Registration Dossier

Administrative data

Description of key information

No alert for protein binding was obtained in silico (OECD Toolbox). In chemico testing of Anthrachinon-2-sulfonsäure, Na-Salz in the DPRA (OECD 442C) and in vitro testing in the h-CLAT (OECD 442E) was technically not feasible. A positive result was obtained in vitro in the KeratinoSens assay (OECD 442D). Based on these results in vivo testing was initiated to gain information on potential in vivo skin sensitization activities. Anthrachinon-2-sulfonsäure, Na-Salz did not reveal any skin sensitising properties in the mouse local lymph node assay (OECD 429) at the maximal technically feasible concentration of 10% and can thus be regarded as non-sensitizer under the conditions of this test. A low sensitizing potential at higher concentrations cannot be fully excluded because the maximal technically feasible concentration in the in vivo test was 10%. Based on the available data, including the fact that no skin sensitizing effects were reported in occupational settings, no classification for skin sensitization is warranted.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
Deviations:
yes
Remarks:
Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2012
Principles of method if other than guideline:
The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Based on the content of 91.80% a correkction factor of 1.09 was considered in test formulation preparation.
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 29 - 38 g
- Housing: The animals were kept singly in MAKROLON cages (type II). Animals were not group-housed to prevent contact of the application sites.
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Photoperiod (hrs dark / hrs light): 12hrs dark / 12 hrs light
- IN-LIFE DATES: From: 03 JULY 2018 To: 14 SEPTEMBER 2018
Vehicle:
dimethyl sulphoxide
Concentration:
Three concentrations of Bayscript Gelbkomponente (2.5%, 5% and 10% (w/w)), dissolved/suspended in dimethyl sulfoxide (DMSO, w/w) were tested in six female NMRI mice per group and compared to a vehicle control group. A 10% (w/w) concentration was the highest feasible concentration of the test item in DMSO (w/w), see Table 1.
No. of animals per dose:
6 females per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: solution in in DMSO (w/w), see Table 1
- Irritation: no effects
- Systemic toxicity: no effects
- Ear thickness measurements: no effects
- Erythema scores: 0
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations out of the technically feasible concentrations (see Table below). Three concentrations of 2.5%, 5%, and 10% (w/w) dissolved in DMF were examined. Doses were selected based on the OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% (w/w) etc.
The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used. Both ears of each mouse were observed for erythema. The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.
No irritating properties were observed in this preliminary experiment at concentrations of 2.5%, 5%, and 10% (w/w), and no differences in ear weight and ear thickness were noted.


MAIN STUDY
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European inter-laboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item.
By additionally measuring simple inflammatory parameters such as ear thickness or ear weight, it is possible to reliably determine the degree of response that is attributable to irritation (Vohr and Ahr, 2005 ).
Hence, in addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group to the test item treated animals by the vehicles treated ones. The cut-off threshold value for ear weight was set at 1.1.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified LLNA
- Criteria used to consider a positive response:
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
- Day 1:
The weight of each animal was individually identified. The weights and any clinical signs were recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

Test formulation analysis was carried out non-GLP in 2 steps:
1) The analytical method was validated by LPT before in vivo testing. The following parameters were determined:
- Linearity, Accuracy, Precision, Sensitivity, Specificity and Stability (6 hours at room temperature)
2) 3 Samples of approximately 5 mL were taken during the in-life phase from the prepared formulations of 2.5%, 5%, and 10% (w/w). The samples were stored at ≤ 20°C until analysis for concentration.

Observations of the animals daily for clinical signs, local irritation or systemic toxicity and body weight (day 1 and day 4) were included in the study.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
Positive control results:
The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid (see Table 2).
Key result
Parameter:
SI
Value:
1.311
Test group / Remarks:
2.5% test item in DMSO: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4
Remarks on result:
other: no indication of skin sensitization
Key result
Parameter:
SI
Value:
1.072
Test group / Remarks:
5% test item in DMSO: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4
Remarks on result:
other: no indication of skin sensitization
Key result
Parameter:
SI
Value:
1.038
Test group / Remarks:
10% test item in DMSO: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4
Remarks on result:
other: no indication for skin sensitization
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
In the main study treatment with Bayscript Gelbkomponente at concentrations of 2.5%, 5% and 10% in DMSO (w/w) did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. (see Table 2)

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. (see Table 2)

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. (see Table 2)

DETAILS ON STIMULATION INDEX CALCULATION
The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

EC 1.4 CALCULATION
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones. No values were above the threshold of 1.4 (lymph node cell count) or 1.1 (ear weight). Thus, under the present test conditions, the test item at concentrations of 1%, 2.5% and 25% (w/w) in DMSO did not reveal any skin sensitising properties in the local lymph node assay. A 5% (w/w) concentration of the test item in DMSO was the maximum feasible concentration.

CLINICAL OBSERVATIONS and BODY WEIGHTS
No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In a preliminary experiment, concentrations of 2.5%, 5% and 10% (w/w) of the test item, employing 1 animal per concentration, were examined. No irritating properties were observed in this preliminary experiment and no differences in ear weight and ear thickness were noted. A 10% (w/w) concentration was the highest feasible concentration of the test item in DMSO.

Table 2: Stimulation indices (SI) in the main experiment (mean of 6 animals per group):

Parameter  negative control (DMSO)  2.5% (w/w) test item in DMSO  5% (w/w) test item in DMSO  10% (w/w) test item in DMSO  positive control (20% alpha-hexyl cinnamic aldehyde (v/v) in A/O)  vehicle of positive control
 Lymph node cell count  1.000  1.311  1.072  1.038  1.695*  1.000
 Lymph node weight  1.000  1.088  1.140  1.070  1.340*  1.000
 Ear weight  1.000  1.042*  1.066*  1.000  1.057  1.000
 Ear thickness  1.000  1.016 1.008  1.085  1.134  1.000

* significantly different from control at p ≤ 0.01

The analysis of the test item vehicle solutions of the 2.5%, 5% and 10% concentrations for the actual test item levels was carried out (non-GLP) under conditions employing a validated analytical method. The analysis resulted in actual levels of 100.1% (2.5% (w/w) concentration), 99.7 (5% (w/w) concentration) and 100.5% (10% (w/w) concentration) of the nominal concentration.

Interpretation of results:
GHS criteria not met
Executive summary:

The purpose of this study was to determine the skin sensitising potential of Anthrachinon-2-sulfonsäure, Na-Salz in the modified local lymph node assay in mice. The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of the test item (2.5%, 5% and 10% (w/w)), suspended in dimethyl sulfoxide (DMSO) were tested in six female NMRI mice per group and compared to a vehicle control group. DMSO was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 10% (w/w) concentration was the highest feasible concentration in DMSO. Acetone/olive oil (4:1, v/v), propylene glycol and N,N-dimethylformamide, other recommended vehicles, did not provide higher concentrated suitable suspensions or solutions. Methyl ethyl ketone was not applicable for analytics.

In the main study treatment at concentrations of 2.5%, 5% and 10% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, Anthrachinon-2-sulfonsäure, Na-Salz at concentrations of 2.5%, 5% and 10% (w/w) in DMSO did not reveal any skin sensitising properties in the local lymph node assay. A 10% (w/w) concentration of the test item in DMSO was the maximum technically feasible concentration.

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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
of February 2015
GLP compliance:
yes
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Based on the content of 91.8% a correction factor of 1.09 was included in the preparation of formulations.
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D (batch number 42, passage 12 for Experiment 1 and batch number 45, passage 12 for Experiment 2).

Preparation of Cultures:
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 9% foetal bovine serum and 1% Geneticin.

Treatment:
In 96-well plates, incubated at 37±1°C, 5% (v/v) CO2, for 48±1 hours in medium with serum but without Geneticin. For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.

Cytotoxicity Assessment:
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements:
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive controls:
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

Negative controls:
The negative control was diluted into culture medium containing serum so that the final concentration was 1%. An aqueous solvent was used, therefore DMSO was added to the treatment solutions at 1% (final concentration).

Positive control results:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 μM in Experiment 1 and 2 and at concentrations of 32 to 64 μM in Experiment 3.

The EC1.5 value for the positive control was 9.01, 9.79 and 16.78 μM in Experiments 1, 2 and 3, respectively.

The average inductions in the two replicates for the positive control at 64 μM were 16.51, 8.99 and 2.38 in Experiments 1, 2 and 3, respectively. The values for Experiments 1 and 2 were above the range specified in the protocol, however luciferase induction values showed clear dose responses, therefore it was considered that these were valid experiments.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 14.67, 10.42 and 9.01% in Experiments 1, 2 and 3, respectively.
Parameter:
other: Imax in experiment 1
Run / experiment:
experiment 1
Value:
17.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Imax in experiment 2
Run / experiment:
experiment 2
Value:
5.99
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no statistical significance
Parameter:
other: Imax in experiment 3
Run / experiment:
experiment 3
Value:
16.13
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: proven

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 14.67, 10.42 and 9.01% in Experiments 1, 2 and 3, respectively.

The acceptance criteria were met in each experiment with the exception that in Experiments 1 and 2, the EC1.5 for the positive control was above the range specified in the protocol. However, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction with the positive control.

The EC1.5 value for Experiment 2 could not be calculated as although the Imax was above 1.5, it was not a statistically significant increase.

The four conditions for a positive prediction prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in Experiments 1 and 3.

The test article, Anthrachinon-2-sulfonsäure, Na-Salz, was thus considered to be positive in the ARE-Nrf2 Luciferase Test.

Results table:

 Criteria  Experiment 1  Experiment 2  Experiment 3
 I max  17.60  5.99  16.13
 Cell viability  79.99% not determined 150.51%
 EC1.5  96.51 µM  not calculated  70.43 µM
 Dose response  Yes Yes   Yes
Executive summary:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed according to OECD 442D. The test article was dissolved in DMSO to the final concentration of the stock solution (200 mM) with further dilution in water and cell culture medium to obtain final concentrations of 0.98 to 2000 µM. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test. Treatment of the cell for 48 hours with the test item led to the following results: The maximal average fold increases (Imax) were 17.6 and 16.13 for experiments 1 and 3, respectively. In experiment 2 the EC 1.5 value could not be calculated as althouth the Imax was above 1.5 (5.99), no statistically significant increase was observed.

The four conditions required for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in two experiments, therefore, Anthrachinon-2-sulfonsäure, Na-Salz was considered to be positive in the ARE-Nrf2 Luciferase Test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No human data are available for the substance to assess the endpoint skin sensitization. However, no skin irritating or skin sensitizing effects were reported in occupational settings.

An assessment of potential skin sensitization properties based on defined approaches and individual sources was conducted for Anthrachinon-2-sulfonsäure, Na-Salz within an IATA. The outcome of this assessment is given below:

Anthrachinon-2-sulfonsäure, Na-Salz is a solid with a MW of 310 g/mole and a calculated log Pow of -1.6. The respective monohydrate is soluble in water (5.29 g/L). Based on these physico-chemical properties dermal uptake of the substance cannot be fully excluded.

Anthrachinon-2-sulfonsäure, Na-Salz was investigated in silico for structural activity relationship (QSAR), in chemico for peptide reactivity, and in vitro for keratinocyte activation (KeratinoSens). Due to positive results in vitro in vivo testing was performed in the LLNA as last option to assess the endpoint skin sensitization.

OECD Toolbox 4.0:

The structure activity relationship of the substance was investigated by using the OECD Toolbox 4.0 (released 2017). No protein binding alerts for skin sensitization were identified in silico (Schlecker, 2017).

DPRA; OECD TG 442C: (no result)

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

As solvent dimethyl sulfoxide/acetonitrile (1:1) was used since the test item was insoluble in all other solvents that are acceptable for the DPRA. However, the dimethyl sulfoxide / acetonitrile 1:1 mixture itself had significant impact on the percent peptide depletion. The mean cysteine concentration of the reference control C (solvent control) was reduced by > 40%. Furthermore, co-elution of the test item with the cysteine-peptide was observed. Thus, the acceptance criteria for the DPRA were not fulfilled and no DPRA result for the test item could be obtained.

KeratinoSens™; OECD TG 442D: (WoE)

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed according to OECD 442D. The test article was dissolved in DMSO to the final concentration of the stock solution (200 mM) with further dilution in water and cell culture medium to obtain final concentrations of 0.98 to 2000 µM. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test. Treatment of the cell for 48 hours with the test item led to the following results: The maximal average fold increases (Imax) were 17.6 and 16.13 for experiments 1 and 3, respectively. In experiment 2 the EC 1.5 value could not be calculated as although the Imax was above 1.5 (5.99), no statistically significant increase was observed.

The four conditions required for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in two experiments, therefore, Anthraquinone-2 -sulfonic acid sodium salt was considered to be positive in the ARE-Nrf2 Luciferase Test.

h-CLAT; OECD 442E (waiver):

The substance was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines (OECD 442E) for the h-CLAT. Thus, the test article was outside the applicability domain for this study and the performance of the study was technically not feasible.

In vivo testing in the mouse LLNA; OECD TG 429: (key study)

Based on the results obtained in silico (negative), in chemico (technically not feasible), and in vitro (positive results in the KeratinoSens, h-CLAT technically not feasible) no conclusion can be drawn for the endpoint skin sensitization. Thus, in vivo testing using the mouse LLNA was initiated to gain information on potential in vivo skin sensitization activities.

Anthrachinon-2-sulfonsäure, Na-Salz was thus tested in the modified local lymph node assay in mice. The study was performed according to OECD 429. An alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitization) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of the test item (2.5%, 5% and 10% (w/w)), suspended in dimethyl sulfoxide (DMSO) were tested in six female NMRI mice per group and compared to a vehicle control group. DMSO was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 10% (w/w) concentration was the highest feasible concentration in DMSO. Acetone/olive oil (4:1, v/v), propylene glycol and N,N-dimethylformamide, other recommended vehicles, did not provide higher concentrated suitable suspensions or solutions. Methyl ethyl ketone was not applicable for analytics.

In the main study treatment at concentrations of 2.5%, 5% and 10% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, Anthrachinon-2-sulfonsäure, Na-Salz at concentrations of 2.5%, 5% and 10% (w/w) in DMSO did not reveal any skin sensitising properties in the local lymph node assay. A 10% (w/w) concentration of the test item in DMSO was the maximum technically feasible concentration.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No alert for protein binding was obtained in silico (OECD Toolbox). In chemico testing of Anthrachinon-2-sulfonsäure, Na-Salz in the DPRA (OECD 442C) and in vitro testing in the h-CLAT (OECD 442E) was technically not feasible. A positive result was obtained in vitro in the KeratinoSens assay (OECD 442D). Thus, in vivo testing was initiated to gain information on potential in vivo skin sensitization activities. Anthrachinon-2-sulfonsäure Na-Salz was tested in the modified local lymph node assay in mice (OECD 429). Solubility trials with all recommended vehicles for the LLNA were performed that revealed a 10% (w/w) concentration of the test item in DMSO as the maximum feasible concentration. Anthrachinon-2-sulfonsäure Na-Salz did not reveal any skin sensitising properties in the mouse local lymph node assay and can thus be regarded as non-sensitizer under the conditions of this test. A low sensitizing potential at higher concentrations cannot be fully excluded because the maximal technically feasible concentration in the in vivo test was 10%. Based on the available data, including the fact that no skin sensitizing effects were reported in occupational settings, no classification for skin sensitization is warranted.