Reaction product of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium chloride and Naphthalenesulfonic acids, reaction products with formaldehyde, ammonium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-24 to 2013-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD/EU guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

08 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tris(9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-9H-xanthen-9-ylium) 6-[(3-sulfonatonaphthalen-2-yl)methyl]-3-[(6-sulfonatonaphthalen-2-yl)methyl]naphthalene-2-sulfonate

EC Number:

940-448-2

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

tris(9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-9H-xanthen-9-ylium) 6-[(3-sulfonatonaphthalen-2-yl)methyl]-3-[(6-sulfonatonaphthalen-2-yl)methyl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals / tissue source

Species:

other: Isolated chicken eye from ROSS 308 and COBB 500

Strain:

other: Isolated chicken eye from ROSS 308 and COBB 500

Details on test animals or tissues and environmental conditions:

not appliable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The three positive control eyes were treated with Imidazole (30 mg) solution and one negative control eye was treated with NaCl (9 g/L saline) solution.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Three test item treated eyes

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature
- Time after start of exposure: 10 seconds

TOOL USED TO ASSESS EYE CORROSION: corneal opacity, swelling, fluorescein retention, morphological effects and histopathology

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Maximum corneal swelling

Basis:

mean

Time point:

other: at up to 75 min

Remarks on result:

other: 4 % relative to control

Results 2

Irritation parameter:

other: Maximum corneal swelling

Basis:

mean

Time point:

other: at up to 240 min

Remarks on result:

other: 4 % relative to control

Results 3

Irritation parameter:

other: Maximum corneal opacity

Basis:

mean

Time point:

other: 240 min

Remarks on result:

other: Corneal opacity score: 0.0

Results 4

Irritation parameter:

other: fluorescein retention

Basis:

mean

Time point:

other: 30 min

Remarks on result:

other: Fluorescein retention score: 2.7

Irritant / corrosive response data:

Based on the mean fluorescein retention value of 2.7 an ICE Class of IV was determined for this endpoint. Since values for corneal swelling and opacity showed no indication for severe eye damage (ICE Class I) fluorescein retention is assumed to be influenced by the test item stuck on the cornea surface after post-treatment rinse. In an additional experiment a possible fluorescence of the test item itself was investigated. Further three eyes were treated with the test item in the same manner as in the main study and fluorescein retention was measured without using fluorescein solution.
In the additional experiment fluorescein retention measurement revealed that the test item stuck on the cornea surface is non-fluorescent and does not directly influence fluorescein retention measurement. However retention of fluorescein solution at the test item stuck on the cornea surface was taken into consideration and might explain the high fluorescein retention in the absence of elevated values for corneal swelling and opacity. Therefore fluorescein retention parameter was assessed as not appropriate in this ICET study and is not considered in the evaluation of ocular corrosion and severe irritation potential of the test item.
Based on the data for corneal swelling and opacity the test item has no ocular corrosion and severe eye irritation potential.

Other effects:

Histopathology
The results of measurement by ocular micrometer method revealed slight thickening of substantia propria in two test item treated eyes and slight thickening of anterior epithelium in three test item treated eyes as compared with the negative control and regarding the positive controls.
The thickness of other DOCU RED 116 treated samples regarding the substantia propria and regarding the anterior epithelium was the same as the negative control.
No thickness of other layers (subepithelial basement membrane, posterior limiting membrane, posterior epithelium) was observed in the treated or control samples.
Desquamation (focal complete loss) of anterior epithelium was observed in serious form (6-8 focal area/ specimen) in the positive control samples indicating for eye corrosion and severe irritation however no significant desquamation occurred in the DOCU RED 116 treated samples.
No other pathological lesion were detectable in the all investigated (treated and control) specimens.
In conclusion it can be stated that the test item DOCU RED 116 did not cause histologically detectable ocular corrosion and severe irritation. Corneas of positive and negative control showed the expected histological response.

Any other information on results incl. tables

Result tables

 

Test Item: DOCU Red 116

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	4 %	I
Mean maximum corneal swelling at up to 240 min	4 %	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	2.7	IV *
Other Observations	The test item was stuck on the cornea surface all test item treated eyes at 30 minutes after the post-treatment rinse. It cannot be removed from cornea surface by extra rinse (20 mL/eye saline solution).
Overall ICE Class	2xI, 1xIV

 

Results of the additional experiment are shown in the table below.

 

Test Item: DOCU Red 116 (Additional Experiment)

Observation	Value
Mean fluorescein retention at 30 min	0.3
Mean fluorescein retention at 75 min	0.3
Mean fluorescein retention at 120 min	0.3
Mean fluorescein retention at 180 min	0.2
Mean fluorescein retention at 240 min	0.2
Other Observations	The test item was stuck on the cornea surface and cornea surface was painted by test item all eyes at 30 minutes after the post-treatment rinse. The cornea surfaces were not totally cleared, little volume of test item was stuck on the cornea surfaces at 240 min after the post-treatment rinse.

 

Positive Control: Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	15 %	III
Mean maximum corneal swelling at up to 240 min	31 %	III
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse
Overall ICE Class	1xIII, 2xIV

 

Positive Control: Imidazole (Additional Experiment)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9 %	II
Mean maximum corneal swelling at up to 240 min	21 %	III
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse
Overall ICE Class	1xIII, 2xIV

 

Negative Control: NaCl (9 g/L saline)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

 

Negative Control: NaCl (9 g/L saline) (Additional Experiment)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

 

Applicant's summary and conclusion

Interpretation of results:

other: not severely eye damaging

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In this In vitro eye corrosives and severe irritants study using the Isolated Chicken Eye model with DOCU Red 116 according to OECD guideline No. 438 ocular corrosion and severe irritation potential was not observed.

Executive summary:

The purpose of the Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item DOCU Red 116 by its ability to induce toxicity in enucleated chicken eyes. The study was conducted in accordance with OECD guideline No. 438 (26 July 2013) and EU guideline B.48 (08 December 2010). The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes.

The purpose of the additional experiment was to examine, whether DOCU Red 116 can cause false positive fluorescein retention.

Based on the data for corneal swelling and opacity DOCU Red 116 did not cause ocular corrosion and severe eye irritation in the enucleated chicken eyes.

However, a mean fluorescein retention value of 2.7 was observed indicating severe eye damage (ICE Class IV). Since values for corneal swelling and opacity showed no indication for severe eye damage (ICE Class I) fluorescein retention is assumed to be influenced by the test item stuck on the cornea surface after post-treatment rinse. Although DOCU Red 116 is not fluorescent itself as shown in the additional experiment retention of fluorescein solution at the test item stuck on the cornea surface was taken into consideration and might explain the high fluorescein retention in the absence of elevated values for corneal swelling and opacity.

Therefore fluorescein retention parameter was assessed as not appropriate in this ICET study and is not considered in the evaluation of ocular corrosion and severe irritation potential of the test item.

Histopathology evaluation of retained corneas was performed as an additional endpoint to further characterize the scope of the corneal damage suggested by the fluorescein retention parameter. DOCU RED 116 did not cause histologically detectable ocular corrosion and severe irritation.

Positive and negative controls showed the expected results. The experiments were considered to be valid.

Conclusion

In this in vitro eye corrosives and severe irritants study using the Isolated Chicken Eyes model with DOCU Red 116 according to OECD guideline No. 438 ocular corrosion and severe irritation potential was not observed.