Reaction products of naphthalene, benzyl alcohol, sulphuric acid, formaldehyde and ammonia

Endpoint summary

Description of key information

As the percentage tissue viability of 25 mg Aryl sulphonate condensate when applied to Reconstructed Human Epidermis (EpiDerm™) was not greater than 50 % of the negative control, the registered substance has been categorised as a skin irritant (Category 2) under the UN Globally Harmonised System.

An experiment for eye irritation was conducted using Reconstructed Human Cornea-like Epithelium (RhCE) (EpiOcular™model) to ascertain the eye irritation potential of aryl sulphonate condensate. Given a resulting percentage tissue viability of 13.1 ± 0.47 (less than 60 % of the negative control), a classification of irritant (UN GHS Category 1 or Category 2) was recommended. A further experiment for eye damage was subsequently performed, in which Aryl sulphonate condensate was concluded to be a Category 1 eye damage (irreversible effects on the eye). This was based on a calculated In Vitro Irritancy Score (IVIS) of 68.9 following 4-hour exposure in a Bovine Corneal Opacity and Permeability (BCOP) test (CLP Regulation (EC) No 1272/2008).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 25 - 28, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

Reconstructed Human Epidermis - EpiDerm™ (EPI-200-SIT)

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: 00267

Justification for test system used:

Reconstructed Human Epidermis - EpiDerm™ (EPI-200-SIT) has been selected as the test system for in vitro skin irritation as it is recommended in OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT).
- Tissue lot number: 25850.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes and the remaining period of the 60 minute exposure within a sterile hood (temperature not specified).
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 43 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Sterile DPBS was used to rinse the tissue by filling and emptying the tissue insert fifteen times in order to remove any residual test item. The constant stream of DPBS was applied from the nearest distance to the tissue surface. After fifteen rinses, the inserts were completely submerged three times in approximately 50 mL of DPBS and shaken to remove all traces of test item/NC/PC. Finally, each tissue was rinsed once from the inside and once from the outside with sterile DPBS. The excess of DPBS was removed by gentle shaking of the insert and blotting the insert on sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution was prepared by thawing the MTT concentrate (MTT-100-CON) of 1 mL (5 mg/mL) and diluting this to 5 mL by adding 4 mL of MTT diluent (MTT-100-DIL) to obtain the final concentration of 1 mg/mL.
- Incubation time: 3 hours ± 5 minutes.
- Spectrophotometer: 96-well plate spectrophotometer.
- Wavelength: 570 nm (OD570).

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Pass - 1.448 ± 0.05.
- Barrier function: Pass - 6.16 hours.
- Morphology: Pass - Tissue viability and the barrier function test indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers. Well differentiated epidermis consisting 11 layers. Tissue had an average thickness of 119.2 µm.
- Contamination: Pass - sterile and no contamination.

NUMBER OF REPLICATE TISSUES: Triplicate.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be an irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be a non-irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg.

NEGATIVE CONTROL
- Amount applied: 30 µl.

POSITIVE CONTROL
- Amount applied: 30 µl.

Duration of treatment / exposure:

60 ± 1 minutes.

Duration of post-treatment incubation (if applicable):

22 hours of incubation and an additional 19 hours after a media change. The bottom of all tissues was then blotted and transferred into MTT solution and incubated for 3 hours. Incubation occurred over 43 hours in total.

Number of replicates:

Triplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Did not reduce MTT.
- Colour interference with MTT: No colour change.

DEMONSTRATION OF TECHNICAL PROFICIENCY: All kit components were examined for integrity. All information about supplied material was recorded and documented. MTT diluent and vial containing the MTT concentrate were stored in the refrigerator (2 - 8 °C) and in the deep freezer (-20 ± 5 °C).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - Mean OD570 was 1.302, which is within the range of ≥1 and ≤2.5.
- Acceptance criteria met for positive control: Yes - Mean viability of tissues was 6.7 %, which is <20 % of the negative control tissue. The standard deviation of the three tissue replicates was 0.27 (below the 18.27 %).
- Acceptance criteria met for variability between replicate measurements: Yes - The standard deviation calculated from individual % tissue viabilities of the three identically treated replicates was <18.27 %, i.e. in the range of 0.27 - 2.27.

Percentage viability of the negative (DPBS) and positive control (5 % aqueous SDS) was 100 ± 2.27 and 6.7 ± 0.27, respectively.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Aryl sulphonate condensate has been concluded to be an irritant (Category 2) to Reconstructed Human Epidermis (EpiDerm™) under the UN Globally Harmonised System as mean percentage tissue viability was less than 50 % of the negative control.

Executive summary:

An experiment was performed using Reconstructed Human Epidermis - EpiDerm™ (EPI-200-SIT) to ascertain the skin irritation potential of aryl sulphonate condensate according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). Triplicate tissue inserts were incubated prior to the experiment for a 20-hour and 10-minute period before being exposed to 30 µl each of Dulbecco's Phosphate Buffered Saline (DPBS) (negative control) and 5 % Aqueous Sodium Dodecyl Sulfate (SDS) (positive control), and 25 mg of the test item. After 60 minutes of exposure, the tissues were washed with DPBS thoroughly and blotted and transferred to fresh medium. The tissues were then incubated as part of a post-treatment incubation period for an additional 43-hour period, interspersed by an assay medium change and later transfer to MTT solution. An extraction of any resultant purple-blue formazan salt, formed predominantly by mitochondrial metabolism, was achieved using an MTT extraction solvent (MTT-100-EXT). A 96-well plate spectrophotometer was used to measure the optical density of the extracted formazan at 570 nm (OD570) and the viability of the tissues was calculated by entering OD values into a MatTek spreadsheet.

Percentage viability scores of 100 ± 2.27, 6.7 ± 0.27, and 3.1 ± 0.57 were determined for the negative control, positive control, and test item, respectively. All experiment acceptance criteria were satisfied. Given that the percentage viability of the test item was not greater than 50 % of the negative control, aryl sulphonate condensate has been concluded to be an irritant (Category 2) to Reconstructed Human Epidermis under the UN Globally Harmonised System.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 30, 2018 to February 14, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Isolated corneas from the eyes of freshly slaughtered cattle.
- Age at study initiation: 3.5 to 4.8 years.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL / VEHICLE
- Amount applied: Volume of 750 µL applied to each cornea consisting of 20% w/v of test item in distilled water (Mysore Research Chemical Laboratories; Batch number: 7787).

Duration of treatment / exposure:

3 hours and 58 minutes.

Observation period (in vivo):

After the incubation period the corneas were washed with EMEM without phenol red and incubated in a horizontal position for 95 minutes.

Number of animals or in vitro replicates:

Nine eyes were selected, with three corneas apportioned to each of the three treatment groups (positive control, negative control, and test item).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment. Before the start of experiment, empty cornea holder’s opacity was measured and the mean opacity value obtained was considered as I0. Corneas free of defects were dissected with 2 to 3 mm rim of sclera.

QUALITY CHECK OF THE ISOLATED CORNEAS
Isolated corneas were mounted in designated corneal holders by placing the endothelial side of the cornea against the O-ring of the posterior chamber. The anterior chamber was placed over the cornea and both the chambers were joined together by tightening the chamber screws. The corneal holders were equilibrated for at least 1 hour by allowing the corneas to equilibrate and to achieve normal metabolic activity with Minimum Essential Medium (MEM) with 1% Fetal Bovine Serum and supplemented with 1% antibiotics - Penicillin and Streptomycin (MEM) in an incubator at 32±1ºC. Fresh pre-warmed MEM was added to both the chambers of cornea holders after completion of equilibrium period and baseline opacity were recorded for each cornea.
Opacity of each cornea was calculated by using a formula I0/I and opacity value was calculated for initial readouts (Before Treatment) by using the formula [(I0/I-b)/a] where a=0.0251, b=0.9894.
Corneas lesser than 7 opacity units, after an initial 1-hour equilibration period were used for the experiment.

NUMBER OF REPLICATES: Triplicate.

NEGATIVE CONTROL USED: Distilled water only from Mysore Research Chemical Laboratories.

POSITIVE CONTROL USED: Imidazole from VETEC.

APPLICATION DOSE AND EXPOSURE TIME
750 µL of distilled water, imidazole (20 % w/v), and test item (20 % w/v).

TREATMENT METHOD: Sealed chamber during exposure.

POST-INCUBATION PERIOD: Yes, 95 minutes at 32 ± 1 ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Treated corneas were washed with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was measured with an aid of opacitometer.
- Corneal permeability: The passage of sodium fluorescein dye was measured after 95 minutes of incubation with the aid of VIS spectrophotometry as optical density at 490 nm (OD490).
- Others (e.g, pertinent visual observations, histopathology): Histopathological examination.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
- Test item of IVIS score, ≤ 3 would be considered as UN GHS no category.
- Test item of IVIS score, >3 and ≤ 55 would be considered as 'no prediction can be made', subsequently testing with any other adequate method remains at the discretion of the sponsor.
- Test item of IVIS score, > 55 would be considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.

Irritation parameter:

cornea opacity score

Run / experiment:

4-hour exposure

Value:

46.54

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: corneal permeability

Run / experiment:

4-hour exposure

Value:

1.488

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

4-hour exposure

Value:

68.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Opacity and permeability values were less than the established upper limits of background opacity and permeability values for bovine corneas.
- Acceptance criteria met for positive control: Yes. The IVIS score was within two standard deviations of the historical mean.

Summary of In Vitro Opacity Scores

Group & Treatment	Initial Opacity Reading (I)	Opacity Reading (After Treatment)	Change in Opacity Value	Mean Change in Opacity Value	Corrected Opacity Value	Average of Permeability Value	Corrected Permeability Value	Mean of Corrected Opacity Value	Corrected Permeability Value	IVIS Value
G1 & Vehicle Control	3.6	6.9	3.3	2.4	2.4	0.078	0.099	-	-	-
	3.0	4.2	1.2			0.124
	1.2	3.9	2.6			0.094
G2 & Positive Control	3.5	100.9	97.4	129.4	95.0	1.767	1.668	127.00	1.634	151.5
	3.4	156.1	152.8		150.4	1.710	1.611
	1.2	139.2	138.0		135.6	1.720	1.622
G3 & Test Item	3.4	56.2	52.8	48.9	50.5	1.679	1.580	46.54	1.488	68.9
	3.2	47.0	43.8		41.4	1.656	1.557
	2.8	52.9	50.1		47.7	1.427	1.328

IVIS: In Vitro Irritancy Score.

 

Note:

Change in Opacity Value = Opacity reading (after treatment) - Initial opacity reading (I)

Corrected Opacity Value = Change in opacity value of positive/negative control/test item – mean change in opacity value of vehicle control.

Corrected Permeability Value = Average of permeability value of positive/negative control/test item - mean average of permeability value of vehicle control.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Following a Bovine Corneal Opacity and Permeability (BCOP) test for aryl sulphonate condensate, a mean corneal opacity and permeability score of 46.54 and 1.488 was calculated, respectively, and an In Vitro Irritancy Score (IVIS) of 68.9. The IVIS score is greater than the threshold value of 55 and indicative of corrosivity and severe irritancy to bovine corneas. Subsequently, the registered substance can be regarded as a Category 1 eye irritant (irreversible effects on the eye) (CLP Regulation (EC) No 1272/2008).

Executive summary:

A study was undertaken for aryl sulphonate condensate to ascertain its eye irritation potential. This was achieved in line with Good Laboratory Practice (GLP) and OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage). Nine cattle eyes collected from a slaughter house that were free of defects and with opacity units <7 were selected for use in the experiment. Aryl sulphonate condensate was applied to corneas within a distilled water vehicle at a volume of 750 µL (20% w/v) (n = 3). A negative control consisting of 750 µL distilled water and a positive control consisting of 750 µL imidazole (20 % w/v) was prepared (n = 3). All corneas were incubated within anterior chambers, in designated cornea holders, for a period of approximately 4 hours at 32 ± 1 ºC. Opacity was measured with an opacitometer and permeability was measured spectrophotometrically at 490 nm (OD490) following an addition post-exposure incubation of 90 minutes at 32 ± 1 ºC. Baseline opacity and permeability values obtained for blank (media) treated corneas were used for correction.

All acceptance criteria were fulfilled. The mean corrected opacity and permeability values of the test were determined to be 46.54 and 1.488, respectively. An In Vitro Irritancy Score (IVIS) of 68.9 was calculated in addition, which is suggestive of a severe irritating / corrosive effect to the corneas. Histopathological examination revealed that the registered substance had resulted in mild and minimal focal vacuolation that was especially pronounced in the wing layer of the epithelium. A classification of serious eye damage / irritation with irreversible effects on the eye (Category 1) has subsequently been assigned to aryl sulphonate condensate (CLP Regulation (EC) No 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)