentration differences of these charged atoms cause a difference in electric potential between the inside and outside of cells, known as the membrane potential. The balance between potassium and sodium is maintained by ion pumps in the cell membrane. The cell membrane potential created by potassium and sodium ions allows the cell generates an action potential—a "spike" of electrical discharge. The ability of cells to produce electrical discharge is critical for body functions such as neurotransmission, muscle contraction, and heart function. Potassium is also an essential mineral needed to regulate water balance, blood pressure and levels of acidity.
The normal levels of potassium in the human blood are between 3.6-5.2 mmol/L. Potassium is non-sensitizer, but values of potassium higher than 5.5 mmol/L are considered critical for health, causing kidney disease and heart problems.
Sodium ion is a naturally occurring cation in the body with a blood plasma concentration of 140 mmol/L. It is excreted with the urine and does not cause any toxic effects when administered in low concentrations. Sodium is not a skin sensitizer.
Lithium is readily absorbed from the gastrointestinal tract and excreted via the kidneys. Dermal absorption of the cation is insignificant. Lithium is not a skin sensitizer and not genotoxic but is known to cause nephro- and neurotoxicity and is used as psychiatric medication (3).
As a conclusion of the evaluation, the cationic part of the substances (Na+ or Li+, K+ and Na+) is not contributing significantly to the sensitization potential. The organic part is the main driver of the sensitization potential On the other hand, with the sensitisation studies conducted on the source substance CAS No. 2196165-14-5 Direct Black 155-NaKLi salt, the contribution to sensitisation of the K+, Li+ and Na+ together with the organic part of the substance was tested (Table3).
In conclusion, the only difference between both substances is the counter ion, and an increase of sensitizing effects due to the presence of higher content of Na+ in the target chemical CAS No. 68877-33-8 is not expected.
Consequently, the read across from the studies conducted on the source chemical CAS No. 2196165-14-5 is regarded as feasible for this endpoint.

Genetic toxicity
Both the source substance Direct Black RBK and the target substance Direct Black RBB are assumed to be rapidly dissociated in the blood to anionic components and free cations (Na+, K+, Li+ cations and Na+ cations, respectively), which are then readily available in the body.
- Contribution of the cations
The contribution of K+, Li+ and Na+ to genetic toxicity has been evaluated based on available information.
The genotoxicity of sodium chloride has been assessed from different sources considered in the “Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part VI: When Salt and Sugar and Vegetables Are Positive, How Can Genotoxicity Data Serve to Inform Risk Assessment” review(3). The conclusion is that “genotoxicity of sodium chloride is a conditional property, in that under certain conditions (high doses leading perturbations in osmotic strength) it can elicit a genotoxic response, while under a different set of conditions (lower dose levels) such a response is not induced nor is it biologically plausible.” The effects were seen at concentrations of 5 mg/mL in different in vitro studies.
The source of K+ is in the form of KCl (potassium chloride). In the SIDS Initial Assessment Report of KCl(4), “no gene mutations were reported in bacterial tests, with and without metabolic activation. However, high concentrations of KCl showed positive results in a range of genotoxic screening assays using mammalian cells in culture. The action of KCl in culture seems to be an indirect effect associated with an increased osmotic pressure and concentration. Therefore KCl, do not have any direct relevance in the intact body were such concentrations cannot occur.” The lowest concentration where effects were seen is 5.5 mg/mL a Chromosome aberration test.
In the Version 02; December 2020 of the CLH report of lithium compounds(5) it is stated that “In summary, lithium compounds have been tested for mutagenicity, chromosome aberrations, sister chromatid exchanges, DNA damage in a number of in vitro and in vivo studies. Mainly negative results were obtained, but positive results were also reported, usually at high cytotoxic doses. According to Lagerkvist and Lindell (2002) a possible explanation for the observation of genotoxic effects at higher doses may be increased cell survival, since lithium inhibits apoptosis by inhibiting the enzyme glycogen synthase kinase-3 (GSK3). However, an aneugenic potential of lithium salts could not be excluded considering positive results obtained in in vitro micronucleus test associated with an increase of kinetochore positive micronuclei and an increase of damage mitosis. Moreover, no micronucleus test was performed in vivo to investigate this aneugenic potential.”
Lithium is a trace element in the environment and is present in soil, fresh water, salt water and plants. Lithium is readily absorbed from the gastrointestinal tract and excreted via the kidneys and it is known to cause nephron- and neurotoxicity and is used as psychiatric medication.
Li concentrations in the blood of rats range from 0.03 to 0.05 mmol/L (Neiri et al, 2012)(7). In the disseminated information in the ECHA website, different studies conducted with LiCl were reported(8). In a 2-year drinking water study with Wistar rats (1958, OECD 452) animals with a Li+ plasma level up to 2 mmol/L were not distinguishable from control animals.
The systemic NOAEL value of 80 mg/kg bw for repeated dose and reproduction/developmental toxicity is stablished for Direct Black RBK based on read-across from the OECD 422 study conducted with the analogue source substance Direct Black 19 ([REACH&colours Kft]; 2012; OECD 422 ; 70% of DYE; GLP; K=1; NOAEL 80 mg/kg bw/day)(9). This results in a NOAEL of approximately 24 mg/rat (rats weight ca. 330 g). The rats blood volume is 60 mL/Kg with a haematocrit of 43% resulting in 34 mL plasma/Kg (Wistar rat, Delerenco et al, 2002)(10) or 11 mL/rat, respectively 11 g/rat (assuming a specific density of 1). This results in 24 mg target chemical/L plasma assuming the test substance is readily absorbed via the gastrointestinal tract (worst case) and well distributed.
The Li+ concentration in Direct Black RBK is ≤0.2% (w/w). Referring to the NOAEL of 80 mg/Kg bw, this would result in 0.023 mmol/L (Mw of Li: 6.94) plus the naturally occurring Li+ plasma concentration of 0.05 mmol/L (Neiri et al, 2012) in a final Li+ plasma concentration of 0.073 mmol/L.
However, since in this estimation clearance parameter are not considered, it must be assumed that actually the Li+ concentration in plasma would be significantly lower than 0.073 mmol/L due to excretion process. The potential Li+ concentration in plasma is therefore regarded to be well within the safe range considering the NOAEL of 2 mmol/L plasma in the 2 year study (8).
In view of the quantities of K+, Li+ and Na+ present in the composition of Direct Black RBK, it is not expected that there is an effect produced by the cations on the results of the in vitro studies conducted on the substance. On the other side, the higher quantity of Na+ present in the composition of the target substance Direct Black RBB (2.2% vs. 1.85% w/w in Direct Black RBK) is neither expected to have an effect on the genotoxicity potential of the target substance. The main driver for genetic toxicity response will be the organic part.
- Contribution of the anions
The toxicity of source and target substances is expected to be driven by the organic anionic parts.
The organic anions of the target and source substances are identical and therefore they will have the same behavior in regards of absorption, distribution and interaction in the body, with the same amine metabolites. The expected toxicity effects of both substances are regarded as very similar
The potential metabolites are amines, as a result of the reductive cleavage of the azo bonds. Generally, the aromatic amines are moderately to highly soluble in water (6.4–238000 mg/L) due to the presence of one or multiple solubilizing functional groups, such as the amino functional group. Most of the aromatic amines are weak bases (pKa values of less than 5.5) that will be protonated at low pH but will be found in their neutral form under environmentally relevant pH (7–9). Given their hydrophilicity and ionic character tend to have low to very low experimental log K and distribution coefficient (log D) values. In our case some of the degradation products have simultaneously the occurrence in the whole structure of amine and sulfonate, which usually favours a diminution of the toxicity in almost all the target organs or aquatic organisms.
Generally stated, genotoxicity is associated with all aromatic amines with benzidine moieties, as well as with some aromatic amines with toluene, aniline and naphthalene moieties. The toxicity of aromatic amines depends strongly on the spatial structure of the molecule or –in other words– the location of the amino-group(s). For instance, whereas there is strong evidence that 2-naphthylamine is a carcinogen, 1-naphthylamine is much less toxic. The toxicity of aromatic amines depends furthermore on the nature and location of other substituents. As an example, the substitution with nitro, methyl or methoxy groups or halogen atoms may increase the toxicity, whereas substitution with carboxyl or sulphonate groups generally lowers the toxicity. As most soluble commercial azo dyestuffs contain one or more sulphonate groups, insight in the potential danger of sulphonated aromatic amines is particularly important. In an extensive review of literature data on genotoxicity and carcinogenicity of sulphonated aromatic amines, it was concluded that sulphonated aromatic amines, in contrast to some of their unsulphonated analogues, have generally no or very low genotoxic and tumorigenic potential(3).
The available tests and literature on Benzene-1,2,4-triyltriamine (CAS 615-71-4) and CAS 615-47-4 (as HCl salt) show that there is a light positivity on strain TA98 and strain TA 1538iIn the Ames test, but this positivity seems to be proved wrong by the Mouse sperm morphology test and by the IARC evaluation on the metabolic precursor 2-nitro-para-phenylenediamine (CAS 5307-14-2).
The available tests on p-phenylenediamine conclude that the substance is not mutagenic, although the Ames test showed mutagenic effect in strain TA98 with metabolic activation.
Metabolite CAS 69762-07-8 is a derivative of H Acid (EC 226-736-4, CAS 5460-093 Sodium hydrogen 4-amino-5-hydroxynaphthalne-2,7-disulphonate). The H acid monosodium salt is registered under REACH and is not classified. Several azo-colourants permitted as food additives like E110 (Sunset Yellow), E122 (Azorubine), E123 (Amaranth), E124 (Ponceaux R), E129 (Allura Red), E151 (Brilliant Black), E154 (Brown FK), are based on naphthalene mono-di-sulphonic acids with amino and(or hydroxy derivatives and none of them gave concern for genotoxicity. Other derivatives with existing negative data on bacteria gene mutation are: acid red 131 (CAS 70210-37-6), Acid Red 249 (CAS 6416-66-6), Acid Red 252 (CAS 70209-97-1), Acid Violet 54 (CAS 70210-05-8) and others. The capability of sulphonation to eliminate the activation to carcinogenic products is noted by Jung et al (Jung, 1992) and is illustrated by the fact that a property of most permitted synthetic azo dyes is sulphonation on all component rings. The article describes the toxicological main principle metabolic pathway of sulphonation as natural detoxification phase II pathway in the liver. The general aim of sulphonation is to make the substrate more soluble in water and usually less active pharmacologically. Sulphonated molecules are more readily eliminated in bile and urine.

In conclusion, due to the high structural similarity and physico-chemical properties of the source and target substances, their toxicokinetic behaviour is regarded as highly similar. In the genetic toxicity studies conducted on the source substance CAS No. 2196165-14-5, Direct Black RBK, the contribution to the genotoxicity of the K+, Li+ and Na+ was tested together with the contribution of the organic part, which is the main driver of mutagenicity. The potential metabolites that may lead to a mutagenic effect are the same for both substances, and therefore, read-across from available mutagenicity studies with Direct Black 155_NaKLi salt is considered adequate to predict the same behaviour and results for Direct Black 155_Na salt, as shown in Table 4.

In view of the quantities of K+, Li+ and Na+ present in the composition of Direct Black 155-NaKLi salt, it is not expected that there is an effect produced by the cations on the results of the in vitro studies conducted on the substance. The main driver for genetic toxicity response will be the organic part.
On the other side, the higher quantity of Na+ present in the composition of the target substance Direct Black 155-Na salt (2.2% vs. 1.85% w/w in Direct Black 155-NaKLi salt) is neither expected to have an effect on the genotoxicity potential of the target substance.

In conclusion, the only difference between the source structure (CAS No. 2196165-14-5) and the target chemical sodium salt (CAS No. 68877-33-8) is the counter ion, and they are not expected to contribute and an increase or change of genotoxic effects due to the presence of Na+ in the target chemical CAS No. 68877-33-8 is not expected. Consequently, read across to the source chemical CAS No. 2196165-14-5 is regarded as feasible.


4. Data matrix
See document attached to Section 13.

5.Conclusions on analogue approach hypothesis, C&L and PBT/vPvB assessment

As it has been discussed above, the similarity between the source and target substances is very high: molecular structure, functional groups, degradation products, physico-chemical properties and toxicological data are comparable.
The solubility, partition coefficient and pKa are similar, as expected for substances that are so similar from a structural and chemical point of view. Therefore, it could be assumed that the biodegradation in water can be assessed from the source substance, Direct Black 155-NaKLi salt.
On the other side, the likelihood of absorption, interaction in the body, degradation pathways and metabolites are expected to be similar for the source and target substances and lead to similar toxicity.
- Results of skin irritation studies with the source and the target substance show comparable results, they are not skin irritants. They also show similar behaviour for acute toxicity. This similar behaviour and the close structural similarity are the basis for considering adequate to assess the sensitisation potential of Direct Black 155-Na salt from the available data on Direct Black 155-NaKLi salt.
- The results on acute toxicity tests with the source and the target substances are comparable (LD50 > 2000 mg/Kg bw). The repeated dose toxicity and screening for reproduction toxicity is assessed from another structural analogue: LD50 = 80 mg/Kg bw for repeated dose toxicity and LD50 ) 80 mg/Kgbw for developmental toxicity. Due to the analogue structures and chemistry, the assessment of mutagenicity of Direct Black 155-Na salt from the available studies with Direct Black 155-NaKLi salt is considered feasible.

C&L: source and target substances are not classified for acute toxicity and repeated dose toxicity, they are not skin irritants and are classified for eye corrosion hazard (Eye Dam 1).The source substance shows a skin sensitizing potential that triggers classification as Skin Sens. 1 and the same classification is applied to the target substance. The source substance was found positive for mutagenicity in an Ames test, while negative results were obtained in a chromosome aberration and a micronucleus test. The same behaviour is assumed for the target substance (read-across).
In addition to the in vitro studies, to conclude on C&L for Direct Black 155_Na salt and Direct Black 155_NaKLi salt, an available in vivo mutagenicity study with the analogue substance Direct Black 19 (CAS 6428-31-5) is taken into account. Direct Black 19 showed negative results in an in vivo mammalian erythrocyte micronucleus test (OECD 474). Based on the structural and chemical similarity, as well as the composition of Direct Black 19 and Direct Black 155 (Na and NaKLi salts), read-across from the in vivo mutagenicity study with Direct Black 19 is used to predict the same behaviour for Direct Black 155 (Na and NaKLi salts).
Based on the available information, Direct Black 155_Na salt and Direct Black 155_NaKLi are not classified as mutagenic.

Both substances are not PBT and not vPvB; they both have a low potential for bioaccumulation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE : QSAR Toolbox and others

2. MODEL (incl. version number) : TOPKAT 4.5, CAESAR (VEGA 1.1.4), Derek 6.0.1, Toxtree 2.6.13.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL : NC1=CC=C(\N=N\C2=CC=C(C=C2)\N=N\C2=C(C=C3C=C(C(\N=N\C4=CC=CC=C4)=C(O)C3=C2N)S([O-])(=O)=O)S([O-])(=O)=O)C(N)=C1.[Na+].[Na+].[K+].[K+].[Li+].[Li+]

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See report with explanations for each model

5. APPLICABILITY DOMAIN
See report with explanations for each model

6. ADEQUACY OF THE RESULT
Only with the results of the in silico assessement it is not possible to conclude on classification and labelling.

Principles of method if other than guideline:

QSAR has been applied

Key result

Parameter:

other: QSAR prediction

Remarks on result:

other: likelihood of skin sensitizing property for the substance

Interpretation of results:

study cannot be used for classification

Conclusions:

Taking into account the consistency in the skin sensitization alerts triggered for the query compound by Derek and Toxtree, and for simulated autoxidation and skin metabolites by the OECD QSAR Toolbox profiler, there is a likelihood that the substance could be a skin sensitizer.

Executive summary:

According to Annex VII (Regulation (EC) No 1907/2006), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step. Due to the complexity of the skin sensitisation endpoint, a combination of alternative test methods (e.g. in silico, in chemico and in vitro) in a weight of evidence approach needs to be considered to increase confidence in the final assessment of skin sensitisation (ECHA Guidance, Chapter R.7a, 2017).

The present in silico assessment was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox.

In the Appraisal of (Q)SAR Modelling, result of each computational tool was discussed if applicable including statistical performance, mode of action, eventual metabolism and reliability. The results were summarized in the appraisal section table. For each tool, the QSAR Prediction Reporting Format (QPRF) section, if any, and the respective software printout section were provided.

Weight of evidence of this in silico assessment suggested that consistency in the skin sensitization alerts triggered for the query compound by Derek and Toxtree, and for simulated autoxidation and skin metabolites by OECD QSAR Toolbox profiler might indicate the likelihood of skin sensitizing property for the substance.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

TEST ITEM PREPARATION
On the day of the experiment (prior to start) DIRECT BLACK RBK was dissolved in culture
medium.
As tested by a solubility test, 5000 µg/mL in culture medium (the OECD 442E guideline
recommended maximal to be tested test item concentration) was used as highest test item
concentration in the cytotoxicity tests. Due to a technical error in the preparation of the test
item stock solution, 4910 µg/mL in culture medium was used as highest test item
concentration in the first cytotoxicity test.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were
analysed. For this, dilutions were prepared by 1:2 serial dilutions.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.
THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1
cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS
GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 106 cells/mL) allowing the
repeated use of the same cell culture batch in experiments. Therefore, the parameters of the
experiments remain similar, because of the reproducible characteristics of the cells. Thawed
stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice
weekly. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is
incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to
two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 11, 19 and 24 in the cytotoxicity tests and
25, 26, 28, 21, 26 and 22 in the h-CLAT for runs 1 to 6, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented
with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium
pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin)
is used to culture the cells during the assay. Medium with supplements has to be stored at
2 - 8 °C and used within one month. The culture medium has to be warmed to room
temperature just before use.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of
the cells, a volume of 500 µL with a cell density of 1.8 - 2 × 106 THP-1 cells/mL was seeded
in each corresponding well of a 24-well flat bottom plate.

Experimental Design and Procedures of the Cytotoxicity Test
Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined
with three cytotoxicity tests, but only the results of the first and the third test were used for
the calculation of the mean CV75. The tests were performed on different days. The test item
was prepared separately for each run.
-Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item and culture medium was added to the
cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested
in one replicate for each cytotoxicity test. At the end of the incubation period, the cell
cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the
7-AAD staining.
- Staining of the Cells
Each test item-treated and not test item treated cells were collected in sample tubes
centrifuged (approx. 250 × g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS
with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At
least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added
in each sample tube.
-Flow Cytometry Acquisition (Cytotoxicity Test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was
calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The
7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD
fluorescence signal.
Preparation of the acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for
the analysis, but the FSC/SSC plot should be checked to make sure that a single population
appears without contamination or excessive debris. The FL-3 voltage was set and compensate
to appropriate position (FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0).
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of
10,000 living cells were analysed.
The maintenance of the flow cytometer was in accordance with the manufacturer’s
instructions. The process of washing was conducted very carefully since insoluble chemicals
could flow in the flow line.
-Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) or is calculated
according to the following equation:
Cell Viability [%] = ( Number of living cells /Number of acquired cells )× 100

The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is
calculated by log-linear interpolation using the following equation:
Log CV75 = (75 −𝑐) × 𝐿og (𝑏) − (75 −𝑎) × 𝐿og (𝑑) / (𝑎 −𝑐)

Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively

-Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
• The cell viability of the medium control should be more than 90%.

-Calculation of the Test Doses for the Main Experiment (h-CLAT)
The mean of two CV75 values (first and third cytotoxicity experiment) was used to determine
the dose-range for the main experiment (h-CLAT).
Eight final concentrations (µg/mL) were used for the test item in the main experiment
(h-CLAT). The highest concentration used was 1.2 × mean CV75 and seven further
concentrations of the test item were prepared by serial 1:1.2 dilution.
Due to strong cytotoxicity (cell viability < 50%) observed in all test item treated cells of the
first h-CLAT run, the test item concentrations were adjusted for the following h-CLAT runs.

Experimental Design and Procedures of h-CLAT
The test item was tested in six independent runs. The tests were performed on different days.
The test item was prepared separately for each run.
- Treatment of the Cells
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO
control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At
the end of the incubation period, the cell cultures were microscopically evaluated for
morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was
prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54
antibody or mouse IgG1).
- Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and
equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5
min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA).
Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking
solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and
the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-
labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis
procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected
for 30 ± 5 min. at 2 - 8 °C (on ice).
-Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS
buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes
before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

-Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was
calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using
the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal
detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set
for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC
are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single
population appears without contamination or excessive debris. The FL-1 and FL-3 voltage
were set and compensate to appropriate position. The FL-1 voltage was set using the FITC
labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set
in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo
Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson
GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining
with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between
the peak of the negative fraction and the positive fraction in the FL-3 histogram using
DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the
subsequent analyses. For each control and all test item concentrations, the cell viability was
recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the
CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability
evaluation.
Since the peak in the FL-3 histogram showed a shift to the right side (due to possible
interference of the test item) and no clear cytotoxicity could be observed in the 2D plot
consisting of FSC (Forward Scatter) versus SSC (Side Scatter), the R1-gate was shifted to the
right side to obtain a reliable result for the cell viability of the test item treated cells. The
R1-gate in the h-CLAT runs was set comparable to the R1-gate set in the dose range finder
tests (cytotoxicity tests).
The maintenance of the flow cytometer was in accordance with the manufacturer’s
instructions. The process of washing was conducted very carefully since insoluble chemicals
could flow into the flow line.
Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC
(side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence
intensity (MFI) of viable cells and viability for each sample were used for analysis. The other
tubes were acquired without changing the settings of the cytometer. The MFI was recorded
for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded
from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of
cytoplasmic structures that could be generated due to cell membrane destruction). for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded
from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of
cytoplasmic structures that could be generated due to cell membrane destruction).

Vehicle / solvent control:

cell culture medium

Negative control:

other: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD86 RFI value of the positive control (2.0 µg/mL DNCB) in the third h-CLAT run did not exceed the positive
criterion (CD86 ≥ 150%) and the CD54 RFI value of the positive control (2.0 µg/mL DNCB)
in the first, third, fourth and sixth h-CLAT run did not exceed the positive criterion (CD54 ≥
200%). However, this is considered to be acceptable since the CD86 and CD54 RFI value of
the positive control (3.0 µg/mL DNCB) in the first, third, fourth and sixth h-CLAT run
exceeded the positive criteria.

Group:

test chemical

Run / experiment:

other: run/experiment:4

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

>83%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

>72%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

<50%

Remarks on result:

other: not valid

Key result

Group:

test chemical

Run / experiment:

other: run/experiment: 6

Parameter:

RFI CD54>150 [442E]

Value:

215.2 %

Cell viability:

73.89%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: run /expeiment: 5

Parameter:

RFI CD54>150 [442E]

Value:

263.4 %

Cell viability:

88.56%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

RFI CD54>150 [442E]

Value:

245.8 %

Cell viability:

68.

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium
control of both CD86 and CD54 should not exceed the positive criteria (CD86
≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86
and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the
positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should
be > 50% in at least one concentration of the two tested positive control
concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested
concentratins in each run.

ACCEPTANCE CRITERIA OF THE h-CLAT
Acceptance Criteria of the first h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 94.00%
DMSO = 93.69%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 154.0%#
2 µg/mL DNCB (CD 86): 579.8%
3 µg/mL DNCB (CD 54): 261.5%
3 µg/mL DNCB (CD 86): 538.0%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 114.1%
CD86: 93.7%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 244.5%
Medium CD 86: 331.4%
DMSO CD 54: 265.0%
DMSO CD 86: 316.8%
# CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Acceptance Criteria of the second h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 96.51%
DMSO = 96.39%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 360.7%
2 µg/mL DNCB (CD 86): 384.8%
3 µg/mL DNCB (CD 54): 519.7%
3 µg/mL DNCB (CD 86): 408.9%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 66.3%
CD86: 83.1%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 138.8%
Medium CD 86: 237.1%
DMSO CD 54: 125.7%
DMSO CD 86: 213.9%

Acceptance Criteria of the third h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 96.11%
DMSO = 95.96%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 163.6%#
2 µg/mL DNCB (CD 86): 132.4%#
3 µg/mL DNCB (CD 54): 329.1%
3 µg/mL DNCB (CD 86): 175.9%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 114.6%
CD86: 112.8%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 140.9%
Medium CD 86: 263.0%
DMSO CD 54: 145.6%
DMSO CD 86: 279.3%
# The CD86 and CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Acceptance Criteria of the fourth h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 96.69%
DMSO = 97.35%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 89.2%#
2 µg/mL DNCB (CD 86): 255.2%
3 µg/mL DNCB (CD 54): 305.4%
3 µg/mL DNCB (CD 86): 332.4%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 94.9%
CD86: 84.8%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 153.2%
Medium CD 86: 264.1%
DMSO CD 54: 147.6%
DMSO CD 86: 231.3%
# CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Acceptance Criteria of the fifth h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 93.87%
DMSO = 93.15%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 350.0%
2 µg/mL DNCB (CD 86): 565.0%
3 µg/mL DNCB (CD 54): 245.5%
3 µg/mL DNCB (CD 86): 500.0%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 130.7%
CD86: 85.4%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 147.9%
Medium CD 86: 291.9%
DMSO CD 54: 172.5%
DMSO CD 86: 290.1%

Acceptance Criteria of the sixth h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 93.24%
DMSO = 92.91%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2.7 µg/mL DNCB (CD 54): 181.6%#
2.7 µg/mL DNCB (CD 86): 292.9%
4 µg/mL DNCB (CD 54): 287.7%
4 µg/mL DNCB (CD 86): 340.6%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 107.6%
CD86: 99.5%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 260.2%
Medium CD 86: 422.0%
DMSO CD 54: 282.8%
DMSO CD 86: 439.7%
# CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Interpretation of results:

other: the study is part of a WoE assessment on skin sensitisation

Conclusions:

The test item DIRECT BLACK RBK with a log Pow of -1.84 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of DIRECT BLACK RBK dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of DIRECT BLACK RBK was previously calculated from two cytotoxicity tests.
Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2455 µg/mL up to the highest tested concentration (4910 µg/mL) in the first cytotoxicity test, starting with the concentration of 625 µg/mL up to the highest tested concentration (5000 µg/mL) in the second cytotoxicity test and starting with the concentration of 1250 µg/mL) up to the highest tested concentration (5000 µg/mL) in the third cytotoxicity test (threshold of cytotoxicity: < 75%). Due to a calculation error in the preparation of the test item stock solution for the first cytotoxicity test, observed at the end of the study, the calculated CV75 value was 1835.66 µg/mL instead of 1802.61 µg/mL.
Subsequently, the mean CV75 calculated with the CV75 of the first and third cytotoxicity test was 1411.01 µg/mL instead of 1394.49 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):
472, 567, 680, 816, 980, 1176, 1411 and 1693 µg/mL in the first main experiment, 132, 158, 190, 228, 273, 328, 393 and 472 µg/mL in the second to sixth main experiments.

The test item with a log Pow of -1.84 was tested in 6 independent runs. Since the peak in the FL-3 histogram showed a shift to the right side (due to possible interference of the test item) and no clear cytotoxicity could be observed in the 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter), the R1 gate of all six h-CLAT runs was shifted to the right side to obtain a reliable result for the cell viability of the test item treated cells.

The first h-CLAT run was not valid, since all test item treated concentrations showed a cell viability < 50%. Therefore, the test item concentrations were adjusted for the following h-CLAT runs. In the second h-CLAT run the RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively, at any test item concentration. In the third h-CLAT run the RFI of CD54 was equal or greater than 200% at the highest tested test item concentration.

The first to third h-CLAT run were conducted with THP1 cells which were slightly older than two month, therefore additional h-CLAT runs were performed and used for the evaluation.

The RFI of CD86 and CD54 in the fourth h-CLAT run was not equal or greater than 150% and 200%, respectively, at any test item concentration. In addition, the RFI of CD54 was equal or greater than 200% in at least one concentration of the fifth and sixth h-CLAT run. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT. However, a color interference by the intrinsic color of the test item cannot be fully excluded.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD86 RFI value of the positive control (2.0 µg/mL DNCB) in the third h-CLAT run did not exceed the positive criterion (CD86 ≥ 150%) and the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first, third, fourth and sixth h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD86 and CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first, third, fourth and sixth h-CLAT run exceeded the positive criteria.

In conclusion, the test item DIRECT BLACK RBK with a log Pow of -1.84 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) Protocol: KeratinoSens™, 2009/vers.6.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

1. Cell culture: The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan. The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10270) and 5.5 mL Geneticin (Gibco 10131).
1.1. Cell Culture from Frozen Stocks: Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in a total of 10 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.
1.2. Cell Passage: Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks may have been pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages.
1.3. Preparation of Test Cell Cultures: The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except well H12 of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. Well H12 of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.
2. Positive control: Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using a formula where the purity of the chemical in % is used. The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.
Results from the positive control were shared with other studies performed in the same assay.
3. Test Item Solubility: Prior to commencing testing, the solubility of the test item, Direct Black RBK, in DMSO was assessed.
The test item, Direct Black RBK, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.
4. Test Item Solubility: Prior to commencing testing, the solubility of the test item, Direct Black RBK, in DMSO was assessed.
The test item, Direct Black RBK, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.
5. Preparation of the Test Item: A stock solution of the test item, Direct Black RBK, was prepared by weighing between 20 - 40 mg into a tared glass container and diluting to 200 mM in DMSO using the same formula as for the positive control. As the test item had a range for molecular weight, the mid-point (705 g/mol) was used for calculating the volume of DMSO required to dilute the test item to 200 mM.
6. Test procedure:
6.1. Preparation of the 100x Solvent Plate: A 100x solvent plate was set up by adding 100 µL of DMSO to all wells of a 96 well plate except wells in column 12 and well H11 of the plate. 200 µL of the stock solution of the test item, Direct Black RBK, was added to one well in column 12. The test item was serially diluted across the plate by transferring 100 µL from column 12 to column 11 and then mixed by repeat pipetting (at least 3 times) and then 100 µL was transferred from column 11 to
column 10 and so forth across the plate.
200 µL of the 6.4 mM stock solution of cinnamic aldehyde was added to well H11 and serially diluted from column 11 to column 7.
6.2. Preparation of the Dilution Plate: The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.
6.3. Treatment of Cultured Plates: Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring
cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.
6.4 Cell viability measurement: A kit (Molecular Probes Vybrant MTT kit V13154) was used to determine cell viability. 1 vial from the kit was reconstituted by adding 1 mL of sterile PBS (Gibco 10010) and vortexed mixed until dissolved to give 5 mg/mL MTT in DPBS. After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution was added to each well of the 96-well plate. The plate was incubated at 37 ± 2C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes.
The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.
6.5. Luciferase measurement: Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for test 1. Frozen reconstituted reagent was used for both tests
and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Positive control results:

Positive Control (cinnamic aldehyde)
Results- test 1: Imax 3.64; EC1.5 16.09;
Results - test 2: Imax 4.92; EC1.5 8.88

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

2.99

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

3.79

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Value:

2.11

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Value:

0.98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
Test1: As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 50% and approximately 30% at concentrations 125 – 500 µM. The cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.

Test 2: As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 60% and approximately 50% at concentrations 125 – 250 µM. The confluency of the cells treated with a concentration of 500 µM of Direct Black RBK was approximately 30% and the cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
yes, The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 14.8% and 11.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.
- Acceptance criteria met for positive control:
yes. Luciferase activity induction with the positive control is statistically significant above the threshold of 1.5 in at least one of the test concentrations. Average induction in the three replicates of positive control at 64 µM is between 2 and 8. EC1.5 of positive control is within two standard deviations of the historical mean of the testing facility (4.94 – 50.00). The aveerage coefficient of variatton of the luminescence reading for the negative solvent control (DMSO) was 14.8% and 11.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Direct Black RBK results test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.16	1.42	2.47	2.99	2.45	2.31	1.02	0.89	0.55	0.24	0.00	0.00
Statistically significant	No	No	Yes	Yes	Yes	Yes	No	No	No	No	No	No
Viability (%)	90.49	87.22	78.01	81.53	73.58	77.68	71.82*	75.25*	82.11*	70.23*	52.57*	76.84*
Imax	2.99
EC1.5	2.11
IC30	506.48
IC50	N/A

* As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 50% and approximately 30% at concentrations 125 – 500 µM. The cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with  concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.

 

Direct Black results test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.81	1.90	2.55	3.17	3.68	3.79	3.09	1.64	1.42	0.61	0.30	0.07
Statistically significant	yes	yes	Yes	Yes	Yes	Yes	yes	yes	No	No	No	No
Viability (%)	116.03	107.74	102.03	98.91	96.32	89.09	120.59*	115.65*	112.15*	112.15*	112.15*	123.64*
Imax	3.79
EC1.5	<0.98
IC30	N/A
IC50	N/A

* As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 60% and approximately 50% at concentrations 125 – 250 µM. The confluency of the cells treated with a concentration of 500 µM of Direct Black RBK was approximately 30% and the cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.

 

Foorr both Direct Black RBK tests (1 and 2):

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	Yes
Is the EC1.5value <1000µM	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

Interpretation of results:

other: the study is part of a WoE assessment on skin sensitisation

Conclusions:

It was concluded that Direct Black RBK gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that Direct Black RBK is a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Direct Black RBK, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for Direct Black RBK was 2.99 in test 1 and 3.79 in test 2. The Imax for both tests was >1.5 fold and statistically significant as compared to the DMSO control.  

The cellular viability was 52.57% at 1000 µM in test 1 and the IC30 was 506.48 µM. In test 2 the cellular viability did not fall below 89.09% and therefore the IC30 and IC50 could not be calculated. However, the cellular viability results were unreliable as Direct Black RBK gave a coloured solution which interfered with MTT and therefore the cells were visually assessed for cytotoxicity prior to the addition of MTT. In test 1, the confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 50% and approximately 30% at concentrations 125 – 500 µM. The cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. In test 2 the confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 60% and approximately 50% at concentrations 125 – 250 µM. The confluency of the cells treated with a concentration of 500 µM of Direct Black RBK was approximately 30% and the cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent.  In both tests the DMSO control produced approximately 70% confluency.

The EC1.5 for Direct Black RBK was 2.11 µM and <0.98 µM for tests 1 and 2, respectively. Graphs for Direct Black RBK showed an overall dose-response for luciferase induction.

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.

The EC1.5 values of the positive control, cinnamic aldehyde were 16.09 μM and 8.88 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory.  

The average induction in the three replicates for cinnamic aldehyde at 64 µM were 3.64 and 4.92 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 14.8% and 11.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

It was concluded that Direct Black RBK gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is a skin sensitizer.

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

272-559-0

EC Name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68877-33-8

Molecular formula:

C28H21N9O7S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Results and discussion

In vitro / in chemico

Results

Key result

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The predicted behavior of the substance as skin sensitizer was assessed in an weight-of-evidence approach by in vitro / in silico methods, taking into account the key events of skin sensitisation and using read-across from a similar substance. The substance is predicted to be a skin sensitizer. As the classificacion in category 1A or 1B can not be derived, the substance is classified in category 1.