[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Endpoint summary

Description of key information

Skin irritation:

An in vitro-study was performed to assess the irritation potential of C.I. Pigment Orange 68 by means of the Human Skin Model Test.

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 81.3 % after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Eye irritation:

An Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the corneal damage potential of C.I. Pigment Orange 68 resulting in an in vitro irritation score of >/= 31.91 to </= 47.09.

The test item was not identified to be a chemical that induces serious eye damage or that is not requiring classification for eye irritation or serious eye damage.

In an OECD guideline Acute Eye Irritation/Corrosion study in rabbits with C.I. Pigment Orange 68 moderate conjunctival irritation was observed in both animals. All signs of irritation were fully reversible within 72 h. Therefore, the test item (C.I. Pigment Orange 68) can be regarded as not eye irritating.

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. No abnormal findings were observed in the cornea of any animal at any of the examinations. No clinical signs of toxicity were observed. The test item did not induce significant or irreversible damage to the rabbit eye.

Respiratory tract:

Based on available data  the substance is considered likely to behave like an inert dust. Consequently the substance is considered not to exert any local irritative effects. Therefore, it is concluded, that testing is not necessary to reach the scientific conclusion that classification is not warrantable.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 439, GLP compliant 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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended standard in vitro test system

Vehicle:

other: DPBS

Details on test system:

Test For Direct MTT Reduction and Colour Interference:

For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. For this purpose, approximately 25 mg of the test item were added to 1 mL of MTT solution (MTT (Sigma, Germany) concentrate diluted with MTT diluent (DMEM, Gibco, Germany) (resulting: 1 mg/mL)). This mixture was incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 60 minutes.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose approximately 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2.
Since the colour of the test item / water mixture was intensive, an additional functional check was performed in parallel to the main experiment. The coloured test item was applied to one viable tissue, which underwent the entire skin irritation test (exposure period: 60 minutes; recovery period: about 41.5 hours; extraction period: 70 hours), but was incubated with medium instead of MTT solution during the MTT incubation step.
Since the OD of the tissue treated by the coloured test item was > 30% (173.6%) of the DPBS treated control tissue, the test item could have been considered as incompatible with the test. But according to an expert judgment test item particles could have been trapped in the tissue and could not be removed in the washing step completely. The gained high absorption value was most probably caused by reflection of the remaining particles.
Therefore, the real MTT OD (unaffected by interference with the coloured test items) was calculated using following formula:
OD = OD coloured tissue (MTT assay) – OD coloured tissue (no MTT assay)

Performance:
Pre-warming of EpiDerm™ Tissues
One day prior to the performance, the plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with steriles cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.

0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22.5 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

Treatment:
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, the vehicle control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for approximately 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another 19 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was nearly 42 hours.

MTT Assay:
On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for 70 hours without shaking in the refrigerator.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3  200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 ± 1 nm filter. Mean values were calculated from the 3 wells per tissue.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 to 26 mg (~ 39 mg/cm exp.2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

VEHICLE: No vehicle used

Negative Control
30 µL DPBS (MatTek) was used as negative control per tissue.

Positive Control
30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue,

Duration of treatment / exposure:

60 min.

Species:

other: reconstituted human epidermis model

Irritation / corrosion parameter:

other: absorbance

Run / experiment:

mean of all

Value:

81.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Corrected Mean Rel. Absorbance [% of Negative Control]: 81.3

Not irritant

Results after treatment with the test item and the controls

Dose Group		Treat-ment Interval	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*			Absor-bance 570 nm Tissue 3*		Mean Absor-bance of 3 Tissues			Rel. Absor-bance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Negative Control		60 min	1.869	1.894			1.949		1.904			98.2 99.5 102.3	2.1	100.0
Positive Control		60 min	0.102	0.105			0.074		0.094			5.4 5.5 3.9	18.0	4.9
Test Item		60 min	1.624	1.493			1.540		1.552			85.3 78.4 80.9	4.3	81.5
Colour interference pre-experiment: Functional Test without MTT
			Absorbance 570 nm Well 1			Absorbance 570 nm Well 2		Absorbance 570 nm Well 3			Mean Absorbance of 3 Wells		Mean Absorbance subtracted by blank	Mean Rel. Absorbance [% of Negative Control]***
Negative Control		60 min	0.0409			0.0408		0.0402			0.0406		0.0042	100.0
Test Item without MTT		60 min	0.0463			0.0424		0.0424			0.0437		0.0072	173.6
Data correction procedure
	Mean Absorbance of 3 Tissues (main experiment)				Mean Absorbance of 3 Wells (Functional Test without MTT)					ODMTT– ODno MTT			Corrected Mean Rel. Absorbance [% of Negative Control]***
Negative Control	1.904				0.0042					1.8996			100.0
Test Item	1.552				0.0072					1.545			81.3

*       Mean of three replicate wells after blank correction

**        relative absorbance per tissue [rounded values]:

***      relative absorbance per treatment group [rounded values]:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. Due to the intrinsic colour of the test item, an additional test with viable tissues (entire test procedure) but without MTT was performed. A correction from the mean relative absorbance value of the test item, corresponding to the cell viability was necessary.

 

After correction of absorbance (OD = absorbance_{with MTT}– absorbance_{without MTT}) the mean relative absorbance of the test item, corresponding to the cell viability was 81.3% (threshold for irritancy:≤50%), consequently the test item was not irritant to skin.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study and under the experimental conditions reported, the test item 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.

The test item passed the MTT- pre-test. Due to its intrinsic colour, an additional test with one viable tissue (entire test procedure), but with medium instead of MTT addition was performed.

Since the OD of the tissue treated by the coloured test item was > 30% (173.6%) of the DPBS treated control tissue in this test,the test item could have been considered as incompatible with the test. But according to an expert judgment test item particles were most probably trapped in the tissue and could not be removed in the washing step. The gained high absorption value was most probably caused by reflection of the remaining particles.

Therefore, the real MTT OD (unaffected by interference with the coloured test items) was calculated by subtracting the OD_{no MTT}from the OD_{with MTT.}

The test item, the negative control (DPBS), and the positive control (5% SLS) were applied to each of triplicate tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for nearly 42 hours the tissues were treated with the MTT solution for 3 hours following 70 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD >=0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.9% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were 18% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: ≤ 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 81.3% after exposure of the skin tissues to the test item. 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:

Between 10 November 2015 and 19 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147, amended 10 December 2002

Deviations:

no

GLP compliance:

yes

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 2.71 or 2.79 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 19 to 25 °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 study.

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 60 mg

Duration of treatment / exposure:

single instillation without subsequent rinse out

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours following treatment

Number of animals or in vitro replicates:

2

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 60 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 (Annex 1).
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.

Irritation parameter:

cornea opacity score

Basis:

animal: 75273 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: Reversibility does not apply since no effects occured

Irritation parameter:

cornea opacity score

Basis:

animal: 75262 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: Reversibility does not apply since no effects occured

Irritation parameter:

iris score

Basis:

animal: 67732 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: Reversibility does not apply since no effects occured

Irritation parameter:

iris score

Basis:

animal: 75262 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: Reversibility does not apply since no effects occured

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 75273 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 75262 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: 75273 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: 75262 Male

Time point:

other: 24, 48 and 72 hours

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Appendix 1 and Appendix 2.
Orange colored staining of the fur around the treated eye was noted in both animals at all observations. The staining was attributed to the color of the test item.
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 48 Hour observation.
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 3.
Both animals showed expected gain in body weight during the study.

Data Evaluation

Data were summarized in tabular form, showing for each animal the irritation scores for the designated observation time, a description of the degree and nature of irritation, the presence of serious lesions and non‑ocular effects. The scores of each animal at the following reading times (24, 48 and 72 hours) were used in calculating the respective mean values for each type of lesion.

 

The results were interpreted according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Appendix 1     Eye Irritation Scores

Individual Scores for Eye Irritation

Rabbit Number and Sex	IPR	Evaluation interval	Corneal Opacity	Area of Corneal Opacity	Iris	Conjunctivae
						Redness	Chemosis	Discharge
75273Male	0	1 Hour	0	0	0	2	1	2Sf
75262Male	0		0	0	0	2	1	1Sf
75273Male		24 Hour	0	0	0	2	1	1Sf
75262Male			0	0	0	2	1	0Sf
75273Male		48 Hour	0	0	0	1	0	0Sf
75262Male			0	0	0	1	0	0Sf
75273Male		72 Hour	0	0	0	0	0	0Sf
75262Male			0	0	0	0	0	0Sf

IPR = Initial pain reaction

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

Mean Values after 24, 48 and 72 Hours

Rabbit Number and Sex	Number of available data points	Corneal Opacity	Iris	Conjunctivae
				Redness	Chemosis
75273Male	3	0.0	0.0	1.0	0.3
75262Male	3	0.0	0.0	1.0	0.3

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

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

 

Appendix 2     Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75273Male	2.71	2.76	0.05
75262Male	2.79	2.83	0.04

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

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 1.0 for reddening and 0.3 for chemosis for both animals. 

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

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

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 Bensheim, 64625 Bensheim, 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.

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:

Experimental Performance
Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes.

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, 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).
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Exposure of the Corneae to the Test Groups

The corneae were distributed as follows:
Number of Corneae:
Negative Control: 3
Positive Control: 3
Test Item: 3

The anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the corneae was determined.

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 INTERPRETATION
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 (t130 – 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:

all 3

Value:

>= 31.91 - <= 47.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

The calculated mean in vitro irritancy score (IVIS) was 40.35
According to OECD 437 no prediction can be made.

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed classification
		Mean		Mean
Negative Control	0	0.00	0.067	0.068	1.01	1.02	No category
	0		0.068		1.02
	0		0.069		1.04
Positive Control	118.00		0.106		119.59	117.15	Category 1
	114.00		0.122		115.83
	114.00		0.135		116.03
test item	29.00		0.194		31.91	40.35	No prediction can be made
	39.00		0.205		42.08
	45.00		0.137		47.06

*corrected values

Interpretation of results:

other: No prediction can be made

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item was not identified to be a chemical that induces serious eye damage or that is not requiring classification for eye irritation or serious eye damage.

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 = 1.02).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed a clearly increased opacity and a distinctive increase in permeability of the corneae (mean IVIS = 117.15) corresponding to a classification as serious eye damaging (CLP/GHS (Cat 1)).

Relative to the negative control, the test item caused a noticeable increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score (IVIS) was 40.35. Thus PV-Echtorange 6RL meets the decision criteria of OECD 437 as given below:

 

IVIS	Classification (CLP / GHS)	Compliance
≤ 3	No Category	no
> 3; ≤ 55	No prediction can be made	yes
> 55	Category 1 Serious eye damage	no

 

According to OECD 437 no prediction can be made.

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

Skin irritation/corrosion:

The test item is not considered to possess an irritant potential.

Therefore, the test item has not to be classified for skin irritation/corrosion according to Regulation (EC) No 1272/2008.

Eye irritation/corrosion:

Mean scores (mean of the 24/48/72 h reading calculated for each animal) for conjunctivae, cornea, and iris were below the threshold for classification.

Therefore, test item has not to be classified for eye irritation/corrosion according to Regulation (EC) No 1272/2008.