Benzene-1,4-diammonium sulphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

The living tissues treated with the test item were applied less than 8 mg of test item. As it was noted that all tissues were evenly covered by the test item, this deviation was not considered to comprise the outcome of the study.

Principles of method if other than guideline:

To evaluate the skin irritation potential of the test item Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) /E507469 using the EpiskinTM reconstructed human epidermis model.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzene-1,4-diammonium sulphate

EC Number:

240-357-1

EC Name:

Benzene-1,4-diammonium sulphate

Cas Number:

16245-77-5

Molecular formula:

C6H8N2.H2O4S

IUPAC Name:

benzene-1,4-diammonium sulphate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 60810- Expiration date of the lot/batch: 09 November 2018- Purity test date:20 July 2017STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature, protected from humidity- Stability under test conditions:not applicable- Solubility and stability of the test substance in the solvent/vehicle: not applicable- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: the test item was not treated before testing and was used neat- Preliminary purification step (if any): not applicable- Final dilution of a dissolved solid, stock liquid or gel: not applicable- Final preparation of a solid: no final preparationFORM AS APPLIED IN THE TEST (if different from that of starting material) neat

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Human reconstructed epidermis

Cell source:

other: not applicable

Source strain:

other: not applicable

Justification for test system used:

Based on the peer review of the results of an inter-laboratory study with the EpiskinTM model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the EpiskinTM model can be used for distinguishing between skin irritant and non-irritant chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: EpiskinTM- Tissue batch number(s):Not specified- Production date: not specified- Shipping date: not specified- Delivery date:not specified- Date of initiation of testing: 5 March 2018TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 27°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items.- Observable damage in the tissue due to washing: No damage- Modifications to validated SOP: No modificationsMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 0.3 mg/mL- Incubation time: 3 hours (±5 minutes) - Spectrophotometer: plate reader- Wavelength: 570 nm- Filter: no filter was used- Filter bandwidth: no filter was used- Linear OD range of spectrophotometer: not specifiedFUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATAhistorical data were not mentioned in the reportNUMBER OF REPLICATE TISSUES: triplicates per condition were used CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- water-killed tissues- Procedure used to prepare the killed tissues (if applicable): untreated EpiskinTM tissues were placed in a 12 well plate containing 2 mL of water for injections per well. The 12-well plate was placed in an incubator (set at +37°C, 5% CO2) for 48 hours (± 1 hour). At the end of the incubation period, the tissues were dried before being placed in a freezer (-20°C) for at least one night. Once killed, the tissues were stored in the freezer for up to 6 months. Before using, the water-killed tissues were thawed at room temperature (for at least 1 hour ± 5 minutes in 2 mL of maintenance medium).- N. of replicates : triplicates were used- Method of calculation used:-Non-Specific MTT reduction (NSMTT) calculation:-cOD Untreated Killed tissues = ODUK - mean ODblank,-cOD Test Item-treated Killed tissues = ODTIK - mean ODblank.NSMTT = [(mean cODTIK - mean cODUK) / mean cODNC] x 100True MTT metabolic conversion (TODTI):- TODTI = [mean cODTI - (mean cODTIK - mean cODUK)].Then, the relative mean viability is calculated as follows:Relative mean viability = (TODTI / mean cODNC) x 100NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Two independent sequences : pretest and main test QUANTIFICATION OF IL-1 AlphaThe concentration of the IL-1α in the culture medium retained following the 42-hour recovery period was determined only for test items found with a mean relative viability > 50% after the MTT reduction. For these test items, culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA according to our internal procedures. The assay for the determination of IL-1α concentration in EpiskinTM culture medium is an ELISA (Enzyme Linked Immunosorbent Assay) in which anti-IL-1α antibodies are coated in wells of a 96 wells plate. The IL 1α present in the tested samples bind to the immobilised anti-IL-1α antibodies and are revealed using a peroxidase (HRP) conjugated anti-IL-1α antibody.Enzymatic reaction is performed by using a substrate which is transformed into a colored product, which intensity is measured in OD value using a spectrophotometer. The analyte concentration concentrations can be then determined by interpolation from a calibration curve in which OD values are plotted against IL 1α concentrations of calibration standards. PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post exposure period is less than or equal to 50% or higher than 50M associated with a Il-1 alpha higher than 60 pg/mL- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure and 42 hours post exposure period is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit):10 mg ± 2mg- Concentration (if solution): pureVEHICLENo vehicle was usedNEGATIVE CONTROL- Amount(s) applied (volume or weight): 10 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 10µL- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (true MTT metabolic conversion)

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

other: the IL-1α concentrations (pg/mL)

Run / experiment:

Test item

Value:

< 5

Negative controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:- Visible damage on test system: no damage- Direct-MTT reduction: The MTT solution containing the test item turned blue/purple when compared with the negative control. The test item was therefore considered to have direct MTT reducing properties. As a result, additional controls were performed on water-killed tissues in parallel to the main test.- Colour interference with MTT: During this test, as the water solution containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.DEMONSTRATION OF TECHNICAL PROFICIENCY: Positive control condition showed proficiency of the test system to quantify tissue viabilityACCEPTANCE 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-

Any other information on results incl. tables

Table 1 :Results

	cOD		Viability(%)		NSMTT
Groupe	Mean	SD	Mean	SD
Negativecontrol	0.738	0.031	100	4
positive control	0.026	0.004	4	1
test item	0.739	0.085	100	12
test item (deadtissues)	0.079	0.013	11	2	6
untreated(deadtissues)	0.036	0.015	5	2
Test item (true MTT metabolic conversion)	0.697		94

cOD=blankcorrectedopticaldensity

MTT = « -(4,5-dimethylthiazol-2-ylà-2,5-diphenyltetrazoliumbromide

NSMTT= nonspecificMTTreduction

SD = standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental condition of the study, the test item did not induced decrease of tissue viability (94%) and no IL1-alpha release in medium (under 5pg/mL) when applied on EpiskinTM. Hence, , the test item, E507469, is considered to be non-irritant to skin.According to the results of this study, the classification of the test item should be: No Category (UN GHS and Regulation (EC) No. 1272/2008).

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item, E507469, using the EpiskinTM reconstructed human epidermis model.

The study design was based upon the international guideline OECD No. 439. The study was conducted in compliance with the principles of Good Laboratory Practice.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).

In addition, the concentration of the inflammatory mediator IL-1α was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary tests, the test item was found to have direct MTT reducing properties but no colouring properties. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 94%.

As the mean relative viability > 50% after the MTT reduction, the IL-1α concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1α concentration value of two tissues was found below the limit of quantification (< 5.00 pg/mL). Consequently, the mean IL-1α concentration from the three test item-treated tissues was not calculated. The IL-1α concentration value of the other test item-treated tissue was found < 60 pg/mL (11.2 pg/mL). Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

Under the experimental conditions of this study, the test item, E507469, is considered to be non-irritant to skin.

According to the results of this study, the classification of the test item should be:      No Category (UN GHS and Regulation (EC) No. 1272/2008).