Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Endpoint summary

Description of key information

No alert for protein binding was obtained in silico (OECD Toolbox). However, due to a weak positive result in the DPRA (in chemico testing) and inconsistent results in vitro (KeratinoSens negative and h-CLAT positive), in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Bayscript Gelb BR did not reveal any skin sensitising properties in the mouse local lymph node assay at the maximal technically feasible concentration of 5% 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. Additionally, no skin sensitizing effects were reported in occupational settings. 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 5%.

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:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on 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.

Positive control results:

The positive control cinnamic aldehyde led to a depletion of 71.49% cysteine peptide and 61.72% lysine peptide. The mean cysteine/lysine peptide depletion was calculated with 66.61% leading to a positive result (high reactivity class according to the cysteine 1:10/lysine 1:50 prediction model).

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean % cysteine depletion

Value:

25.3

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

reactive versus cysteine peptides

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean % lysine depletion

Value:

0.04

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

not reactive versus lysine peptides

Run / experiment:

other: mean of 2 runs; percent peptide depletion

Parameter:

other: reactivity in the cysteine 1:10/lysine 1:50 prediction model

Value:

12.67

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

DPRA prediction: positive (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

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 visually appeared a clear solution in water at the test concentration of 100 mmol/L this solvent was used in the DPRA. No precipitaton of the test item occured before or after incubation. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. Slight depletion of cysteine but not lysine peptides became obvious in the DPRA. The mean cystein/lysine peptide depletion was calculated with 12.67%. According to the prediction model “low reactivity” was derived for the test item in water, 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:

of February 2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Calculations of all test article concentrations stated in this report include a correction for content (purity) using a factor of 1.161.
Test formulations were prepared using dimethyl sulfoxide (DMSO). This was the first of the listed vehicles that produced a visually clear solution at a concentration of 200 mM.

Details on the 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:

The assay aceptance criteria for the positive controls were met: 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 Experiments 1 and 2.
The EC1.5 values for the positive control were 12.10 and 9.36 µM in Experiments 1 and 2, respectively. The average induction for the positive control at 64 µM was 5.69 in Experiment 1 and 9.41 in Experiment 2. Although the latter value was above the range specified in the protocol, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 10.55% and 14.43% in Experiments 1 and 2, respectively.

Run / experiment:

other: experiment 1

Parameter:

other: Imax

Value:

1.03

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: experiment 2

Parameter:

other: Imax

Value:

1.16

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: experiment 1 and 2

Parameter:

other: EC1.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5 > 2000 µM in both experiments

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 10.55% and 14.43% in Experiments 1 and 2, respectively.

All assay acceptance criteria were met.

Since the Imax in both experiments was less than 1.5-fold, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test.

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 acording to OECD 442D. The test article was dissolved in water 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 1.03 and 1.16 for experiments 1 and 2, respectively. There were no EC1.5 values for Experiments 1 or 2 determinable as there were no statistically significant increases in induction (EC1.5 > 2000 µM). In Experiments 1 and 2, there was no apparent overall dose response for luciferase and the dose response curves were not biphasic.

All assay acceptance criteria were met. Since the Imax in both experiments was less than 1.5-fold, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test.

Reference 3

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:

other: OECD Guideline 442E (In vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT))

Version / remarks:

adopted 09 October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Calculations of all test article concentrations stated in this report thus include a correction for content (86.1%) using a factor of 1.161.

Details on the study design:

The test item was examined for sensitising properties in the human Cell Line Activation test (h-CLAT). The h-CLAT is an in vitro assay proposed to address the third key event of the skin sensitisation AOP (Adverse Outcome Pathway) by quantifying changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.
The h-CLAT has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The test article was soluble in cell culture medium at 116.1 mg/mL giving a maximum test concentration of 1000 µg/mL.
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value is used to determine the concentration of test item for the CD86/CD54 expression measurement. No effect on viability was noted, therefore the dose finding assay was repeated using the maximum attainable concentration of 150 mg/mL in saline, giving a maximum test concentration of 1292 μg/mL. No reduction in viability was observed.
For the expression measurements, test concentrations in a range from 361 to 1292 μg/mL (after dilution in medium) were used.
DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.
Following cell staining with fluorochrome-tagged antibodies the expression of CD86 and CD54 is analysed via flow cytometry with the aquisition chanel FL-1. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control. The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) is also calculated.

Positive control results:

RFI values for positive control with regard to CD86 were all > 150 : 286 and 489
RFI values for positive control with regard to CD54were all > 200: 873 and 1038

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity (RFI) for CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity (RFI) for CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: 1 and 2

Parameter:

other: relative fluorescence intensity (RFI) for CD86

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

at all tested concentrations; EC200 < 375.1 µg/mL

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
All assay acceptance criteria were met.
The cell viability of the solvent controls (medium) was higher than 90% in each independent run.
In the solvent control (medium), RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For solvent control (medium), the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Relative fluorescence intensity (RFI) values:

Concentration (µg/mL)	RFI CD86 - 1st Exp.	RFI CD86 - 2nd Exp.	RFI CD54 - 1stExp.	RFI CD54 - 2nd Exp.
360.6	128	139	293	897
432.7	111	145	293	831
519.3	97	118	242	594
623.1	117	122	257	515
747.7	165	128	56	442
897.3	268	121	-188	420
1076.6	109	130	249	412
1292.1	157	127	120	288
sovent control (medium)	100	100	100	100
positive control (DNCB)	286	489	873	1038

In Experiment 1, RFI values were >150% for CD86 and were >200% for CD54. In Experiment 2, RFI values were <150% for CD86, but were >200% for CD54.

The RFI values for CD54 were >200% in both experiments, it was considered that the test article gave a positive prediction in this assay.

Conclusions:

The test article was considered to be positive in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of the test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). 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 human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

The test article was soluble in cell culture medium at 116.1 mg/mL, giving a maximum test concentration of 1000 µg/mL. Dose finding assays were conducted to determine a concentration showing 75% THP-1 cell survival (CV75) compared to the solvent control. No effect on viability was noted, therefore the dose finding assay was repeated using the maximum attainable concentration of 150 mg/mL in saline, giving a maximum test concentration of 1292 μg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 361 to 1292 μg/mL (after dilution in medium) were used. In Experiment 1, the relative fluorescence intensity (RFI) values of the test article were >150% for CD86 and were >200% for CD54. In Experiment 2, the RFI values were <150% for CD86, but were >200% for CD54.

As the results were concordant for CD54 it was considered that the test article gave a positive prediction in this assay.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment. The test article, Bayscript Gelb BR, was considered to be positive in the human Cell Line Activation Test.

Reference 4

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 86.1% 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 10 weeks
- Weight at study initiation: 28 - 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: 28 August 2018 To: 11 October 2018

Vehicle:

dimethylformamide

Concentration:

Three concentrations of Bayscript Gelb BR (1%, 2.5%, and 5% (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 5% (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 1%, 2.5%, and 5% (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 1%, 2.5%, and 5% (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 1%, 2.5%, and 5% (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:

1.226

Test group / Remarks:

1% 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:

1.24

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:

1.355

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 for skin sensitization

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In the main study treatment with Bayscript Gelb BR at concentrations of 1%, 2.5% and 5% 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 1%, 2.5% and 5% (w/w) of Bayscript Gelb BR, 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 5% (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)	1% (w/w) test item in DMF	2.5% (w/w) test item in DMF	5% (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	1.226	1.240	1.355	1.643*	1.000
Lymph node weight	1.000	1.136	1.136	1.295*	1.875*	1.000
Ear weight	1.000	0.952	0.979	0.994	1.076*	1.000
Ear thickness	1.000	0.968	0.992	0.976	1.093	1.000

* significantly different from control at p ≤ 0.01

The analysis of the test item vehicle solutions of the 1%, 2.5% and 5% 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 101.6% (1% (w/w) concentration), 99.1 (2.5% (w/w) concentration) and 101.9% (5% (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 Bayscript Gelb BR 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 Bayscript Gelb BR (1%, 2.5% and 5% (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 5% (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 1%, 2.5% and 5% (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, Bayscript Gelb BR at concentrations of 1%, 1.5% and 5% (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 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. However, no skin irritating or skin sensitizing effects were reported in occupational setting.

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

Bayscript Gelb BR is a solid with a high MW of 1067 g/mole, is well soluble in water (71.33 g/L) and a log Pow of -2.88 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.

Bayscript Gelb BR was investigated in silico for structural activity relationship (QSAR), in chemico for peptide reactivity, and in vitro for keratinocyte activation (KeratinoSens) and dendritic cell activation (h-CLAT). 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 visually appeared a clear solution in water at the test concentration of 100 mmol/L this solvent was used in the DPRA. No precipitaton of the test item occured before or after incubation. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. Slight depletion of cysteine but not lysine peptides became obvious in the DPRA. The mean cystein/lysine peptide depletion was calculated with 12.67%. According to the prediction model “low reactivity” was derived for the test item in water, 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 water 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 1.03 and 1.16 for experiments 1 and 2, respectively. There were no EC1.5 values for Experiments 1 or 2 determinable as there were no statistically significant increases in induction (EC1.5 > 2000 µM). In Experiments 1 and 2, there was no apparent overall dose response for luciferase and the dose response curves were not biphasic.

All assay acceptance criteria were met. Since the Imax in both experiments was less than 1.5-fold, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test.

h-CLAT; OECD TG 442E:

The study was conducted to investigate the potential of the test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). 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 human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

The test article was soluble in cell culture medium at 116.1 mg/mL, giving a maximum test concentration of 1000 µg/mL. Dose finding assays were conducted to determine a concentration showing 75% THP-1 cell survival (CV75) compared to the solvent control. No effect on viability was noted, therefore the dose finding assay was repeated using the maximum attainable concentration of 150 mg/mL in saline, giving a maximum test concentration of 1292 μg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 361 to 1292 μg/mL (after dilution in medium) were used. In Experiment 1, the relative fluorescence intensity (RFI) values of the test article were >150% for CD86 and were >200% for CD54. In Experiment 2, the RFI values were <150% for CD86, but were >200% for CD54. As the results were concordant for CD54 it was considered that the test article gave a positive prediction in this assay.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment. The test article, Bayscript Gelb BR, was considered to be positive in the human Cell Line Activation Test.

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, h-CLAT positive) 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.

Bayscript Gelb BR 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 Bayscript Gelb BR (1%, 2.5% and 5% (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 5% (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. Methyl ethyl ketone was not applicable for analytics.

In the main study treatment at concentrations of 1%, 2.5% and 5% (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, Bayscript Gelb BR at concentrations of 1%, 1.5% and 5% (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.

Conclusion:

No protein binding alert was obtained in silico (OECD Toolbox). However, due to inconsistent results in chemico and in vitro, with a weak positive outcome of the Direct Peptide Reactivity Assay (DPRA, OECD 442C),  a negative outcome of the KeratinoSens assay (OECD 442D) and a positive outcome of the h-CLAT (OECD 442E), in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Bayscript Gelb BR 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 5% (w/w) concentration of the test item in DMF after vortexing and ultrasound treatment as the maximum feasible concentration. The concentrations tested were chosen according to OECD Guideline 429 with 1%, 1.5% or 2% (w/w) in DMF. The test item did not reveal any skin sensitising properties in the mouse local lymph node assay. Bayscript Gelb BR can thus be regarded as non-sensitizer under the conditions of the mouse LLNA. This result is in line with the physico-chemical properties of Bayscript Gelb BR that limit dermal uptake of the substance. Additionally, no skin sensitizing effects were reported in occupational settings. 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 5%.

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). However, due to inconsistent results in chemico (DPRA weak positive) and in vitro (KeratinoSens negative, h-CLAT postitive) in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Bayscript Gelb BR was thus tested in the modified local lymph node assay in mice. Bayscript Gelb BR 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 Bayscript Gelb BR that limit or even prevent dermal uptake of the substance. Additionally, no skin sensitizing effects were reported in occupational settings. Consequently, no classification is warranted for skin sensitization.