Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Endpoint summary

Description of key information

Skin Irritation

The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt, CAS No. 129-17-9 was determined to be 94.1%. Thus, Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt, CAS No. 129-17-9 was considered to be not irritating to the human skin.

Eye Irritation

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The mean % tissue viability of hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt [CAS: 129-17-9] was determined to be 66.8%. Thus, hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt [CAS: 129-17-9] was considered to be not irritating to MatTek EpiOcular Tisssue Model OCL-200.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

data is from safety assessment reports

Justification for type of information:

data is from safety assessment reports

Qualifier:

equivalent or similar to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

To assess the skin irritation potential of the test chemical in rabbits

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Details on test animals or test system and environmental conditions:

no data available

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

other: saline

Controls:

not specified

Amount / concentration applied:

0. 5 ml of saturated solution in saline

Duration of treatment / exposure:

no data available

Observation period:

no data available

Number of animals:

no data available

Details on study design:

no data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: not mentioned

Score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No irritation was observed

Interpretation of results:

other: not irritating

Conclusions:

Patent Blue V was observed to be not irritating to rabbit skin.

Executive summary:

A skin irritation study was conducted in rabbits to observe the irritation potential of the Patent Blue V.

0. 5 ml of a saturated solution of the test chemical in saline was applied to rabbit skin (site not specified) and effects were observed (duration not specified).

Patent Blue V was observed to be not irritating to rabbit skin.

 

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:

December 06, 2017 to March 24, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Principles of method if other than guideline:

The purpose of this study is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2. This assay is not intended to differentiate between UN GHS Category 1 / HazardCode 318 and UN GHS Category 2 / Hazard Codes 319 and 320.

GLP compliance:

yes

Specific details on test material used for the study:

IUPAC name: hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium saltMolecular weight:566.672 g/molMolecular formula:C27H31N2NaO6S2physical state: Purple powderSmiles:C(\c1c(cc(S(=O)(=O)[O-])cc1)S(=O)(=O)[O-])(c1ccc(N(CC)CC)cc1)=C1/C=C\C(=[N+](\CC)CC)C=C1.[Na+]InChI:1S/C27H32N2O6S2.Na/c1-5-28(6-2)22-13-9-20(10-14-22)27(21-11-15-23(16-12-21)29(7-3)8-4)25-18-17-24(36(30,31)32)19-26(25)37(33,34)35;/h9-19H,5-8H2,1-4H3,(H-,30,31,32,33,34,35);/q;+1/p-1LOT Number: I/13-14/1002Product Code: 016P200Manufacture date: 01/2014RADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature or Fridge storage- Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test articles is tested as provided (neat).- Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data availableFORM AS APPLIED IN THE TEST: SolidOTHER SPECIFICS: No data available

Species:

other: MatTek EpiOcular Tisssue Model OCL-200

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

-Test System: MatTek EpiOcular™ Tissue Model OCL-200Storage:EpiOcular™ tissues and assay medium will be refrigerated at approximately 2-8°C upon arrival and until use.Supplier:MatTek Corporation, Ashland, MA- Justification of the test method and considerations regarding applicabilityThe EpiOcular™ Tissue Model closely parallels human ocular tissue, thus providing a useful in vitro means to assess ocular irritancy and toxicology

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL - Amount(s) applied (volume or weight with unit): 50 mg VEHICLE (no vehicle) - Amount(s) applied (volume or weight with unit): none - Concentration (if solution): none - Lot/batch no. (if required): none - Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 50 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 50 µL- Concentration (if solution): neat

Duration of treatment / exposure:

Tissues will be topically exposed to the test article and control articles for 6 hours ± 15 minutes.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Following the post soak,Tissues will be incubated in 1 ml fresh assay medium in a humidified 37±1°C, 5±1% CO2 incubator.

Number of animals or in vitro replicates:

2 tissues were used for test compound and control.

Details on study design:

-Plate Reader Linearity Check:The linearity of the plate reader or spectrophotometer used for optical density (OD) determination will be verified prior to its use the same week the EIT assay is beingperformed.A dilution series of trypan blue or thiazolyl blue tetrazolium bromide (MTT) formazan will be prepared and 200 μl aliquots will be pipetted into a 96-well plate.The optical density of the plate wells will be measured at a wavelength of 570 nm (OD570), with no reference wavelength.A regression line and an R-squared value will be generated using Microsoft Excel®. Verification will be considered acceptable if the R-squared value is >0.999.Assessment of Direct MTTReduction:No assessment of the direct MTT (methyl thiazole tetrazolium) reduction potential of each test article will be made.-Assessment of Coloring or Staining Materials:No assessment of each test article’s ability to absorb light at the wavelength (570 nm) used for MTT determination will be made.- Pre-Incubation:EpiOcular™ tissues will be placed in six-well plates containing warmed assay media and will be equilibrated in a humidified 37±1°C, 5 ±1% CO2 incubator for at least one hour. The media will then be changed and the tissues incubated overnight (16-24 hours).Any tissues not being incubated the same day will be allowed to re-equilibrate at 37±1°C, 5±1% CO2 and will be stored at approximately 2-8°C..-Pre-Treatment:After the overnight incubation, the tissues will be moistened with 20 μl of phosphatebuffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.-Dosing:Whenever possible, solids should be ground to a fine powder before application. 50 mg of a solid test article will be applied topically to duplicate tissuesand incubated at 37±1°C, 5±1% CO2 for 6 hours ± 15 minutes.After dosing and incubation, the tissues will be thoroughly rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for the appropriate amount of time.Tissues will be soaked for 25±2 minutes.-MTT Extraction:Following the three-hour MTT incubation period, each tissue will be removed individually and gently rinsed with PBS to remove any residual MTT solution.The extraction plate will be covered and sealed to reduce evaporation of extractant.For solid, colored, or staining test articles, 2.0 ml of extractant solution will be used in a six-well plate, allowing extraction to occur through the bottom of the insert. Extraction Conditions:The extraction will be allowed to proceed overnight at room temperature in the dark.Alternatively, the extraction can proceed for at least two hours, with shaking, at room temperature.-Decant Extractant:Tissues immersed in extractant solution in a 24-well plate: After the extraction period is complete, the liquid within each tissue insert will be decanted back into the wellfrom which it was taken, i.e., the solution will be mixed with the extractant in the well.The tissue inserts will then be discarded.-Transferring to 96-Well Plate:Two 200 μl aliquots from each well of the extraction plate(s) will be pipetted into a 96- well microtiter plate.-Measuring Optical Density:The optical density of the extracted samples will be determined at a single wavelength of 570 nm and using eight 200 μl aliquots of the Extractant as blanks.Calculating Percent Viability:The percent viability of the test tissues will be determined using the following formula:% Viability = 100 x (ODsample / ODNegative Control)Quality Controls:Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.Tissue Variability: The difference in viability between identically treated tissues must be less than 20%. This applies to tissues treated with the same test article as well as living and killed controls.Ocular Irritation Potential:An irritant is predicted if the mean relative tissue viability of two individual tissues exposed to the test substance is less than or equal to 60% of the mean viability of thenegative control-treated tissue viability.In Vitro Result In Vivo Prediction (GHS3)Mean tissue viability ≤ 60% Category 1 / Hazard Code 318, or Category 2 / Hazard Codes 319 and 320Mean tissue viability > 60% No Category (Non-Irritating)Borderline Results:If the test article-treated tissue viability is 60±5%, a second EIT should be performed. If the results of the second test disagree with the first, then a third test should be performed. The conclusion will be based on the agreement of two of the three tests.Duration:The duration of the EpiOcularTM Eye Irritation Test is approximately five days.

Irritation parameter:

other: mean % tissue viability

Run / experiment:

Run 1

Value:

66.8

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.Tissue Variability: The difference in viability between identically treated tissues must be less than 20%.

Test and Control Article Identity	Tissue Viability		Irritancy Classification	GHS Category
	Mean	SD
hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt [CAS: 129-17-9]	66.8	8.51%	Non-Irritant	No Category

 

Test and control article identity	Tissue no.	Raw data		Blank corrected data		Mean of aliquots	% viability	OD		Viabilities (%)
								MEAN	SD	Mean	SD
		Aliq 1	Aliq 2	Aliq 1	Aliq 2
hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt [CAS: 129-17-9]	1	1.088	1.105	1.043	1.060	1.051	62.6	1.123	0.143	66.8	8.51
	2	1.238	1.241	1.193	1.196	1.194	71.1

 

Interpretation of results:

other: not irritating

Conclusions:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The mean % tissue viability of hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt[CAS: 129-17-9] was determined to be 66.8%. Thus, hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt[CAS: 129-17-9] was considered to be not irritating to MatTek EpiOcular Tisssue Model OCL-200.

Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% of negative control (for 6 hour exposures with solids) in run one.

The mean % tissue viability of hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt[CAS: 129-17-9]was determined to be 66.8.%.

Hence, under the experimental test conditions it was concluded that hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt[CAS: 129-17-9] was considered to be not irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as “Not classified’’.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 3.67 passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of theHydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt, CAS No. 129-17-9was determined to be 94.1%.

Hence, under the experimental test conditions it was concluded that test substanceHydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt, CAS No. 129-17-9was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

A skin irritation study was conducted (Scientific Committee on Cosmetology (seventh series), 1988) in rabbits to observe the irritation potential of the Patent Blue V. 0. 5 ml of a saturated solution of the test chemical in saline was applied to rabbit skin (site not specified) and effects were observed (duration not specified). Patent Blue V was observed to be not irritating to rabbit skin.

Studies were conducted on similar substances 16470-24-9, 4193-55-9 and 67786-25-8 by U.S. Environmental Protection Agency, March 2012 to determine their dermal irritation potential.

A skin irritation study of C.I. Fluorescent Brightener 220 (CAS- 16470-24-9) was performed in rabbits to determine its irritation potential. The test sample was applied dermally to the ear. 2 rabbits/ group were used. Since, there was no evidence of any dermal irritation, the test sample, C.I. Fluorescent Brightener 220 (CAS No: -16470-24 -9) was considered to be not irritating.

A skin irritation study of Cellufluor (CAS No:-4193-55-9) was conducted in 6 rabbits/ group for the observation period of 14 days. The test sample was administered in water at 50% concentration on intact or abraded skin and covered with occlusive bandages for 24 hours exposure period.  The test animals did not show any cutaneous reactions during the observation period of 14 days following exposure and the skin reactions were scored according to Draize. Hence the test chemical Cellufluor (CAS No: -4193-55-9) was reported to be non irritant on rabbits skin.

A skin irritation study of C.I. Fluorescent Brightener 263, tetrasodium salt (CAS No:-67786-25-8) was carried out in rabbits. The rabbits were administered unspecified quantities of one of three formulations of C.I. Fluorescent Brightener 263, tetrasodium salt: a liquid formulation of sodium/potassium salt, technical grade, ca. 28% water free acid; technical grade (100% purity); or a commercial formulation, ca. 45% C.I. Fluorescent Brightener 263, tetrasodium salt. Test sample were applied to the inner ear under semi-occluded conditions for 24 hours. All formulations were not irritating to rabbit skin and the test animals did not show any cutaneous reactions. Hence the test chemical C.I. Fluorescent Brightener 263, tetrasodium salt(CAS No: -67786-25-8)  was reported to be non irritant on rabbits skin.

 

Based on the available information for target as well as its various read across substances, , hydrogen [4-[4-(diethylamino)-2', 4’-disulphonatobenzhydrylidene] cyclohexa-2, 5-dien-1-ylidene]diethylammonium, sodium salt (Patent Blue V) was not irritating to skin of rabbits.

 

Eye Irritation:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% of negative control (for 6 hour exposures with solids) in run one.

The mean % tissue viability of hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt[CAS: 129-17-9]was determined to be 66.8.%.

Hence, under the experimental test conditions it was concluded that hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt[CAS: 129-17-9] was considered to be not irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as “Not classified’’.

An eye irritation study was conducted in rabbits (Scientific Committee on Cosmetology (seventh series), 1988) to observe the irritation potential of the Patent Blue V. 0.25 ml of a saturated solution of the test chemical in saline was instilled into the eyes of rabbits and effects were observed (duration not specified).

Patent Blue V was observed to be not irritating to rabbit eyes.

Studies were conducted on similar substances 68921-42-6 and 3844-45-9 by S.D. Gettings et al, 1992 to determine their ocular irritation potential.

The ocular irritation, staining, and embedding potential of FD & C color Blue No. 1 Aluminum Lake (68921-42-6) produced by repeated topical application to rabbit eyes was studied. Test materials (3%,wt./vol. in aqueous vehicle) was administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits (6 of each sex per group) at a dose vol. of 30 µl. Control animals (6 of each sex) received 30 µl of the vehicle daily. All animals survived and were free of significant clinical signs of toxicity throughout the study. Ophthalmoscopic examinations revealed that all animals were free of abnormalities considered to be of clinical importance; all animals were free of significant signs of ocular irritation, staining and particle embedment suggesting that Brilliant Blue FCF lake was not irritating to eyes.

Ocular irritation was determined according to a modification of the Draize test (Draize, 1959).FD&C Blue 1 (3844-45-9) was prepared daily as a 3% (w/v) suspension in aqueous vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose and 0.25% (w/v) laureth -10 acetate. FD&C Blue 1 (3% w/v in aqueous vehicle) was administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits (6 of each sex/ group) at a dose volume of 30µl. Control animals (6 of each sex) received 30/µl of the vehicle daily. Ocular irritation was determined according to a modification of the Draize test (Draize, 1959). Interpretation of observations and assignment of scores were consistent with those described by the Consumer Product Safety Commission (1972).All eyes were scored for ocularirritation pretest (8 days, 24 hr and immediately prior to the initial dose) and approximately 24 hr after eachtreatment, prior to the next instillation of test material; on days 1, 3, 7, 14 and 21, the eyes were also evaluated for irritation 1 hr after treatment. In addition, all readily observable ocular structures were evaluated for eye stain and particle embedment 24 hr after each treatment. All animals survived and were free of significant clinical signs of toxicity throughout the study Ophthalmoscopic examinations revealed that all animals were free of abnormalities, all animals were free of significant signs of ocular irritation, staining and particle embedment. Thus it was concluded that the test substance FD&C Blue 1 (Brilliant Blue FCF) was not irritating to eyes.

Based on the available information for target as well as its various read across substances,, hydrogen [4-[4-(diethylamino)-2', 4’-disulphonatobenzhydrylidene] cyclohexa-2, 5-dien-1-ylidene]diethylammonium, sodium salt (Patent Blue V) was not irritating to rabbit eyes.

 

Justification for selection of skin irritation / corrosion endpoint:

Data is from in vitro k1 study report

Justification for selection of eye irritation endpoint:

Datais from in vitro k1 study report

Justification for classification or non-classification

Available studies on hydrogen [4-[4-(diethylamino)-2', 4’-disulphonatobenzhydrylidene] cyclohexa-2, 5-dien-1-ylidene]diethylammonium, sodium salt (Patent Blue V) indicates that it is not irritating to both skin and eyes. It can be classified as 'Not classified' .