Hydrogen [4-[[4-(diethylamino)phenyl][4-[ethyl[(3-sulphonatobenzyl)amino]-o-tolyl]methylene]-3-methylcyclohexa-2,5-dien-1-ylidene](ethyl)(3-sulphonatobenzyl)ammonium, sodium salt

Endpoint summary

Description of key information

No alert for protein binding was obtained in silico (OECD Toolbox). A weak positive result in the DPRA (in chemico testing) and a negative result in vitro (KeratinoSens negative following the 2 of 3 approach) was obtained. Thus, in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Special Brilliant Blue FFR did not reveal any skin sensitising properties in the mouse local lymph node assay at the maximal technically feasible concentration of 10% and can thus be regarded as non-sensitizer under the conditions of this test. This negative result is in line with the interpretation of the physico-chemical data of the substance that predict low to negliglible dermal absorption in vivo. Overall the compound is considered to be not sensitizing to skin. A low sensitizing potential at higher concentrations cannot be fully excluded because the maximal technically feasible concentration in the in vivo test was 10%.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The Direct Peptide Reactivity Assay (DPRA) 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.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).
The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5ºC. Relative peptide concentration is measured by reversed phase (C18) high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. The synthetic peptides contain phenylalanine to aid in the detection. The test item was dissolved and tested according to the given test procedure. Cinnamic aldehyde was used as positive control at a concentration of 100 mmol/L in acetonitrile.

Cysteine and lysine peptide solutions were incubated at 1:10 and 1:50 ratio in glass auto sampler vials with the test item solution for 24 hours at 25±2.5°C in the dark. Samples were visually inspected for precipitation or phase separation before HPLC analysis. The test item was analyzed in triplicate for both peptides. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. HPLC analysis for the cysteine and lysine peptides were performed on separate days with the test item solutions freshly prepared for both assays on each day. The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards. Percent peptide depletion is calculated according to OECD Guideline.

The following criteria should be met for a test chemical’s results to be considered valid: a) the maximum standard deviation for the test chemical replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion, b) the mean peptide concentration of three injections of the reference control C in the appropriate solvent should be 0.50±0.05 mM.

According to the study protocol the mean percent cysteine and percent lysine depletion value was calculated for the test item and the positive control. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion can be used to support the discrimination between skin sensitizers and non-sensitizers in the framework of an IATA. (see also table under 'any other information on materials and methods')

Positive control results:

The positive control cinnamic aldehyde led to a depletion of cysteine and lysine peptides (67.9%). According to the cysteine 1:10/lysine 1:50 prediction model “high reactivity” was derived for the positive control, leading to a DPRA prediction of “positive“.

Run / experiment:

other: Cysteine/Lysine (filtrate tested)

Parameter:

other: Mean cyst/lys peptide depletion for the filtrate (%)

Value:

9.65

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Reactivity class: low

Run / experiment:

other: Cysteine/Lysine (suspension tested)

Parameter:

other: Mean cyst/lys peptide depletion for the suspension (%)

Value:

11.36

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Reactivity class: low

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. Since the test item was poorly soluble in any solvent acceptable for the DPRA the required test concentration of 100 mmol/L could not be reached. The test was therefore performed with a suspension at the concentration of 100 mmol/L in acetonitrile and with the filtrate of this suspension (saturated solution). In both cases the concentration of solved test item was < 100 mmol/L.
A depletion of cysteine and lysine peptides became obvious in the DPRA for the filtrate (9.65%) and for the suspension (11.36%). According to the cysteine 1:10/lysine 1:50 prediction model “low reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “positive“.

Table 1: Summary of results

	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	Reactivity Class (CYS + LYS)	Reactivity Class (CYS)	DPRA Prediction
Positive Control	72.08	63.63	67.86	High	n.a.	Positive
Test Item (filtrate)	19.29	0	9.65	Low	n.a.	Positive
Test item (suspension)	20.25	2.47	11.36	low	n.a.	Positive

SD: Standard deviation

CV: Coefficient of variation

Executive summary:

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.

Since the test item was poorly soluble in any solvent acceptable for the DPRA the required test concentration of 100 mmol/L could not be reached. The test was therefore performed with a suspension at the concentration of 100 mmol/L in acetonitrile and with the filtrate of this suspension (saturated solution). In both cases the concentration of solved test item was < 100 mmol/L.

The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. A depletion of cysteine and lysine peptides became obvious in the DPRA (11.36 % for the suspension / 9.65% for the filtrate). According to the cysteine 1:10/lysine 1:50 prediction model “low reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “positive”.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

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:

adopted Feb 2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the 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 was statistically significant above the threshold of 1.6 at concentrations of 16, 32 and 64 µM in Experiments 1 and 2 and at 64 µM in Experiment 3. These met the assay acceptance criteria. The EC1.5 values were 14.17, 36.90 and 9.54 µM in Experiments 1, 2 and 3, respectively. The value for Experiment 2 was outside the range specified in the protocol (7 to 30 µM).
The average induction values in the three replicates at 64 µM were 3.05, 1.99 and 6.53 in Experiments 1, 2 and 3, respectively. These met the assay acceptance criteria.

Run / experiment:

other: Experiment 1

Parameter:

other: I(max) in Exp 1:

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: Imax in Exp 3:

Value:

1.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: I(max) in Exp 2:

Value:

2.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: based on the results of 3 Experiments on a 2 out of 3 basis: the final result is negative

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
The average coefficient of variation of the luminescence readings for the negative control were 3.83%, 14.14% and 6.83% for Experiments 1 and 2. These met the assay acceptance criteria.
- Acceptance criteria met for positive control: yes (only the EC1.5 value in experiment 2 was outside the range specified)
- Acceptance criteria met for variability between replicate measurements: yes

As all four conditions for a positive prediction were only met in Experiment 2, it was considered that the test article gave a negative prediction in this assay based on the 2 of 3 mode of outcome.

Results table:

Criteria	Experiment 1	Experiment 2	Experiment 3
I max	1.40	2.57	1.47
Cell viability	not applicable	121.36%	not applicable
EC1.5	not calculated	54.47 µM	not calculated
Dose response	Yes	Yes	Yes

Executive summary:

The study was conducted to investigate the potential of Special Brilliant Blue FFR to induce genes that are regulated by the antioxidant response element (ARE).

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final concentration of the stock solution (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium romide (MTT).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was then 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.

The acceptance criteria were met in each experiment, with the exception that in Experiment 2 the EC1.5 for the positive control was 36.90 and as such outside the range specified in the protocol (7 to 30 µM). However, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction with the positive control.

None of the four criteria specified in the prediction model for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in Experiment 1 and 3, whereas all four criteria were met in Experiment 2. It is therefore considered that the test article is predicted as negative in the assay based on 2 out of the 3 experiments giving negative results.

The test article Special Brilliant Blue FFR was considered to be negative in the Luciferase Test ARE-Nrf2 cells derived from HaCaT human keratinocytes.

Reference 3

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

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

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. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

To account for the content of 86.4% a correction factor of 1.16 was included in the dose formulations.

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 8 weeks
- Weight at study initiation: 28 - 34 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: 11 July 2018 To: 07 September 2018

Vehicle:

dimethylformamide

Concentration:

Three concentrations of Special Brilliant Blue FFR (2.5%, 5% and 10% (w/w)), dissolved in N,N-dimethylformamide (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 N,N-dimethylformamide (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 N,N-dimethylformamide (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:

1.068

Test group / Remarks:

2.5% test item in DMF: 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:

1.102

Test group / Remarks:

5% test item in DMF: 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:

0.989

Test group / Remarks:

10% test item in DMF: 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 Special Brilliant Blue FFR at concentrations of 2.5%, 5% and 10% in DMF (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 Tabel 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, Bayscript Gelb BR at concentrations of 1%, 2.5% and25% (w/w) in DMF did not reveal any skin sensitising properties in the local lymph node assay. A 5% (w/w) concentration of the test item in DMF 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 Special Brilliant Blue FFR, 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 DMF.

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

Parameter	negative control (DMF)	2.5% (w/w) test item in DMF	5% (w/w) test item in DMF	10% (w/w) test item in DMF	positive control (20% alpha-hexyl cinnamic aldehyde (v/v) in A/O)	vehicle of positive control
Lymph node cell count	1.000	1.068	1.102	0.989	2.083*	1.000
Lymph node weight	1.000	1.140	1.093	1.070	2.324*	1.000
Ear weight	1.000	0.981	1.068	1.043	1.105*	1.000
Ear thickness	1.000	1.004	1.025	1.008	1.008	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 96.4% (2.5% (w/w) concentration), 95.3 (5% (w/w) concentration) and 105.7% (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 Special Brilliant Blue FFR 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 Special Brilliant Blue FFR (2.5%, 5% and 10% (w/w)), dissolved in N,N-dimethylformamide (DMF) were tested in six female NMRI mice per group and compared to a vehicle control group. DMF 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 DMF after vortexing and ultrasound treatment. Acetone/olive oil (4:1, v/v), propylene glycol and dimethyl sulfoxide, 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, Special Brilliant Blue FFR at concentrations of 2.5%, 5% and 10% (w/w) in DMF did not reveal any skin sensitising properties in the local lymph node assay. A 10% (w/w) concentration of the test item in DMF was the maximum technically feasible concentration.

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.

An assessment of potential skin sensitization properties based on defined approaches and individual sources was conducted for Special Brilliant Blue FFR within an IATA. The outcome of this assessment is given below:

Special Brilliant Blue FFR is a solid with a MW of 790 g/mole, is well soluble in water (> 292 g/L) and a log Pow of -2.49 was dermined for the substance. Based on these physico-chemical properties dermal uptake of the substance can be predicted to be low (ECHA Guidance on Information Requirements, Chapter R7c, 2017) or even negliglible.

Special Brilliant Blue FFR 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 by the OECD QSAR Toolbox (Schlecker, 2017).

DPRA; OECD TG 442C:

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.

Since the test item was poorly soluble in any solvent acceptable for the DPRA the required test concentration of 100 mmol/L could not be reached. The test was therefore performed with a suspension at the concentration of 100 mmol/L in acetonitrile and with the filtrate of this suspension (saturated solution). In both cases the concentration of solved test item was < 100 mmol/L.

The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. A depletion of cysteine and lysine peptides became obvious in the DPRA (11.36 % for the suspension / 9.65% for the filtrate). According to the cysteine 1:10/lysine 1:50 prediction model “low reactivity” was derived for the test item in acetonitrile, leading to a DPRA prediction of “positive”.

KeratinoSens™; OECD TG 442D:

The objective of the study was 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 acording to OECD 442D.

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final concentration of the stock solution (200 mM) with further dilution in DMSO. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test.

None of the four criteria specified in the prediction model for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in Experiment 1 and 3, whereas all four criteria were met in Experiment 2. It is therefore considered that the test article is predicted as negative in the assay based on 2 out of the 3 experiments giving negative results. The test article Special Brilliant Blue FFR was thus considered to be negative in the Luciferase Test ARE-Nrf2 cells derived from HaCaT human keratinocytes.

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

Based on inconsistent results in chemico (DPRA positive) and in vitro (KeratinoSens negative) in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. No information on skin sensitization potency can be derived from skin sensitization hazard reported in the in vitro studies.

Special Brilliant Blue FFR 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 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 Special Brilliant Blue FFR (2.5%, 5% and 10% (w/w)), dissolved in N,N-dimethylformamide (DMF) were tested in six female NMRI mice per group and compared to a vehicle control group. DMF 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 DMF after vortexing and ultrasound treatment. Acetone/olive oil (4:1, v/v), propylene glycol and dimethyl sulfoxide, 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, Special Brilliant Blue FFR at concentrations of 2.5%, 5% and 10% (w/w) in DMF did not reveal any skin sensitising properties in the local lymph node assay. A 10% (w/w) concentration of the test item in DMF was the maximum technically feasible concentration.

Conclusion:

No alert for protein binding was obtained in silico (OECD Toolbox). A weak positive result in the DPRA (in chemico testing) and a negative result in vitro (KeratinoSens negative following the 2 of 3 approach) was obtained. Thus, in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Special Brilliant Blue FFR did not reveal any skin sensitising properties in the mouse local lymph node assay at the maximal technically feasible concentration of 10% and can thus be regarded as non-sensitizer under the conditions of this test. This negative result is in line with the interpretation of the physico-chemical data of the substance that predict low to negliglible dermal absorption in vivo. Overall the compound is considered to be not sensitizing to skin. A low sensitizing potential at higher concentrations cannot be fully excluded because the maximal technically feasible concentration in the in vivo test was 10%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A weak positive result in the DPRA (in chemico testing) and a negative result in vitro (KeratinoSens negative following the 2 of 3 approach) was obtained. Thus, in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Special Brilliant Blue FFR was thus tested in the modified local lymph node assay in mice. Solubility trials with all recommended vehicles for the LLNA were performed that revealed a 10% (w/w) concentration of the test item in DMF as the maximum feasible concentration. Special Brilliant Blue FFR 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. This result is in line with the physico-chemical properties of Special Brilliant Blue FFR that limit or even prevent dermal uptake of the substance. Consequently, no classification is warranted for skin sensitization.