Bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-)

Endpoint summary

Description of key information

 Skin irritation

The dermal irritation potential of target chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) was assessedin various experimental studies which were conducted on rabbits and rats for target chemicalbis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) and its structurally similar read across substances[4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 84434-47-9) and [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 83803-79-6). The predicted data usingQSAR toolboxhas also been compared with the experimental data.Based on the available data for the target and read across substances and applying the weight of evidence approach, it can be concluded that chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) is unable to cause skin irritation and thus considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye Irritation:

Since there were no detailed information available for target chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9), an ocular irritation potential of target chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) was estimated on the basis of in vitro and in vivo experimental studies available for its structurally similar read across substances. These experimental studies were conducted in rabbits for its structurally similar read across substances 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7), [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6), Rhodamine B [CAS: 81-88-9] and C.I Basic Green 1 [CAS: 633-03-4]. Thus on the basis of read across substances and applying the weight of evidence approach, it can be concluded that the target chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) might be able to cause eye irritation and thus can be considered as irritating . Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2”.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

Data is from OECD QSAR toolbox version 3.3 and QMRF report has been attached

Qualifier:

according to guideline

Guideline:

other: estimated data

Principles of method if other than guideline:

Prediction is done using QSAR Toolbox version 3.3

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material : bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-)
- Molecular formula : C33H40N3.1/2C6FeN6.Cu
- Molecular weight : 1296.445 g/mol
- Smiles notation : CCNc1ccc(c2c1cccc2)C(=C3C=CC(=[N+](CC)CC)C=C3)c4ccc(cc4)N(CC)CC.CCNc1ccc(c2c1cccc2)C(=C3C=CC(=[N+](CC)CC)C=C3)c4ccc(cc4)N(CC)CC.C(#N)[Fe-4](C#N)(C#N)(C#N)(C#N)C#N.[Cu+].[Cu+]
- InChl : 1S/2C33H39N3.6CN.2Cu.Fe/c2*1-6-34-32-24-23-31(29-13-11-12-14-30(29)32)33(25-15-19-27(20-16-25)35(7-2)8-3)26-17-21-28(22-18-26)36(9-4)10-5;6*1-2;;;/h2*11-24H,6-10H2,1-5H3;;;;;;;;;/q;;;;;;;;2*+1;-4/p+2
- Substance type: Organic
- Physical state: Solid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

No data available

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

No data available

Duration of treatment / exposure:

24 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

No data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

72 h

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No skin reaction was observed.

Estimation method: Takes mode value from the 5 nearest neighbours
Domain  logical expression:Result: In Domain

((((((((("a" and ("b" and ( not "c") )  )  and ("d" and ( not "e") )  )  and ("f" and ( not "g") )  )  and "h" )  and ("i" and ( not "j") )  )  and "k" )  and ("l" and ( not "m") )  )  and ("n" and ( not "o") )  )  and ("p" and "q" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Amine OR Aromatic compound OR Cation OR Secondary amine OR Secondary mixed amine (aryl, alkyl) OR Tertiary amine OR Tertiary mixed amine by Organic functional groups, Norbert Haider (checkmol) ONLY

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as No alert found by Protein binding by OASIS v1.3

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Michael Addition OR Michael Addition >> alpha,beta-Unsaturated carbonyl compounds OR Michael Addition >> alpha,beta-Unsaturated carbonyl compounds >> alpha,beta-Aldehydes  OR Michael Addition >> Michael addition on conjugated systems with electron withdrawing group OR Michael Addition >> Michael addition on conjugated systems with electron withdrawing group >> alpha,beta-Carbonyl compounds with polarized double bonds  OR Michael Addition >> Polarised Alkenes OR Michael Addition >> Polarised Alkenes >> Polarised Alkene - alkenyl pyridines, pyrazines, pyrimidines or triazines  OR Nucleophilic addition OR Nucleophilic addition >> Addition to carbon-hetero double bonds OR Nucleophilic addition >> Addition to carbon-hetero double bonds >> Ketones OR Schiff base formation OR Schiff base formation >> Schiff base formation with carbonyl compounds OR Schiff base formation >> Schiff base formation with carbonyl compounds >> Aldehydes OR SN1 OR SN1 >> Carbenium ion formation (enzymatic) OR SN1 >> Carbenium ion formation (enzymatic) >> Carbenium ion OR SN2 OR SN2 >> Ring opening SN2 reaction OR SN2 >> Ring opening SN2 reaction >> Epoxides, Aziridines and Sulfuranes  by Protein binding by OASIS v1.3

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Non binder, without OH or NH2 group by Estrogen Receptor Binding

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as Non binder, impaired OH or NH2 group OR Non binder, MW>500 by Estrogen Receptor Binding

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as No alert found by DNA binding by OASIS v.1.3

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as Michael addition OR Michael addition >> Quinone type compounds OR Michael addition >> Quinone type compounds >> Quinone methides OR Radical OR Radical >> ROS formation after GSH depletion OR Radical >> ROS formation after GSH depletion >> Quinone methides OR SN1 OR SN1 >> Alkylation after metabolically formed carbenium ion species OR SN1 >> Alkylation after metabolically formed carbenium ion species >> Polycyclic Aromatic Hydrocarbon Derivatives OR SN2 OR SN2 >> Alkylation, direct acting epoxides and related after P450-mediated metabolic activation OR SN2 >> Alkylation, direct acting epoxides and related after P450-mediated metabolic activation >> Polycyclic Aromatic Hydrocarbon Derivatives OR SN2 >> Direct acting epoxides formed after metabolic activation OR SN2 >> Direct acting epoxides formed after metabolic activation >> Quinoline Derivatives OR SN2 >> SN2 at an activated carbon atom OR SN2 >> SN2 at an activated carbon atom >> Quinoline Derivatives by DNA binding by OASIS v.1.3

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as High (Class III) by Toxic hazard classification by Cramer (original) ONLY

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as Not known precedent reproductive and developmental toxic potential by DART scheme v.1.0

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as AhR binders.Polycyclic aromatic hydrocarbons (PAHs) (3b-3) OR Known precedent reproductive and developmental toxic potential OR Toluene and small alkyl toluene derivatives (8a) by DART scheme v.1.0

Domain logical expression index: "k"

Referential boundary: The target chemical should be classified as Not bioavailable by Lipinski Rule Oasis ONLY

Domain logical expression index: "l"

Referential boundary: The target chemical should be classified as Non-Metals by Groups of elements

Domain logical expression index: "m"

Referential boundary: The target chemical should be classified as Metalloids by Groups of elements

Domain logical expression index: "n"

Referential boundary: The target chemical should be classified as Group 14 - Carbon C AND Group 15 - Nitrogen N by Chemical elements

Domain logical expression index: "o"

Referential boundary: The target chemical should be classified as Group 15 - Phosphorus P OR Group 16 - Oxygen O by Chemical elements

Domain logical expression index: "p"

Parametric boundary:The target chemical should have a value of log Kow which is >= 5.21

Domain logical expression index: "q"

Parametric boundary:The target chemical should have a value of log Kow which is <= 6.51

Interpretation of results:

other: not irritating

Conclusions:

The chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9)was estimated to be not irritating to the skin of New Zealand White rabbits. Based on the estimated result bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9)can be considered to be not irritating to skin.

Executive summary:

The dermal irritation potential of bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) was estimated using OECD QSAR toolbox version 3.3 with logPow as the primary descriptor. The chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9)was estimated to be not irritating to the skin of New Zealand White rabbits. Based on the estimated result bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9)can be considered to be not irritating to skin and can be classified under the category ˋ Not Classified’ as per CLP regulation.

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from authoritative database

Qualifier:

equivalent or similar to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

To assess the ocular irritation potential of C.I. Basic Green 1

GLP compliance:

not specified

Specific details on test material used for the study:

Name of the test chemical:N-(4-((4-(diethylamino)phenyl)(phenyl)methylene) cyclohexa -2,5-dien-1-ylidene) -N-ethylethanaminium sulfate [Basic Green 1]
Molecular Formula:C27H33N2.HO4S
Molecular Weight:482.642 g/mol
SMILES notation : C(\c1ccc(N(CC)CC)cc1)(=C1/C=CC(\C=C1)=[N+](/CC)CC)c1ccccc1.S(O)(=O)(=O)[O-]
Substance Type: Organic
Physical State: Solid

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

no data available

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

no data available

Duration of treatment / exposure:

no data available

Observation period (in vivo):

no data available

Duration of post- treatment incubation (in vitro):

no data available

Number of animals or in vitro replicates:

no data available

Details on study design:

no data available

Other effects / acceptance of results:

no data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: no data available

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Irritation was observed

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Basic Green 1 can be considered to be irritating to rabbit eyes.

Executive summary:

A study was performed to assess the ocular irritation potential of Basic Green 1.Basic Green 1 was instilled into rabbit eyes and effects were observed (duration and dose not mentioned). Signs of irritation were observed in animals.

Hence, Basic Green 1 can be considered to be irritating to rabbit eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 05, 2017 to July 12, 2017

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 was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article 4,4'-carbo nimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) may be predicted by measurement of its cytotoxic effect, as reflected in the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model (MatTek Corp., Ashland, MA).

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material identity: 4,4'-carbonimidoylbis[N,N-diethylaniline] monohydrochloride [Ethyl Aluramine (Basic Yellow 37)]
- Source: K. Patel Chemopharma Pvt. Ltd.
- Lot/batch No.of test material: KCP/FS/44/17
- Date of receipt: 15.Jan.2017
- Expiration date of the lot/batch: No data available
- Purity test date: No data available
- Physical Appearance: Bright yellow powder
- Moisture @ 60 degC: 1.47% w/w
- Insoluble (In methanol): 0.46% w/w
- pH (1g + 99 ml water): 6.86
- Absorbance @ 439.0 ± 2 nm (5 mg/l water + 1 ml Acetic acid): 439.7 nm - 0.595 Abs.

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- 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 available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).
- 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 available

FORM AS APPLIED IN THE TEST: Powder

OTHER SPECIFICS: No data available

Species:

human

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

- Description of the cell system used
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

- Justification of the test method and considerations regarding applicability
Human Corneal Epithelia (HCE) by MatTek, Inc.:
The test article and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek Corporation (Ashland, MA). This model consists of normal human keratinocytes cultured on a permeable synthetic membrane at the air-liquid interface in a chemically defined medium. The cells form a stratified, squamous corneal epithelium resembling the corneal mucosa of the human eye. This model has been used with several common tests of cytotoxicity including MTT and interleukin 1-alpha (IL-1α). A growing body of evidence indicates that EpiOcular™ effectively provides a non-animal means to assess potential irritancy. The EpiOcular™ model closely mimics the human corneal mucosa and thus provides an important in vitro approach in the evaluation of ocular irritancy and toxicity. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).

Vehicle:

unchanged (no vehicle)

Controls:

other: See ''Remark" for Control Samples used in the study

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

Duration of treatment / exposure:

Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~18 hours for test articles and controls.

Number of animals or in vitro replicates:

3 tissues were used for test compound and control.

Details on study design:

- Details of the test procedure used
The tissues were exposed to the test article 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~25 minutes for test article and controls. Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~18 hours for solid test articles and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
- MTT Pre-test
~50 mg of test article 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 55 minutes and 02 seconds. 50 µL of ultrapure water was used as a negative control.

- Test Article Color Test
Approximately 50 mg of test article 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a BioTek Synergy H4 (or equivalent) plate reader set to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay
After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours 55 minutes and 00 seconds (solids) of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the tissues were rinsed twice with DPBS. The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 59 minutes and 00 seconds. The tissues were not incubated overnight.

Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 31 minutes 00 seconds. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.

a)Controls:
50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

b)Test Article:
Approximately 50 mg of the test article 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

3. Post exposure treatment
After the exposure, the tissues were rinsed 20 to 25 times with ~1 mL of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to solid test articles (and the respective control) were incubated, submerged in the media for ~25 minutes at room temperature. Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 17 hours 48 minutes 01 seconds at approximately 37 degC, 5% CO2 in a humidified incubator.

- Doses of test chemical and control substances used
a)Test Article:
Approximately 50 mg of the 4,4'-carbonimidoylbis[N,N-diethylaniline] monohydrochloride were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

b)Controls:
50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~18 hours for test article and controls.

- Justification for the use of a different negative control than ultrapure H2O (Not applicable)

- Justification for the use of a different positive control than neat methyl acetate (Not applicable)

- Number of tissue replicates used per test chemical and controls: 3 tissues were used for test compound and control.

- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
·  The OD mean from all replicates for each plate (ODblank).

Negative Controls (NC):
·  The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
·  The OD mean per NC tissue was calculated.
·  The mean OD for all tissues corresponds to 100% viability.
·  The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.

ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.

Positive Control (PC):
·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
·        The OD mean per PC tissue was calculated.
·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.

Tested Articles:
·  Calculate the blank corrected value ODTT= ODTTraw– ODblank.
·  The OD mean per tissue is calculated.
·  The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
·  The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
·  The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.

Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.

Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.

- Evaluation of data
The results of the assay was evaluated and compared to negative control.

Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category

- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
 
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
 
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.

 

Irritation parameter:

other: mean % tissue viability

Run / experiment:

Run 1

Value:

3.1

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.4 passing the acceptance criteria.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The ocular irritation potential of test article 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance 4,4'-carbonimidoyl bis[N,N- diethylaniline] monohydrochloride was determined to be 3.1%. Thus, substance 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) was considered to be an eye irritant.

Executive summary:

The ocular irritation potential of test article 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles 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 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.4 passing the acceptance criteria.

 

The mean % tissue viability of test substance 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride was determined to be 3.1%.

 

Hence, under the experimental test conditions it was concluded that test substance 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) was considered to be an eye irritant and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Eye irritation potential of rhodamine B was assessed by exposing the individuals through acute inhalation route.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Rhodamine B
- Molecular formula : C28H31N2O3.Cl
- Molecular weight : 479.017 g/mol
- Substance type: Organic
- Physical state: Solid

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Age at study initiation: 16 males of mean age 39 years (25-50) and one female 40 year old.

Vehicle:

not specified

Controls:

no

Amount / concentration applied:

4530000 mg

Duration of treatment / exposure:

26 min

Observation period (in vivo):

24hrs

Duration of post- treatment incubation (in vitro):

no data available

Number of animals or in vitro replicates:

17 individuals

Details on study design:

Not applicable

Other effects / acceptance of results:

no data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24 h

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Burning of the eyes (82%), excessive tearing (47%), nasal burning (41%), nasal itching (35%), chest pain/tightness (35%), rhinorhea (29%), cough (29%), dyspnea (29%), burning of the throat (24%). burning/ pruritic skin (24%), chest burning (12%), headache (6%), and nausea (6%).

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

14 patients indicated 82 % eye irritation at the dose of approx (4.53kg) 4530000mg of dose indicating Rhodamine B to be irritating to eyes.
Acute exposure to Rhodamine B caused irritation to the eyes of humans.

Executive summary:

The study reports the evaluation of 17 patients who were exposed to aerosolized Rhodamine B.

Approximately 10 lbs =(4.53kg) 4530000mg of Rhodamine B powder was spilled onto the floor of a vehicle maintenance shop. A cleanup crew began to sweep up the powder from the floor . This resulted in an aerosol of Rhodamine B dust that dispersed throughout the building contaminating the hair , skin, eyes, and clothing of most of the shop employees.

Sixteen o f the patients (94%) complained of symptoms such as redness, burning sensation..etc. One patient (6%) denied any symptoms despite being exposed to the dust for 15 minutes without a surgical mask. The most frequently reported symptom was burning o f the eyes which occurred in 82% of the patients . Four of the patients (24%) were placed on humidified oxygen because of respiratory symptoms. Five of the patients (29%) underwent irrigation of their eyes secondary to eye burning.

After being discharged from the hospital, follow up was maintained. Ten of the patients (59%) reported that their symptoms had resolved within 4 hours of their exposure to the dust. One patient (6%) reported that his symptoms lasted 12 hours and 2 patients (12%) reported their symptoms lasted 24 hours. Three of the patients (18%) could not remember the duration of their symptoms.

Burning of eyes was observed in 82% of the patients. Based on these effects Rhodamine B may be considered as irritating to eyes.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

data is from experimental reports

Qualifier:

according to guideline

Guideline:

other: Fed. Reg. 38, No. 187, § 1500.42, 1973 (Draize test)

Principles of method if other than guideline:

To assess the ocular irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate in rabbits

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material: N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate
- Molecular formula: C33H32N3C2H3O2
- Molecular weight: 529.68 g/mol
- Smiles notation : [N+](=C1/C=CC(\C=C1)=C(\c1ccc(c2ccccc12)Nc1ccccc1)c1ccc(cc1)N(C)C)(\C)C.C(C)(=O)[O-]
- InChl : 1S/C33H31N3.C2H4O2/c1-35(2)27-18-14-24(15-19-27)33(25-16-20-28(21-17-25)36(3)4)31-22-23-32(30-13-9-8-12-29(30)31)34-26-10-6-5-7-11-26;1-2(3)4/h5-23H,1-4H3;1H3,(H,3,4)
- Substance type: Organic
- Physical state: Liquid

Species:

rabbit

Strain:

other: albino rabbit

Details on test animals or tissues and environmental conditions:

TEST ANIMALS

- Weight at study initiation:ca. 3 - 4 kg
- Housing:One animal per cage
- Diet (e.g. ad libitum): a standardized animal laboratory diet, ad libitum
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period:at least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/- 1°C
- Humidity (%):55 +/- 10%
- Photoperiod (hrs dark / hrs light):12 h light, 12 h darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

6

Details on study design:

The test substance was not washed out.
Readings: 1 h, 24 h, 48 h and 72 h after instillation

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24 - 72 hours

Score:

3.3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24 - 72 hours

Score:

1.9

Max. score:

2

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24 - 72 hours

Score:

3.2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Due to substance colouring, conjunctivae score was not assessed.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Conjunctivae score was not assessed due to staining of the test chemcal. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24,72 hours of observation were 3.3, 1.9, 3.2 respectively.
Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

Executive summary:

The ocular irritation potential of N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate was assessed in rabbits according to Fed. Reg. 38, No. 187, § 1500.42, 1973 (Draize test) Guidelines. 100 mg of the undiluted test chemical was instilled into one eye of the 6 albino rabbits.The untreated eye served as control. The animals were exposed to the test chemical for 24 hours, and the treated eyes remained unwashed throughout the study. Rabbits were observed for signs of irritation 1 h, 24 h, 48 h and 72 h after instillation of the test chemical. The ocular reactions at 24 and 72 hours were scored according to method of Draize.

Conjunctivae score was not assessed due to staining of the test chemical. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24,72 hours of observation were 3.3, 1.9, 3.2 respectively.

Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Various studies has been investigated for the test chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) to observe the potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits and rats for target chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) and its structurally similar read across substances [4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 84434-47-9) and [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 83803-79-6).The predicted data using the OECD QSAR toolbox has also been compared with the experimental data and summarized as below;

 

In a prediction done by SSS (2017) using the OECD QSAR toolbox with log kow as the primary descriptor, skin irritation potential was estimated for bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9). The substance bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) is considered to be not irritating to the skin of rabbits.

 

 

The above result was supported by two studies designed and conducted by Sustainability Support Services (Europe) AB, 2017 to determine the dermal reaction profile of read across chemicals [4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 84434-47-9) and [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 83803-79-6)in Sprague Dawley rats. These studies were performed as per OECD Guidelines 402 and complying to the GLP procedures. In both the studies, the test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.    Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment. The overall irritation score of the substance was obtained to be 0 and no erythema and edema (skin irritation) were observed at the end of 14 days observation period after patch removal. Hence, it was concluded that the substances [4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 84434-47-9) and [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS no.: 83803-79-6)were non-Irritating to the skin of rats under the experimental conditions tested .

 

Based on the available data for the target and read across substances and applying the weight of evidence approach, it can be concluded that chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

 

Eye Irritation:

In different studies,bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9)has been investigatedon the basis of in vitro and in vivo experimental studies available forits structurally similar read across substances4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7), [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6), Rhodamine B [CAS: 81-88-9] and C.I Basic Green 1 [CAS: 633-03-4]which have been summarized as follows;

 

 

In vitro experiment, the ocular irritation potential of read across chemical 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) was determined by Sustainability Support Services (Europe) AB (has letter of access from BASF SE) according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles 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 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.4 passing the acceptance criteria. The mean % tissue viability of test substance 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride was determined to be 3.1%. Hence, under the experimental test conditions it was concluded that test substance 4,4'-carbonimidoyl bis[N,N-diethylaniline] monohydrochloride (CAS No.- 6358-36-7) was considered to be an eye irritant.

 

Another ocular irritation study was assessed by [Sustainability Support Services (Europe) AB has letter of access from BASF SE, 1978] for N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate in rabbits according to Fed. Reg. 38, No. 187, § 1500.42, 1973 (Draize test) Guidelines. About 100 mg of the undiluted test chemical was instilled into one eye of the 6 albino rabbits. The untreated eye served as control. The animals were exposed to the test chemical for 24 hours, and the treated eyes remained unwashed throughout the study. Rabbits were observed for signs of irritation 1 h, 24 h, 48 h and 72 h after instillation of the test chemical. The ocular reactions at 24 and 72 hours were scored according to method of Draize. Conjunctivae score was not assessed due to staining of the test chemical. The effects observed in the rabbit eyes were not reversible even after 72 hours. The mean scores for corneal opacity, iris and chemosis after 24, 72 hours of observation were 3.3, 1.9, 3.2 respectively. Based on the scores, N-(4-((4-(dimethylamino)phenyl)(4-(phenylamino)naphthalen-1-yl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate can be considered to be irritating to rabbit eyes.

 

 

These results were supported by the case reported by Daniel J. Dire et.al [Journal of Toxicology: Clinical Toxicology, 25:7, 603-607; 1987] for the read across substance Rhodamine B [CAS: 81-88-9].The study reports the evaluation of 17 patients who were exposed to aerosolized Rhodamine B. Approximately 10 lbs =(4.53kg) 4530000mg of Rhodamine B powder was spilled onto the floor of a vehicle maintenance shop. A cleanup crew began to sweep up the powder from the floor. This resulted in an aerosol of Rhodamine B dust that dispersed throughout the building contaminating the hair, skin, eyes, and clothing of most of the shop employees. Sixteen of the patients (94%) complained of symptoms such as redness, burning sensation..etc. One patient (6%) denied any symptoms despite being exposed to the dust for 15 minutes without a surgical mask. The most frequently reported symptom was burning of the eyes which occurred in 82% of the patients. Four of the patients (24%) were placed on humidified oxygen because of respiratory symptoms. Five of the patients (29%) underwent irrigation of their eyes secondary to eye burning. After being discharged from the hospital, follow up was maintained. Ten of the patients (59%) reported that their symptoms had resolved within 4 hours of their exposure to the dust. One patient (6%) reported that his symptoms lasted 12 hours and 2 patients (12%) reported their symptoms lasted 24 hours. Three of the patients (18%) could not remember the duration of their symptoms. Burning of eyes was observed in 82% of the patients. Based on these effects, Rhodamine B may be considered as irritating to eyes.

 

The above results were further supported by the experimental data summarized in HSDB (Hazardous Substances Data Bank); US national Library of Medicine reviewed by SRC, last updated 2003; for the structurally similar read across substance C.I Basic Green 1 [CAS: 633-03-4].Basic Green 1 was irritating to rabbit eyes.

 

Thus on the basis ofread across substances and applying the weight of evidence approach, it can be concluded that the target chemical bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) might be able to cause severe eye irritation and thus can be considered as irritating . Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2”

 

Justification for classification or non-classification

The skin and eye irritation potential of test chemical Bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) and its structurally similar read across substanceswere observed in various studies. The results obtained from these studies indicate that the chemical Sodium 5-(aminosulphonyl)-2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazolesulphonate is unlikely to cause skin irritation but might cause severe eye damage. Hence Bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-) (CAS No.: 82338-76-9) can be classified under the category “Not Classified” for skin and “Category 2”eye as per CLP.