C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite

Endpoint summary

Description of key information

The read-across test item CAS 68784 -08 -7 containing 39.8% active ingredient was predicted to be irritant to human skin based on the three-dimensional EST 1000 human skin model, showing a cell viability of 1.3% compared to control, which meets the 50% classification threshold). However it was negative for corrosion in the same test model (3 minutes and 1 hour exposure), showing cell viability of 101 and 78.0% versus controls, respectively, which were above threshold value of 50%. CLP classification 2 for skin irritation is proposed for the registered substance. A 5 and 10% solution were previously tested to be non-irritant based on human application to arm skin for 48 hours, and therefore these are concentration limits for non-classification. 

The read-across test item CAS 68784 -08 -7 containing 39.8% active ingredient was predicted to be irritant to human eye based on the in vitro HET-CAM model, showing an in vitro irritation score of 19, which is comparable to positive control value. However it was negative for severe eye irritation and damage in the in vitro BCOP model, showing an in vitro irritation score of 21.13, which was below classification threshold of 55.1. CLP classification 2 for eye irritation is proposed for the registered substance. A 20% solution was previously tested to be mildly irritant based on the Draize method in rabbit eyes. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean; % versus negative control group.

Run / experiment:

test group 3 minutes exposure

Value:

101

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean; % versus negative control group.

Run / experiment:

test group 1 hour exposure

Value:

78

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean; % versus negative control group.

Run / experiment:

positive control group 3 minutes exposure

Value:

4.1

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean; % versus negative control group.

Run / experiment:

positive control group 1 hour exposure

Value:

3.6

Negative controls validity:

valid

Positive controls validity:

valid

The test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the skin surface. Water for injection was used as the negative control. 8 N KOH was used as the positive reference item. Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1-hour exposure. The OD₅₄₀ values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 4.1% (3-minute incubation) and 3.6% (1-hour incubation) of the negative controls and were below the cut-off values and well within the range of historical background data of 3.3 – 47.4% (3-minute incubation) and 0.2 – 4.9% (1-hour incubation) of the negative controls (the last 6 experiments, not audited by the QAU-department). Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts tested at two exposure times of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells which might lead to corrosion by read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The test item containing 39.8% active ingredient was applied to the skin surface. Water for injection was used as the negative control. 8 N KOH was used as the positive reference item.Two exposure times of 3 minutes or 1 hour were employed. In comparison to the negative controls, the mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1 -hour exposure.The OD₅₄₀ values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 4.1% (3 -minute incubation) and 3.6% (1 -hour incubation) of the negative controls and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 replicates; % versus negative control group

Run / experiment:

test group

Value:

1.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Time point: 20 minutes, followed by 42h incubation.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 replicates; % versus negative control group

Run / experiment:

positive control group

Value:

1.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Time point: 20 minutes, followed by 42h incubation.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No interactions of the test item with MTT were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.
All quality criteria required were fulfilled.
No interactions of the test item with MTT were noted.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control (NC): For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries.
- Acceptance criteria met for positive control (PC): Tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 50% for the validated method)
- Acceptance criteria met for variability between replicate measurements: Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value.The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS,was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the present test conditions read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested at an exposure time of 20 minutes, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells, which might lead to irritation by read-across substance Butandioic acid, 2(or 3)-sulfo, 4 - [2 - [(1 -oxo(C12 -C18(even numbered) and C18 unsaturated)alkyl)amino]ethyl], esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST1000 model was employed. The test item containing 39.8% active ingredient was applied. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed, followed by refreshment of the medium and further 42 hours incubation. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

overall irritation score

Basis:

other: individual scores

Time point:

other: 48 hours & 5 days

Score:

0

Irritant / corrosive response data:

No reactions were detected.

Table 1. Irritation

Subject	5% solution		10% solution
	48 hours	5 days	48 hours	5 days
102	-	-	-	-
103	-	-	-	-
104	-	-	-	-
105	-	-	-	-
Result-No reaction detected

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

No reactions were detected with read-across substance CAS 68784-08-7.

Executive summary:

A 1 cm2 of unmedicated white lint was soaked in the 5 and 10% read-across test substance solution in comparison with toilet soaps, and applied to the skin of the left arm of 4 volunteers for 48 hours under waterproof adhesive dressing, followed by an observation period of 72 hours. No reactions were detected.

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:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

other: total cornea score (opacity and area involved)

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

80

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

other: total conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1.77

Max. score:

20

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: total conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3.33

Max. score:

20

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: total conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

20

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

other: total conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

20

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

other: total iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

10

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Table 1. Results Rabbit 3.G : Eye irritation study with [Trade name] (20% active matter)

	Hours							Days
	1	2	4	6	24	48	72	4	7
CORNEA
Opacity	0	0	0	0	0	0	0	0	0
Area	0	0	0	0	0	0	0	0	0
Score	0	0	0	0	0	0	0	0	0
IRIS
Values	0	0	0	0	0	0	0	0	0
Score	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
Redness	2	3	3	3	2	1	0	0	0
Chemosis	1	2	2	2	1	0	0	0	0
Discharge	2	2	2	2	1	0	0	0	0
Score	10	14	14	14	8	2	0	0	0
Total Score	10	14	14	14	8	2	0	0	0

 

 

Table 2. Results Rabbit 3.V : Eye irritation study with [Trade name] (20% active matter)

	Hours							Days
	1	2	4	6	24	48	72	4	7
CORNEA
Opacity	0	0	0	0	0	0	0	0	0
Area	0	0	0	0	0	0	0	0	0
Score	0	0	0	0	0	0	0	0	0
IRIS
Values	0	0	0	0	0	0	0	0	0
Score	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
Redness	2	3	3	3	1	0	0	0	0
Chemosis	2	3	3	2	0	0	0	0	0
Discharge	2	2	2	2	0	0	0	0	0
Score	12	16	16	14	2	0	0	0	0
Total Score	12	16	16	14	2	0	0	0	0

 

Table 3. Results Rabbit 3.R : Eye irritation study with [Trade name] (20% active matter)

	Hours							Days
	1	2	4	6	24	48	72	4	7
CORNEA
Opacity	0	0	0	0	0	0	0	0	0
Area	0	0	0	0	0	0	0	0	0
Score	0	0	0	0	0	0	0	0	0
IRIS
Values	0	0	0	0	0	0	0	0	0
Score	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
Redness	2	2	2	2	1	0	0	0	0
Chemosis	2	2	3	2	0	0	0	0	0
Discharge	2	2	2	2	1	0	0	0	0
Score	12	12	14	12	4	0	0	0	0
Total Score	12	12	14	12	4	0	0	0	0

 

Interpretation of results:

GHS criteria not met

Conclusions:

CAS 68784-08-7: mild irritation reversible within 3 days.

Executive summary:

The objective of the study was to determine the potential eye irritation properties of different samples of which the chemical nature was not disclosed. The method used was generally that of Draize, Calvery and Woodward (Journ. Pharm. Exp. Therap. 82, 377 -390, 1944) and later described in "Appraisal of Safety of Chemicals in Foods, drugs and Cosmetics" (F.D.A. Div. Pharmacology, Draize J.H., 1959). The test solutions were made up by diluting the submitted samples of declared content of active matter with distilled water so that the content of active matter was reduced to 20% in each case. The tap water used for the control was London Tap Water. 0.1mL of the read-across test solution (20% act. ingr.) was instilled into the conjunctival sac of one eye of each rabbit, the other eye serving as a control. In no case was the eye washed after instillation. For the sample of [Trade name], 3 rabbits were used. Readings were made visually and with the aid of an ophthalmoscope at 1, 2, 4, 6, 24, 48, 72 hours and 4 and 7 days after instillation and evaluated according to the Draize scoring method.
For the sample of [Trade name], mild irritation was observed which was reversible within 3 days.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

other: Irritation score

Remarks:

mean

Run / experiment:

test item

Value:

18.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum score 21.0

Irritation parameter:

other: irriatation score

Remarks:

mean

Run / experiment:

0.1% NaOH positive control

Value:

19.4

Remarks on result:

other: maximum score 21.0

Irritation parameter:

other: irritation score

Remarks:

mean

Run / experiment:

1% SDS positive control

Value:

10

Remarks on result:

other: maximum score 21.0

Irritation parameter:

other: irritation score

Remarks:

mean

Run / experiment:

negative control

Value:

0

Remarks on result:

other: maximum score 21.0

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The HET-CAM assay is considered acceptable if the negative controls each induce a response that falls within the classification of nonirritating.
- Acceptance criteria met for positive control: The HET-CAM assay is considered acceptable if the positive controls each induce a response that falls within the classification of severely irritating, respectively.
- Range of historical values: Historical control studies demonstrate that using 1% SDS and 0.1 N NaOH as positive controls results in IS values of approximately 10 and 18, respectively.

The eggs treated with Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)­alkyl))amino]­ethyl]­esters, disodium salts revealed a pronounced effect with an irritation index (IS) of 18.6. The test item was considered to be strong irritant.

The positive control items 0.1% NaOH or 1% SDS caused the expected effect with irritation indices (IS) of 19.4 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution. Hence, the HET-CAM assay is considered to be valid.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the present test conditions, read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts was strong irritant to eyes in an experiment with the chorioallantoic membrane of hens' eggs (HET-CAM).

Executive summary:

The purpose of this study was to determine the eye irritancy potential of read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts by means of the chorioallantoic membrane of hens' eggs (HET-CAM). Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects. The CAM of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex.

Three eggs each were treated with 300 µL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts/egg and with the control items. 0.9% NaCl solution was used as the negative control item. 0.1 N Sodium hydroxide (NaOH) and 1% aqueous sodium dodecyl sulphate (SDS) were used as the positive control items. The administration volume for the control items was 300 µL per egg.

After administration of the test item blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes.

The eggs treated with Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)­alkyl))amino]­ethyl]­esters, disodium salts revealed a pronounced effect with an irritation index (IS) of 18.6. The test item was considered to be strong irritant.

The positive control items 0.1or 1% SDS caused the expected effect with irritation indices (IS) of 19.4 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution. Hence, the HET-CAM assay is considered to be valid.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

test item

Value:

21.9

Negative controls validity:

valid

Remarks:

mean IVIS 0.03

Positive controls validity:

valid

Remarks:

mean IVIS 87.43

Remarks on result:

other: A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Upper limit of acceptance for opacity 1.259; Upper limit of acceptance for permeability 0.049.
- Acceptance criteria met for positive control: A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
Lower limit of acceptance 76.8; Upper limit of acceptance 133.2.

Table 1. In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
NaCl 0.9% NC	1	-0.066	0.0160	0.2	0.03	0.15
	2	-0.235	0.0130	0.0
	3	-0.262	0.0130	-0.1
NaOH 1%	4	53.497	1.851	81.3	87.43	9.11
	5	65.173	2.181	97.9
	6	51.047	2.137	83.1
Active ingredient 1:10#	7	0.628	1.128	17.5	21.90	3.83
	8	2.295	1.479	24.5
	9	4.854	1.256	23.7

 

NC: negative control

SD: standard deviation

#: tested as 10% concentration in 0.9% NaCl solution

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.

Executive summary:

The purpose of this study was to determine if the read-across test item can be classified as “ocular corrosive and severe irritant” employing an in vitro system. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from bovine eyes. The corneas of the eyes were dissected and the remaining sclera was mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as a 3.98-fold dilution in 0.9% NaCl-solution in order to obtain a 10% w/v dilution of active ingredient, which complies with the guideline requirements for surfactants. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder for an exposure time of 10 minutes.

The optical density (OD₄₉₂) or absorbance values were measured at a wavelength of 492 nm. An opacity value of 2.592 and a permeability value of 1.288 compared to the negative control were determined. An IVIS 21.90 was calculated. Hence, the test item was not classified as a severe irritant and not corrosive, based on the results of this test.

The corneas treated with the positive control item 1% NaOH solution revealed an opacity value of 56.572 and a permeability value of 2.056 compared to the negative control. The IVIS value of 87.43 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. 1% NaOH solution was a severe irritant and corrosive to eyes.

Under the present test conditions read-across substance Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Assessment of skin irritation was based on a weight-of-evidence approach:

- In an in vivo (human) study, a 1 cm² of unmedicated white lint was soaked in the 5 and 10% read-across test item solution in comparison with toilet soaps, and applied to the skin of the left arm of 4 volunteers for 48 hours under waterproof adhesive dressing, followed by an observation period of 72 hours (Baker Research Laboratories limited, 1962a). No reactions were detected.

- In a first in vitro study, the corrosive properties of read-across substance CAS 68784 -08 -7 were studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013a). The test item (containing 39.8% active ingredient) was applied for exposure times of 3 minutes or 1 hour. The mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1-hour exposure. These values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this human skin model and is predicted to be non-corrosive to human skin.

- In a second in vitro study, the irritant properties of read-across substance CAS 68784 -08 -7 to skin cells were also studied in an experiment with a three-dimensional EST-100 human skin model (Flügge, 2013b). The test item (containing 39.8% active ingredient) was applied for an exposure time of 20 minutes, followed by refreshment of the medium and further 42 hours incubation. The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

- According to ECHA progress report 2010 (p. 32), it is accepted that in vitro methods for skin irritation represent a full replacement of the in vivo method in a tiered testing strategy and in conjunction with in vitro skin corrosivity tests, if necessary. A positive result in the human skin model for irritation does not need to be confirmed by additional testing and shall lead to classification.

 

Assessment of eye irritation was also based on a weight-of-evidence approach:

- In an in vivo study, the potential eye irritation properties of different samples were tested according to the Draize mehod in rabbits (Baker Research Laboratories limited, 1962b). The test solutions were diluted with distilled water so that the content of active matter was reduced to 20% in each case. 0.1mL of the read-across test solution (20% act. ingr.; CAS 68784 -08 -7) was instilled into the conjunctival sac of one eye of each rabbit, the other eye serving as a control (3 rabbits were used). Readings were made visually and with the aid of an ophthalmoscope at 1, 2, 4, 6, 24, 48, 72 hours and 4 and 7 days after instillation and evaluated according to the Draize scoring method. Mild irritation was observed which was reversible within 3 days, which was below threshold of classification.

- In a first in vitro study the eye irritancy potential of read-across test item containing 39.8% active ingredient (CAS 68784 -08 -7) was tested by means of the chorioallantoic membrane of hens' eggs (HET-CAM) method (Haferkorn, 2012). Three eggs each were treated with 300 µL/egg. After administration of the test item, blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes. The test item treated eggs revealed a pronounced effect with an irritation index (IS) of 18.6, compared to IS of 19.4 or 10.0 for 0.1% NaOH and 1% SDS positive controls and no effects in the negative control 0.9% NaCl solution. The read-across test item CAS 68784 -08 -7 was predicted to be irritating.

- In a second in vitro study severe eye irritancy potential and corrosivity potential of the read-across test item containing 39.8% active ingredient (CAS 68784 -08 -7) was tested by means of the BCOP test method (Leuschner, 2013). Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as a 3.98-fold dilution in 0.9% NaCl-solution in order to obtain a 10% w/v dilution of active ingredient, which complies with the guideline requirements for surfactants. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. An IVIS score of 21.90 was calculated, hence the read-across test item was not classified as a severe irritant and not corrosive, based on the results of this test. The corneas treated with the positive control item 1% NaOH solution revealed an IVIS score of 87.43 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. The read-across test item, containing 39.8% active ingredient was consequently not classified as a severe irritant and is not corrosive.

- According to Column 2, the criteria for classification are met as irritating to eyes, based on weight of evidence, and furtherin vivotesting was therefore not needed.

 

Conclusion

- A subgroup CLP category 2 classification was decided for skin irritation, with concentration limit of 10% for non-classification of registered substance.

- A subgroup CLP category 2 classification was decided for eye irritation, with concentration limit of 20% for non-classification of registered substance.

- More information on the subgroup classification is provided in the read-across justification, separately attached in Section 13.

 

Justification for selection of skin irritation / corrosion endpoint: Although the in vitro study for irritation was selected, the corrosion study was equally valuable in a weight-of-evidence approach. 

Justification for selection of eye irritation endpoint: Although the BCOP study was selected, the HET-CAM and in vivo studies were equally valuable in a weight-of-evidence approach. 

Justification for classification or non-classification

The registered substance is classified for skin irritation according to the CLP regulation (No. 1272/2008 of 16 December 2008) as Category 2, with signal word 'warning' and hazard statement: H315 - Causes skin irritation. However, a concentration limit of 10% for non-classification can be applied.

For the eye, the test substance is classified as irritating to eyes according to CLP regulation as Category 2, with signal word 'warning' and hazard statement: H319 -Causes serious eye irritation. However, a concentration limit of 20% for non-classification can be applied.