Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Endpoint summary

Description of key information

The test item was examined after expoure to skin and eyes. No signs of irritation were indentified in these studies.

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:

21 April 2016 until 06 June 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 94.7% (w/w), dose calculation was not adjusted to purity
Expiry Date: 09 March 2026

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to triplicate tissues wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

Duration of treatment / exposure:

60 minutes

Species:

other: reconstituted human epidermis model

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Amount / concentration applied:

- Amount(s) applied (volume or weight with unit):
-Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL DPBS (MatTek) were used as negative control per tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL of a 5% SLS solution in deionised water (MatTek) were used a positive control per tissue

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

The test item, the negative control (DPBS) and the positive control (5% SLS) were applied to triplicate tissues each.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following approximately 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Irritation / corrosion parameter:

other: Relative Absorbance (%)

Run / experiment:

1

Value:

105.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative Absorbance (%)

Run / experiment:

2

Value:

112.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative Absorbance (%)

Run / experiment:

3

Value:

112.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results after treatment with the test item and the controls:

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Ab-sor-bance of 3 Wells	Mean Ab-sorbance of three wells blank corrected	Mean Absorbance of 3 wells after blank correction*	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Rel. Stand. De-viation [%]	Mean Rel. Absorbance [% of Negative Control]**	Mean Absorbance Blank Corrected Viable Tissue without MTT (Step 2)	Mean Absorbance Blank Corrected Freeze-killed Tissue With MTT (Step 4)	Mean Rel. Absorbance [% of Negative Control] after Complete Correction Procedure
Blank contr.		0.036	0.037	0.036	0.036	0.000
Blank TI		0.037	0.038	0.038	0.037	0.000
Negative Control	1	1.578	1.549	1.558	1.562	1.525	1.811	84.2	13.7	100.0
	2	2.026	1.984	1.971	1.994	1.957		108.1
	3	2.024	1.971	1.965	1.986	1.950		107.7
Positive Control	1	0.098	0.097	0.097	0.097	0.061	0.078	3.4	19.0	4.3
	2	0.118	0.121	0.118	0.119	0.082		4.6
	3	0.125	0.126	0.126	0.126	0.089		4.9
Test Item	1	2.012	1.921	1.929	1.954	1.917	1.996	105.8	3.4	110.2	0.149	0.348	85.8
	2	2.082	2.087	2.051	2.073	2.036		112.4
	3	2.093	2.067	2.059	2.073	2.036		112.4

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Duasyn Acid Violet SD is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

Since the test item was deeply coloured (black), a change of colour in the MTT interference pre-test to detect the test item’s property directly reducing MTT was not visible, if there was any. Therefore, as a precaution, the additional test with freeze-killed tissues was performed for possible data correction in the main experiment. Due to its intensive colour and for the same purpose, also the additional test with one viable tissue but without MTT addition had to be performed (test for colour interference).

Each three tissues of the human skin model EpiDerm^™were treated with the test item, the negative or the positive control for 60 minutes.

Approximately 25 mg of the test item were applied to each tissue and spread to match the surface of triplicate tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each triplicate tissue.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

Taking the results in the additional tests with viable and freeze-killed tissues into consideration, the mean relative absorbance of the test item amounted to 85.8% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.97 or 3.07 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.
Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

0.1 mL of the test item, which was found to weigh approximately 79 mg

Duration of treatment / exposure:

Up to 1 hour

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2 rabbits

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item, which was found to weigh approximately 79 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Annex 2.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Annex 3.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

cornea opacity score

Basis:

animal: 75494 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal: 75501 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal: 75494 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal: 75501 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

other: redness

Basis:

animal: 75494 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: redness

Basis:

animal: 75501 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: 75494 Female

Time point:

other: Mean 24, 48 and 72

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: 75501 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Irritant / corrosive response data:

Ocular Reactions
Individual and mean scores for ocular irritation are given in Appendix 1.
Blue colored staining of the fur around the treated eye was noted in both animals at all observations.
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 Hour observations.
Both treated eyes appeared normal at the 72-Hour observation.
No corrosive effects were noted during the study. The test item did not induce significant or irreversible damage to the rabbit eye.

Other effects:

Body Weight
Individual body weights and body weight change are given in Appendix 2.
One animal showed no body weight gain and the other animal showed expected gain in body weight during the study.

Appendix1     Eye Irritation Scores

Individual Scores for Eye Irritation

											Conjunctivae
Rabbit Number and Sex	IPR			Evaluation interval	Corneal Opacity		Area of Corneal Opacity		Iris		Redness	Chemosis		Discharge
75494Female		0	1 Hour			0		0		0	2	1		1Sf
75501Male		0				0		0		0	2	1		1Sf
75494Female			24 Hour			0		0		0	1	1		0Sf
75501Male						0		0		0	1	0		0Sf
75494Female			48 Hour			0		0		0	1	0		0Sf
75501Male						0		0		0	1	0		0Sf
75494Female			72 Hour			0		0		0	0	0		0Sf
75501Male						0		0		0	0	0		0Sf

IPR=Initial pain reaction

Sf =       Blue colored staining of the fur around the treated eye

Appendix 1 (continued)       Eye Irritation Scores

Mean Values after 24, 48 and 72 Hours

				Conjunctivae
Rabbit Number and Sex	Number of available data points	Corneal Opacity	Iris	Redness	Chemosis
75494Female	3	0.0	0.0	0.7	0.3
75501Male	3	0.0	0.0	0.7	0.0

Assessment According to Regulation (EC) No. 1272/2008

			Conjunctivae
Evaluated Intervals	Corneal Opacity	Iris	Redness	Chemosis
24 Hours	Not classified	Not classified	Not classified	Not classified
48 Hours	Not classified	Not classified	Not classified	Not classified
72 Hours	Not classified	Not classified	Not classified	Not classified

Appendix2     Individual Body Weights and Body Weight Change

	Individual Body Weight (kg)
Rabbit Number and Sex	Day 0	Day 3	Body Weight Change (kg)
75494Female	3.07	3.09	0.02
75501Male	2.97	2.97	0.00

Interpretation of results:

other: does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Conclusions:

According to the findings in this study, the test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Executive summary:

The primary eye irritation potential of the test item was investigated according to a method compatible with OECD test guideline No. 405 and Method B5. The test item was applied by instillation of 0.1 mL into the right eye of each of two young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation. 

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iritis, redness and chemosis of the conjunctivae. The individual mean scores for corneal opacity and iritis was 0.0 for both animals. The individual mean scores for the conjunctivae were 0.7 for reddening and 0.3 or 0.0 for chemosis. 

The instillation of the test item into the eye resulted in conjunctival irritation.

These effects were reversible and were no longer evident 72 hours after treatment in both animals (end of the observation period). No abnormal findings were observed in the cornea of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No clinical signs of toxicity were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

According to the findings in this study, the test item does notmeet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 94.7% (w/w), dose calculation was not adjusted to purity
Expiry Date: 09 March 2026

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was tested as a 20% suspension (w/v) in saline. The test item could not be suspended or solved homogeneously, therefore, each 0.75 mL of the prepared stock was distributed to each cornea.


Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Example 2:
After a first opacity measurement of the fresh bovine corneae (t0), the neat test item Rewoteric QAM 50, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).



SCORING SYSTEM:
Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

8.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

5.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

9.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

Relative to the negative control, the test item Duasyn Acid Violet SD caused a slight increase of the corneal opacity but no relevant increase in permeability compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 7.50. According to OECD 437 (see table in chapter 3.8.3) no prediction can be made whether the test item induces serious eye damaging (UN/GHS (Category 1) or not (UN GHS: no Category)

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0	0.081	0.075	1.22	1.13	Not categorized
	0		0.072		1.08
	0		0.072		1.08
Positive Control	101*		0.111*		102.67	108.57	Category 1
	117*		0.118*		118.77
	103*		0.085*		104.28
Duasyn Acid Violet SD	8*		0.013*		8.20	7.50	No prediction can be made
	5*		0.011*		5.17
	9*		0.010*		9.15

*corrected values

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, no prediction can be made whether Duasyn Acid Violet SD induces serious eye damage (UN GHS: Category 1) or not (UN GHS: no Category).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline of the test item , the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.13).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 108.57) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item caused a slight increase of the corneal opacity but no relevant increase in permeability. The calculated mean IVIS was 7.50 (threshold for Category 1: IVIS > 55, threshold for no Category: IVIS ≤ 3, range for “no prediction can be made”: IVIS > 3; ≤ 55). According to OECD 437 no prediction can be made whether the test item induces serious eye damage (UN GHS: Category 1) or not (UN GHS: no Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

There is no information indicating an irritation hazard on skin or eyes.