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

Endpoint summary

Description of key information

The results obtained from the in vitro skin irritation test indicated that the test item reveals no skin irritation potential under the utilised testing conditions. Based on the results of the isolated chicken eye test a severe eye damaging potential of the test substance can be excluded.

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:

2013-10-09 to 2013-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with GLP regulations and OECD/EU guideline.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiSkinSM

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EpiSkinSM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- Supplier: SKINETHIC Laboratories; 4, rue Alexandre Fleming, 69007 – LYON, France.
- Batch No.: 13-EKIN-035
- Expiry date: 14 October 2013

Type of coverage:

other: EpiSkin model

Preparation of test site:

other: EpiSkin model

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 10 mg of the test item

Duration of treatment / exposure:

15 min

Observation period:

42 hours

Number of animals:

3 replicates per test item
3 replicates negative controls
3 replicates positive controls
3 replicates of additional control for coloured test item (i.e. for non-specific OD evaluation)

Details on study design:

TEST SITE
- Area of exposure: the whole surface of the EpiSkin model

REMOVAL OF TEST SUBSTANCE
- Washing: PBS 1x solution
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Viability of the test item treated EpiSkin model in comparison to negative control.
The test substance is considered to be a skin irritant (R38), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Irritation / corrosion parameter:

other: other: Viability [% control]

Value:

87

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 h. Max. score: 100.0. Remarks: Test item. (migrated information)

Irritation / corrosion parameter:

other: other: Viability [% control]

Value:

26

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 h. Max. score: 100.0. Remarks: Positive control (SDS 5% aq.). (migrated information)

Irritant / corrosive response data:

- Cell viability
All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control, therefore the test item was considered to be non-irritant to skin.

Other effects:

- Validity of the test
The mean OD value of the three negative control tissues was 0.862. The mean OD value obtained for the positive control was 0.224 and this result corresponds to 26 % viability when compared to the results obtained from the negative controls. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

- Indicator for potential false viability
Possible direct MTT reduction with test substance:
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
Colouring potential of test substances:
As the test item has an intrinsic colour (red), three additional chemical-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues were determined as 0.025. The Non Specific Colour% (NSC %) was calculated as 2.9 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, DOCU Red 116 is considered as non-irritant to skin and is therefore not classified.

Executive summary:

The purpose of the study was to determine the skin irritation potential of the test item DOCU Red 116 on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item DOCU Red 116 did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, DOCU Red 116 is considered as non-irritant to skin and is therefore not classified.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

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.

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

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)

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

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

Irritation parameter:

other: Maximum corneal swelling

Basis:

mean

Time point:

other: at up to 75 min

Remarks on result:

other: 4 % relative to control

Irritation parameter:

other: Maximum corneal swelling

Basis:

mean

Time point:

other: at up to 240 min

Remarks on result:

other: 4 % relative to control

Irritation parameter:

other: Maximum corneal opacity

Basis:

mean

Time point:

other: 240 min

Remarks on result:

other: Corneal opacity score: 0.0

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.

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

 

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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The purpose of the study was to determine the skin irritation potential of the test item DOCU Red 116 on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item DOCU Red 116 did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, DOCU Red 116 is considered as non-irritant to skin and is therefore not classified.

 

Eye irritation

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.

 

Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one study available.

Justification for classification or non-classification

Based on the available experimental data the test item is not classified for skin irritation according to Regulation (EC) No 1272/2008 (CLP) and Directive 67/548/EEC (DSD).

Eye irritation study (ICET test) showed that the test item is non-corrosive. Based on these results, DOCU Red116 is not classified and labelled for serious eye damage, category 1 according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP).