Butanedioic acid, sulfo-, 4-[2-[(2-hydroxyethyl)amino]ethyl] ester, N-C18-unsatd. acyl derivs., disodium salts

Endpoint summary

Description of key information

The test item containing 29.9 % active ingredient was predicted to be non-corrosive to human skin based on the three-dimensional EST 1000 human skin model  (3 minutes and 1 hour exposure); mean cell viability was 56.7% and 47.0% after a 20-minutes and 1-hour exposure, which were above the cut-off  values of 50% or 15% for a 3-minute or 1-hour treatment, respectively.  In a second in vitro study, test item  applied for 20 minutes, followed 42 hours incubation, was predicted to be non-irritant to human skin: the mean cell viability  was 110.5% of the mean negative control value. According to ECHA progress report, this weight-of-evidence approach can replace in vivo testing. Nevertheless, a subgroup classification for CLP category 2 for skin irritation  was proposed, with concentration limit of 29.9% for non-classification.
The test substance containing 29.9% active ingredient was predicted to be slightly irritant to human eye based on the in vitro HET-CAM model; the test item treated eggs revealed an effect with an irritation index (IS) of 4.0, compared to IS of 19.0 or 9.3 for 0.1 N NaOH and 1% SDS. However it was not predicted to be a severe eye irritant in the in vitro BCOP model; an IVIS 0.80 was calculated, which was below the threshold value of 15.0, hence the test item was not classified as a severe irritant and not corrosive.  This confirmed the subgroup classification CLP category 2 for eye irritation. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. It is an in vitro study predicting skin irritation, which is considered adequate in combination with the in vitro corrosivity testing.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: a three-dimensional human skin model

Strain:

other: The Skin model EST-1000 was used.

Details on test animals or test system and environmental conditions:

Not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 mg of the supplied test item were dissolved in 1 mL water for injection. 30 µL Butanedioic acid, sulfo-, 4-(2-((2-hydroxyethyl)amino)ethyl) ester, N-C18-unsatd. acyl derivs., disodium salts were applied to the skin model with a surface area of 0.6 cm2

Irritation / corrosion parameter:

other: other: cell viability test group

Value:

110.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 20 minutes, followed by 42h incubation . Remarks: % versus negative control group . (migrated information)

Irritation / corrosion parameter:

other: other: cell viability positive control group

Value:

1.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 20 minutes, followed by 42h incubation . Remarks: % versus negative control group . (migrated information)

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, sulfo-, 4 -(2 -((2 -hydrocyethyl)amino)ethyl)ester, N-C18 -unsatd. acyl derivs., disodium salts, was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SS) was used as the positive reference item.

The mean viability of the cells exposed to the test item was 110.5% of the mean negative control value. The OD₅₄₀ values were well above the cut-off percentage cell viability value (that distinguishes irritant from non-irritant test items) of >50% for a 20-minute exposure.

The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

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:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions Butanedioic acid, sulfo-, 4-(2-((2-hydroxyethyl)amino)ethyl) ester, N-C18-unsatd. acyl derivs., disodium salts, tested at an exposure time of 20 minutes, was non-cytotoxic and not irritant to skin in an experiment with 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 Butanedioic acid, sulfo-, 4 -(2 -((2 -hydroxyethyl)amino)ethyl) ester, N-C-18 -unsatd. acyl derivs., disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin.

The EST1000 model was employed.

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

The test item, Butanedioic acid, sulfo-, 4 -(2 -((2 -hydroxyethyl)amino)ethyl) ester, N-C18 -unsatd. acyl derivs., disodium salts, was applied to 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 the cells exposed to the test item was 110.5% of the mean negative control value. The OD₅₄₀values were well above the cut-off percentage cell viability value (that distinguishes irritant from non-irritant test items) of >50% for a 20-minute exposure.

The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. It is an in vitro study by means of BCOP, predicting severe eye irritation, which is considered adequate in combination with the in vitro irritation testing.

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICCVAM BCOP test method, 2007

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eyes from cattle in the age range of 6 to 12 months were obtained from Hubert Bahlmann GmbH & Co., Lindern.

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL of test or control item were added to completely cover the cornea's epithelium in the anterior chamber.
- Concentration (if solution): The test item was dissolved in 0.9% NaCl-solution to a final concentration of 10% active ingredient, which complies with the requirements for surfactants.

Duration of treatment / exposure:

10 minutes

Irritation parameter:

other: IVIS (in vitro irritancy score)

Score:

0.8

Irritant / corrosive response data:

The optical density (OD492) or absorbance values were measured at a wavelength of 492 nm. An opacity value of -0.336 and a permeability value of 0.075 compared to the negative control were determined. An IVIS 0.80 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 69.199 and a permeability value of 2.150 compared to the negative control. The IVIS value of 101.43 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.

Table 3. In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
NaCl 0.9%	1	0.331	0.036	0.9	0.50	0.36
	2	0.125	0.008	0.3
	3	0.229	0.012	0.4
NaOH 1%	4	65.705	2.147	97.9	101.43	3.66
	5	69.863	2.091	101.2
	6	72.029	2.213	105.2
Active ingredient 1:10#	7	-0.585	0.094	0.8	0.80	0.10
	8	-0.104	0.053	0.7
	9	-0.320	0.078	0.9

NC: negative control
SD: standard deviation
#: tested as 10% concentration in 0.9% NaCl solution

Interpretation of results:

other: predicted non-corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions Butanedioic acid, sulfo-, 4-(2-((2-hydroxyethyl)amino)-ethyl) ester, N-C18-unsatd. acyl derivs., 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 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 dissolved in 0.9% NaCl-solution to a final concentration of 10% active ingredient, which complies with the 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 -0.336 and a permeability value of 0.075 compared to the negative control were determined. An IVIS 0.80 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 69.199 and a permeability value of 2.150 compared to the negative control. The IVIS value of 101.43 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 Butanedioic acid, sulfo-, 4-(2-((2-hydroxyethyl)amino)-ethyl) ester, N-C18-unsatd. acyl derivs., 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 a first in vitro study, the corrosive properties of registered substance were studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013a). The test item (containing 29.9 % active ingredient) was applied for exposure times of 3 minutes or 1 hour. The mean viability of cells exposed to the test item was 56.7% after a 3-minute exposure period and 47.0% 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 to skin cells of registered substance were studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013b). The test item (containing 29.9% 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 110.5% of the mean negative control value. The OD₅₄₀ values were well above the cut-off percentage cell viability value (that distinguishes irritant from non-irritant test items) of > 50% for a 20 -minute exposure. The test item was considered to be non-cytotoxic and predicted to be not irritant to skin. 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 skin irritation.

- 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 negative result in the human skin model for irritation does not need to be confirmed by additional testing.

- In conclusion, the 29.9% solution was predicted to be not irritating. However a (worst case) subgroup classification is proposed: CLP cat. 2 for skin irritation, with concentration limit of 29.9% act.ingr. for non-classification.

 

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

- In a first in vitro study the eye irritancy potential of test item containing 29.9% active ingredient was tested by means of the chorioallantoic membrane of hens' eggs (HET-CAM) method (Haferkorn, 2013c). 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 an effect with an irritation index (IS) of 4.0, compared to IS of 19.0 or 9.3 for 0.1 N NaOH and 1% SDS positive controls and no effects in the negative control 0.9% NaCl solution. The test item was considered to be a slight irritant.

- In a second in vitro study severe eye irritancy potential and corrosivity potential of the test item containing 29.9% active ingredient 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 dissolved in 0.9% NaCl-solution to a final concentration of 10% active ingredient, which complies with the 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. An opacity value of -0.336 and a permeability value of 0.075 compared to the negative control were determined. An IVIS 0.80 was calculated, which was below the threshold value of 15.0, hence the test item was not classified as a severe irritant and not corrosive, based on the results of this test.

- According to Column 2, the criteria for classification are met as irritating to eyes, based on weight of evidence, and further in vivo testing was therefore not needed. The approach followed was a top-down approach based on in vitro testing as described by Scott et al., 2010 (publication attached). 

- In conclusion, the 29.9% solution was predicted to irritant; this complies with the subgroup classification CLP cat. 2 for eye irritation.

 

Conclusion:

- A subgroup CLP category 2 classification was decided for skin irritation, with concentration limit of 30% for non-classification of registered substance.
- A subgroup CLP category 2 classification was decided for eye irritation, with no concentration limit for non-classification.
- 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.

Effects on skin irritation/corrosion: slightly irritating

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

The registered substance is classified for skin irritation according to the Directive 67/548/EEC, Annex VI with symbol Xi and the indication of “irritant”; the following risk phrase shall be assigned: R38 - Irritating to skin.  According to CLP regulation (No. 1272/2008 of 16 December 2008), the substance is classified as Category 2, with signal word 'warning' and hazard statement: H315 - Causes skin irritation. However, a concentration limit of 30% for non-classification can be applied.

For the eye, the test substance is classified as irritating to eyes and the risk phrase R36- Irritating to eyes shall be assigned. According to CLP regulation, the substance is classified as Category 2, with signal word 'warning' and hazard statement: H319 -Causes serious eye irritation. A concentration limit is not applied for eye irritation.