Sodium 2-[[4-(cyclohexylamino)-9,10-dihydro-9,10-dioxo-1-anthryl]amino]-5-ethoxybenzenesulphonate

Endpoint summary

Description of key information

Non sensitising

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:

From June 11th to June 20th, 2019

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

-Purpose: the DPRA is a chemistry-based assay (based on the OECD guideline for the testing of chemicals, In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD document TG 442C) exploiting the fact that most chenmical allergens have electrophilic properties and are therefore able to reacte with the nucleophilic sidechains of amino acids to form covalent bonds. The underLying rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitizer. The endpoint measured in the assay is the percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures following an approximate 24 hour (22-26 hours) incubation with the test item. The percentage of peptide depletion is calculated by High Performance Liquid Chromatography using ultra-violet detection. The DPRA test allows quantification of a chemical's reactivity and is used to categorize a substance in one of four classes of reactivity to allow discrimination between skin sensitizing and non-sensitizing chemicals and thus assess their sensitization potential.

-Apparatus: HPLC = Waters Alliance 2695 separation module and 2487 dual wavelenght detector; balances fitted with printers = capability of weighing to 5 decimal places; general laboratory apparatus and glassware.
-Assessment of test item solubility: the solubility of test item was assessed at a concentration of 100 mM in acetonitrile and acetonitrile/water 50/50 v/v.

-Preparation of peptide stock solutions: stock solutions of each peptide at a concentration of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 ml aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).

-Preparation of peptide calibration standards: calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

-Preparation of stability controls and precision controls: stability controls (reference control B, n=6) and precision control (reference control A) of both peptides were prepared at a concentration of 0.5 mM. Reference control A and reference control B were prepared with buffer and acetonitrile, reference control C (n=3) was prepared with buffer and acetonitrile/water 50/50 v/v.

-Preparation of positive control solution and test item stock solution: the positive control chemical (cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of test item was prepared in acetonitrile/water 50/50 v/v.

-Preparation of positive control solution and cysteine peptide depletion samples and co-elution controls: triplicate solutions each of test item and the positive control stocks were diluted with the cysteine peptide to prepare final solutions containing 0.5 mM cysteine and 5 mM of either test item or the positive control. For the co-elution control, blank buffer solutions was used in place of the cysteine stock solution.

-Preparation of positive control and lysine peptide depletion samples and co-elution controls: triplicate solutions each of test item and the positive control stocks were diluted with the lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either test item or positive control. For the co-elution control, blank buffer solution was used in the place of the Lysine stock solution.

-Incubation: the appareance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run appearance of the samples in the vials was assessed and documented again.

-Analysis: the concentration of each peptide in the presence of the test item and the associated positive controls were quantified by HPLC using UV detection.

-Calculations: the peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - [Peptide peak area in replicate depletion samples (×100)/ Mean Peptide peak area of reference control samples B or C]

Run / experiment:

other: mean of 3 run

Parameter:

other: mean depletion cysteine %

Value:

84.7

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Mean of 3 run

Parameter:

other: mean depletion positive control %

Value:

71.7

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

-Solubility assessment: the solubility of the test item in acetonitrile/water 50/50 v/v at a nominal concentration of 100 mM was confirmed. Test item was insoluble in acetonitrile at 100 mM.

-Reactivity assessment: all analytical acceptance criteria for each peptide run were met, though due to significant co-elution of the test-item with the lysine peptide only the result of the cysteine peptide assay has been used to make the DPRA prediction. The lysine peptide results presented are for information only. Reactivity is classed as "moderate" and the DPRA prediction is positive and test item is thus predicted skin sensitizer by this assay.

	Peptide	Linearity	Positive control depletion ( % )	Reference controls	Test item
Acceptance criteria	Cysteine	>0.99	60.8-100	0.45-0.55 mM	SD <14.9 %
			(SD <14.9 %)	(CV <14.9 %)
	Lysine	>0.99	40.2-69.0	0.45-0.55 mM	SD<11.6 %
			(SD <11.6 %)	(CV <14.9 %)
Achieved results	Cysteine	>0.999	71.7	B: 0.501 mM (CV=0.67 %, n=6)	SD 2.80 %
			(SD 0.44 %, n=3)	C: 0.490 mM (CV=1.23 %, n=3)
	Lysine	>0.999	60.4	B: 0.540 mM (CV=3.56 %, n=6)	SD 1.48 %
			(SD 7.53 %, n=3)	C: 0.539 mM (CV=0.44 %, n=3)

CV = Coefficient of variation

Depletion of peptide in the presence of test item

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide in test samples (µV.sec)	Mean peptide depletion ( % )
Cysteine	B: 898360 (n=6)	134250 (n=3)	84.7
	C: 879920 (n=3)
Lysine	B: 744350 (n=6)	74067^1(n=3)	90
	C: 742560 (n=3)

Note: test item co-elutes with the lysine peptide. The co-elution peak is 10.8 % of the reference control peak area. Therefore, the cysteine 1:10 reactivity prediciton model has been applied.

Depletion

Mean of cysteine % depletion	Reactivity Class	DPRA Prediction
0 %≤ Cys % depletion ≤13.89 %	No or minimal reactivity	Negative
13.89 %< Cys % depletion ≤23.09 %	Low reactivity	Positive
23.09 %< Cys % depletion ≤98.24 %	Moderate reactivity
98.24 %< Cys % depletion ≤100 %	High reactivity

Co-elution occured during the lysine assay

Cysteine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^2(µg/ml)	Peptide Depletion ( % )	Mean Depletion  ( % )	SD ( % )
Positive control^1	250163	104	72.23 ^3	71.7	0.44
	257948	107	71.33 ^3
	255201	106	71.63 ^3
Test Item	160822	66	81.74 ^4	84.7	2.8
	129641	52.9	85.34 ^4
	112290	45.6	87.24 ^4

CV = Coefficient of Variation

¹ Data generated under PV22VQ

² Samples prepared at a concentration of 376µg/ml (0.5 mM)

³ Calculated against a mean reference control B peak area of 898360 µV.sec(n=6)

⁴ Calculated against a mean reference control C peak area of 879920 µV.sec(n=3)

Lysine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^2(µg/ml)	Peptide Depletion^3 ( % )	Mean Depletion  ( % )	CV ( % )
Positive control^1	253561	141	65.93 ^3	60.4	7.53
	271548	151	63.53 ^3
	358374	200	51.93 ^3
Test Item	76716	40.3	89.74 ^4	90	1.48
	83454	44.2	88.84 ^4
	62032	32	91.64 ^4

Data presented for information only

CV = Coefficient of Variation

¹ Data generated under PV22VQ

² Samples prepared at a concentration of 388 µg/ml (0.5 mM)

³ Calculated against a mean reference control B peak area of 744350µV.sec(n=6)

⁴ Calculated against a mean reference control C peak area of 742560µV.sec(n=3)

Interpretation of results:

other: the test result is used in a global assessment to decide on the classification within the CLP Regulation (EC 1272/2008)

Conclusions:

Based on a depletion of cysteine peptide of 85 %, reactivity of test item is "moderate" and hence the DPRA prediction is positive as based on this assay.

Executive summary:

The study was run according to OECD guideline 442C to assess the reactivity and sensitizing potential of test item.

Solutions of test item were successfully analysed by the validated DPRA analytical method (Covance Analytical Method FIA/M101/15) in just the cysteine containing synthetic peptide. There was significant co-elution (ca 11 %) in the lysine containing synthetic peptide and therefore only the result of the cysteine is reported. With depletion of the cysteine peptide of 85 %, reactivity of test item is "moderate" and hence the DPRA prediction is positive as based on this assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 28 to February 14, 2019

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:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol no 155: KeratinoSens™

Version / remarks:

March 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

CELLS
- Cell culture: transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct.
- Supplier: Givaudan (Dubendorf, Switzerland).
- Subculturing: the cells were routinely grown and subcultured in maintenance medium at 37 ± 2 °C in a humidified atmosphere containing 5 % CO2 in air. Maintenance medium was 500 ml Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM), supplemented with 50 ml foetal bovine serum (FBS) and 5.5 ml Geneticin.
- Preparation of test cell cultures: the cell suspension was diluted with maintenance medium without geneticin to give 1 × 10^5 viable cells/ml and 100 µl volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. One well of each plate received 100 µl maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2 °C in a humidified atmosphere of 5 % CO2 in air, to allow the cells to attach.

POSITIVE CONTROL
Cinnamic aldehyde

REPLICATES
Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

TEST ITEM SOLUBILITY
Prior to commencing testing, the solubility of the test item in DMSO was assessed and it was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline.

TEST ITEM PREPARATION
A stock solution of the test item was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO. Serial dilutions were prepared using DMSO to obtain 12 master concentrations. The master concentrations were then further diluted 25 fold in DMEM, supplemented with 5.0 ml foetal bovine serum to give working solutions. The working solutions were for treatment with a further 4 fold dilution factor so that the final concentrations of the test item ranged from 0.98 to 2000 μM (based on a dilution factor of 2).

PREPARATION OF DILUTION PLATES
The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µl of assay medium to each well and then 10 µl solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 ml DMEM, supplemented with 5.0 ml FBS.

TREATMENT OF CULTURE PLATES
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µl of assay medium was added to every well of the 96 well plates. 50 µl from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2 °C, in a humidified atmosphere of 5 % CO2 in air for 48 ± 2 hours.

CELL VIABILITY MEASUREMENT
After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µl fresh assay medium was added to each well.10 µl of MTT solution (5 mg/ml in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2 °C in a humidified atmosphere of 5 % CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µl of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2 °C, in a humidified atmosphere of 5 % CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.

LUCIFERASE MEASUREMENT
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. Frozen reconstituted reagent was used for both tests and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper.
100 µl of fresh assay medium was added to each well before 100 µl of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

INTERPRETATION OF THE RESUULTS
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is ≥1.5 fold and statistically significantly different as compared to the solvent vehicle control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is >70 % at the lowest concentration with induction of luciferase activity ≥1.5 fold (i.e. at the EC1.5 determining concentration);
- the EC1.5 value is <1000 µM;
- there is a dose-response increase for luciferase induction (or a biphasic response).
If in a given test, all of the first three conditions listed above are met, but a clear dose-response for the luciferase induction cannot be observed, then the result of that test should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM and that do not reach cytotoxicity (< 70 % viability) at the maximal tested concentration should also be considered as inconclusive.

ACCEPTANCE CRITERIA
In order for an assay to be accepted the following criteria must be met:
- the luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 (e.g. using a t test) in at least one of the tested concentrations (4 to 64 µM);
- the EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control;
- the average coefficient of variation of the luminescence reading for the negative solvent control (i.e. DMSO) should be below 20 % in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.

Run / experiment:

other: test 1

Parameter:

other: Imax

Value:

0.83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: test 2

Parameter:

other: Imax

Value:

0.88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The Imax for test item was 0.83 in test 1 and 0.88 in test 2.
The Imax for both tests was <1.5 fold compared to the DMSO control and thus the EC1.5 could not be calculated.
The cellular viability fell below 70 % in both tests: the IC30 value was 42.30 µM in test 1 and 43.66 µM in test 2 and the IC50 values were 56.81 µM and 73.63 µM in tests 1 and 2, respectively. No overall dose-response for luciferase induction was recorded.

POSITIVE CONTROL
The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 7.29 μM and 9.95 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 3.75 and 3.65 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

NEGATIVE CONTROL
The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 9.4 % and 11.4 % for test 1 and 2, respectively, which met the acceptance criterion of below 20 %.

Results for test item - test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.83	0.82	0.69	0.7	0.58	0.59	0.66	0.04	0	0	0	0
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	108.12	113.19	110.4	106.35	103.06	85.23	42.15	5.32	4.81	4.9	5.49	5.83
Imax	0.83
EC1.5 (µM)	N/A
IC30 (µM)	42.3
IC50 (µM)	56.81

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax≥ 1.5 fold and statistically significant	No
Is the cellular viability >70 % at the lowest concentration at the EC1.5 determining concentration	N/A
Is the EC1.5 value <1000 µM	N/A
Is there a dose-response increase for luciferase induction	No
KeratinoSens™ prediction	Negative

Results for test item - test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.87	0.88	0.84	0.68	0.69	0.52	0.74	0.15	-0.02	-0.02	-0.01	-0.01
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	117.03	112.95	113.87	103.71	99.46	76.55	60.06	3.58	4.41	4.08	4.75	4.5
Imax	0.88
EC1.5 (µM)	N/A
IC30 (µM)	43.66
IC50 (µM)	73.63

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax≥ 1.5 fold and statistically significant	No
Is the cellular viability >70 % at the lowest concentration at the EC1.5 determining concentration	N/A
Is the EC1.5 value <1000 µM	N/A
Is there a dose-response increase for luciferase induction	No
KeratinoSens™ prediction	Negative

Results for Cinnamic Aldehyde - test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.17	1.57	1.8	2.28	3.75
Statistically significant	No	Yes	Yes	Yes	Yes
Viability (%)	113.28	113.78	116.65	112.09	120.12
Imax	3.75
EC1.5 (µM)	7.29
IC30 (µM)	N/A
IC50 (µM)	N/A

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (3.75)	Pass
EC1.5 of positive control within two standard deviations of the historical mean (‑5.54 to 36.76)	Yes (7.29)	Pass
CV % of blank values < 20 %	Yes (9.4 %)	Pass

Results for Cinnamic Aldehyde - test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.18	1.43	1.71	2.28	3.65
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	105.04	110.04	113.54	118.7	113.95
Imax	3.65
EC1.5(µM)	9.95
IC30 (µM)	N/A
IC50 (µM)	N/A

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (3.65)	Pass
EC1.5 of positive control within two standard deviations of the historical mean (-5.54 to 36.76)	Yes (9.95)	Pass
CV% of blank values < 20 %	Yes (11.4 %)	Pass

Interpretation of results:

other: the test result is used in a global assessment to decide on the classification within the CLP Regulation (EC 1272/2008)

Conclusions:

Negative in this assay.

Executive summary:

The purpose of the study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™). The test was conducted according to OECD guideline 442D (2018) and DB-ALM Protocol no 155: KeratinoSens™ (2018).

The I_max for test item was 0.83 in test 1 and 0.88 in test 2. The I_max for both tests was <1.5 fold compared to the DMSO control and thus the EC1.5 could not be calculated. The cellular viability fell below 70 % in both tests: the IC30 value was 42.30 µM in test 1 and 43.66 µM in test 2 and the IC50values were 56.81 µM and 73.63 µM in tests 1 and 2, respectively. Graphs showed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the substance is not a skin sensitizer.

Conclusion

The substance is not a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From October 9th to 23rd, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Females: nulliparous and non-pregnant
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.5 g -22.4 g
- Housing: one group per cage (4 animals per group) Makrolon type-3 cages with standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

other: ethanol:water, 1:1 v/v

Concentration:

non-GLP pretest: 1 %, 2.5 %, 5 % and 10 % w/v
main study: 2.5 %, 5 %, 10 % w/v
The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

No. of animals per dose:

4

Details on study design:

TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2.5 %, 5 % and 10% w/v in ethanol:water, 1:1 v/v. The application volume 25 µl, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were administered with 250 µl of 82.43 µCi/ml 3HTdR (equalto 20.6 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized.
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing three times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for about 42 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION OF RAW DATA
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- first, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
The decision to select a stimulation index of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

OBSERVATIONS
ln addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: twice daily from acclimatization start to the termination of in-life phase.
Body weights: at acclimatization start and prior to necropsy.
Clinical signs (local/ systemic): daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

SI of 2.9, 2.6 and 7.1 were determined with the test item at concentrations of 5 %, 10 % and 25 % w/v in acetone:olive oil, 4:1 v/v. The positive control showed an allergenic potency when tested at concentration of 25 % w/v.
EC3 is the estimated concentration for a SI of 3. ln this study EC3 of 11.3 % w/v was theoretically calculated with SI of 2.6 and 7.1 at test item concentrations of 10 % and 25 % w/v.

Parameter:

SI

Value:

1.8

Test group / Remarks:

group 2 - 2.5 % w/v

Parameter:

SI

Value:

1.5

Test group / Remarks:

group 3 - 5 % w/v

Parameter:

SI

Value:

1.7

Test group / Remarks:

group 4 - 10 % w/v

Cellular proliferation data / Observations:

An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below Sl value of 3.
VIABILITY / MORTALITY
No deaths occurred during the study period
CLINICAL SIGNS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
BODY WEIGHTS
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

Non sensitising.

Executive summary:

The allergenic potential of test substance was tested in 3 groups of 4 female mice were each treated with the test item at concentrations of 2.5 %, 5 % and 10 % w/v in ethanol:water, 1:1 v/v by topical application to the dorsum of each ear lobe (left and right) on 3 consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 1:1 v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately 5 hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period.

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3 -fold or greater than that recorded in control mice, as indicated by the stimmulation index (SI).

Group 4 - 10 % w/v       SI 1.7

Group 3 - 5 % w/v       SI 1.5

Group 2 - 2.5 % w/v       SI 1.8

An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below SI value of 3.

CONCLUSION

SI of 1.8, 1.5 and 1.7 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % w/v in ethanol:water, 1:1 v/v.

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the SI.

Based on these criteria, test item was found to be a non sensitizer when tested up to 10 % w/v in ethanol:water, 1:1 v/v.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The evaluation of the test skin sensitising potential of Acid Blue 280 relied on both in vitro and in vivo data.

In vitro/in chemico tests were run on Acid Blue 280 to assess the 3 key events of skin sensitisation, contributing to the AOP (adverse outcome pathway) of skin sensitisation. In particular, the following tests were run:

- in chemico Direct Peptide Reactivity Assay (DPRA) according to OECD 442C; the test gave a positive response;

- in vitro ARE-Nrf2 Luciferase Test (KeratinoSens™) according to OCED guideline 442D; the test gave a negative response;

- in vitro Human Cell Line Activation Test (h-CLAT) according to OECD guideline 442E; the test was not reliable as test item interfered with the cytotoxicity interpretation during the test due to its colouration.

Based on these results, no conclusive decision on the skin sensitising potential of Acid Blue 280 could be reached. An in vivo data was needed to clarify the skin sensitising potential of Acid Blue 280.

As no in vivo data on Acid Blue 280 was available, a data on a structural analogue of Acid Blue 280, i.e. Similar Substance 02, was taken into account. Details on the read across are reported in IUCLID section 13.

In particular, Similar Substance 02 was tested in vivo in a Local Lymph Node Assay (LLNA) according to OECD guideline 429. In this test, groups of 4 female mice were treated with test item at concentration of 2.5, 5 and 10 % w/v in a vehicle of ethanol:water 1:1 v/v. Stimulation Index (SI) were measured and found to be 1.8, 1.5 and 1.7 in each group, respectively. As SI were all below the threshold value of 3, Similar Substance 02 was considered as not sensitising and EC3 was not calculated.

Overall, an integrated evaluation of all available data allowed to conclude that Acid Blue 280 is devoid of a skin sensitising potential.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), skin sensitiser means a substance that will lead to an allergic response following skin contact.

Substances shall be classified as skin sensitisers in accordance with the criteria:

Category 1: substances shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub- categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

Sub- category 1A: substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Sub- category 1B: substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Overall, based on available experimental data including in chemico, in vitro and in vivo results, Acid Blue 280 is considered as devoid of a skin sensitising potential and is not classified within the CLP Regulation (EC 1272/2008).