Ethanaminium, N-[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthalenyl]methylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl-, molybdatetungstatephosphate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August - 28 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no 278PE160900 from Client
- Expiration date of the lot/batch: 28 April 2022
- Purity test date: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Roomtemperature in the dark
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Used as supplied
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): N/a
- Final dilution of a dissolved solid, stock liquid or gel: N/a
- Final preparation of a solid: Used as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material) Used as supplied

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories

Source strain:

other: Reconstructed human epidermis

Amount/concentration applied:

Used as supplied = 100% (10 mg)

Duration of treatment / exposure:

15 mins

Number of replicates:

3

Controls:

yes

Details on study design:

An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item. Therefore, an additional procedure using water killed tissues was performed. The results of the water killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
The solution containing the test item was a blue color, therefore additional color correction tissues were incorporated into the testing procedure. The results of the color correction tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
The results of the third set of control tissues were used to adjust the calculated correction factors derived from the color interference and MTT direct reduction controls in order to prevent a double correction from a colored test item that also reduces MTT. Mean OD570 values and correction factor calculations are given in the report.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three runs

Value:

44.7

Negative controls validity:

valid

Positive controls validity:

valid

The relative mean tissue viability for the positive control treated tissues was 10.7% relative to the negative control treated tissues and the standard deviation value of the viability was 2.8%. The positive control acceptance criteria were therefore satisfied.

The mean OD₅₇₀for the negative control treated tissues was 0.847 and the standard deviation value of the viability was 9.8%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.3%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item was classified as irritant. The following classification criteria apply:
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have the same type of toxicological effects based on common underlying mechanisms (ECHA, 2015b). All substances have the same organic structural group (colorant) with different compositions of the inorganic group (metallic salt). Read-across is in this case based on the hypothesis that source and target substances have similar toxicological properties because they degrade to the similar chemical substances (ECHA, 2015c). This prediction is supported by physicochemical and toxicological data on the substances.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Name Target Substance Source Substance
Chemical name Ethanaminium, N-[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthalenyl]methylene]- Bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-
2,5-cyclohexadien-1-ylidene]-N-ethyl-, molybdatetungstatephosphate dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-)
IUPAC name Reaction product of [4-[bis[4-(diethylamino)phenyl]methylidene]naphthalen-1-ylidene]-
ethylazanium chloride with phosphotungstomolybdic acid Unknown
CAS No 1325-87-7 82338-76-9/57485-98-0
EC No 215-410-7 279-935-3
Colourant C.I. Basic Blue 7 (R30200) C.I. Basic Blue 7 (R30200)
Metals Phosphotungstomolybdic acid salt Copper ferrocyanide salt
Molecular weight range ≥ 6600 ≤ 10500 880-1750
Molecular formula (C33H40N3)z.O3.P2O5.(WO3)x.(MoO3)y C33H40N3.1/2C6FeN6.Cu
12 ≤ x+y ≤ 24 (x≥1; y≥1)
z = 6-10
Structural formula

Both substances are UVCB’s and so purity is considered to be 100%

3. ANALOGUE APPROACH JUSTIFICATION
The target substance CAS 1325-87-7is an organometallic multi-constituent substance. The source substance CAS 63022-06-0 has the same colourant (organic constituent) (see table above) as the target substance consisting of a basic [4-[bis[4-(diethylamino)phenyl]methylidene]naphthalen-1-ylidene]-ethylazanium. The inorganic Part B is different between source and target substances, consisting of the elements O, Mo, P and W for the target substance and Cu, Fe and CN for the source substance. These differences are expected to behave very similar. The target and source substances are thus considered suitable for the analogue approach based on structural similarity.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no 278PE160900 from Client
- Expiration date of the lot/batch: 28 April 2022
- Purity test date: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Roomtemperature in the dark
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Used as supplied
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): N/a
- Final dilution of a dissolved solid, stock liquid or gel: N/a
- Final preparation of a solid: Used as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material) Used as supplied

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories

Source strain:

other: Reconstructed human epidermis

Amount/concentration applied:

Used as supplied = 100% (10 mg)

Duration of treatment / exposure:

15 mins

Number of replicates:

3

Controls:

yes

Details on study design:

An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item. Therefore, an additional procedure using water killed tissues was performed. The results of the water killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
The solution containing the test item was a blue color, therefore additional color correction tissues were incorporated into the testing procedure. The results of the color correction tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
The results of the third set of control tissues were used to adjust the calculated correction factors derived from the color interference and MTT direct reduction controls in order to prevent a double correction from a colored test item that also reduces MTT. Mean OD570 values and correction factor calculations are given in the report.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three runs

Value:

44.7

Negative controls validity:

valid

Positive controls validity:

valid

The relative mean tissue viability for the positive control treated tissues was 10.7% relative to the negative control treated tissues and the standard deviation value of the viability was 2.8%. The positive control acceptance criteria were therefore satisfied.

The mean OD₅₇₀for the negative control treated tissues was 0.847 and the standard deviation value of the viability was 9.8%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.3%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item was classified as irritant. The following classification criteria apply:
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jul 1993 - 27 Jul 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0151N/901035
- Storage: room temperature
- Physical Appearance: dark purple powder

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 – 16 weeks old
- Weight at study initiation: 2.20 – 2.42 kg
- Housing: suspended metal cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 56 - 68
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/ 12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL (63mg)

Observation period (in vivo):

1, 24, 48 and 72 hours, 7 and 14 days

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect seen

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Other effects:

Blue – coloured staining was noted on two treated eyes during the study. The staining did not affect evaluation of ocular effects.

	animal #1				animal #2				animal #3
	24 h	48 h	72 h	mean  animal #1	24 h	48 h	72 h	mean  animal #2	24 h	48 h	72 h	mean animal #3	mean
A - Redness	1	0	0	0.3	2	2	2	2	2	2	2	2	1.4
B - Chemosis	0	0	0	0	2	2	2	2	2	2	2	2	1.3
D - Iris	0	0	0	0	1	1	1	1	1	1	1	1	0.7
E - Degree of Opacity	0	0	0	0	1	2	2	1.7	1	1	1	1	0.9

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

The test item is classed as irritating to the eye

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jul 1993 - 2 Aug 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 155N/ 881715
- Expiration date of the lot/batch:
- Physical Appearance: purple powder
- Storage: room temperature

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 – 16 weeks
- Weight at study initiation: 2.56 – 2.84 kg
- Housing: suspend metal cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 56 - 75
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied:0.1mL (83 mg)

Duration of treatment / exposure:

-

Observation period (in vivo):

72 hours (1, 24, 48 and 72 hours after the treatment)

Number of animals or in vitro replicates:

3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: 7days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 7days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Residual test material was noted around the eye of treated animals throughout the study.

Blue – coloured staining was noted in all treated eyes during the study.

Diffuse corneal opacity was noted in all treated eyes at the 24, 48 and 72 hours observations.

Iridial inflammation was noted in all treated eyes one hour after treatment and the 24, 48 and 72 hour observation. No other iridial effects were noted.

Moderate conjunctival irritation was noted in all treated eyes on and 24 hours after treatment with minimal to moderate conjunctival irritation at the 48 and 72 observations. Minimal conjunctival irritation was noted in one of treated eye at the 7-day observation. Petechial haemorrhage of the nictitating membrane was noted in one treated eye at the 24-hour observation.

No ocular effects were noted 7 or 14 days after treatment.

	animal #1				animal #2				animal #3
	24 h	48 h	72 h	mean animal #1	24 h	48 h	72 h	mean animal #2	24 h	48 h	72 h	mean animal #3	mean
Conjunctival Redness	2	2	2	2	2	2	1	1.7	2	2	2	2	1.9
Conjunctival Chemosis	2	2	2	2	2	1	1	1.3	2	2	2	2	1.8
Iritis	1	1	1	1	1	1	1	1	1	1	1	1	1
Corneal opacity	1	1	1	1	1	1	1	1	1	1	1	1	1

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Conclusions:

Test item is classed as irrItating to eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

PB1 is classified as irritating to the eye GHS Cat 2 and irritating to the skin GHS Cat 2