Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 3, 2015 to August 7, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EpiDerm Skin Model (EPI-200, Lot no.: 22625 kit U) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

SOURCE
EpiDerm Skin Model were procured from MatTek Corporation, Ashland MA, U.S.A.

CELL CULTURE
Medium: DMEM (Dulbecco’s Modified Eagle’s Medium); Supplemented DMEM medium, serum-free supplied by MatTek Corporation
MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation
Environmental conditions: Humid atmosphere- 80-100% (actual range 67 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark,
Temperature- 37.0 ± 1.0°C (actual range 36.7 - 37.4°C).

Duration of treatment / exposure:

2 tissues per test substance- 3 min exposure
2 tissues per test substance- 1 h exposure

Details on study design:

Application/Treatment of the test substance
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 h before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for 1.5 h at 37.0 ± 1.0⁰C. The medium was replaced with fresh DMEM medium just before test substance was applied.

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3 min exposure to the test substance and two for a 1 h exposure. The skin was moistened with 25 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and an excessive amount was added into the 6-well plates on top of the skin tissues using a curved spatula and gauze patch. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, Netherlands) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 h at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek Corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Run / experiment:

3 minutes

Value:

ca. 89

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Run / experiment:

1 hour

Value:

ca. 91

Remarks on result:

no indication of irritation

Table 1: Mean absorption in the in vitro skin corrosion test

	3 min application					1 h application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)		Mean (OD570)		SD
Negative control	1.781	1.917	1.849	±	0.096	1.927		1.872	1.899	±	0.039
Test substance	1.770	1.528	1.649	±	0.171	1.748		1.703	1.725	±	0.031
Positive control	0.138	0.156	0.147	±	0.013	0.131		0.138	0.134	±	0.005

SD- Standard deviation

OD- Optical density

Table 2: Mean tissue viability in the in vitro skin corrosion test

	3 min application viability (percentage of control)	1 h application viability (percentage of control)
Negative control	100	100
Test substance	89	91
Positive control	8	7

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3 min exposure to the positive control was 8%. Because the mean relative tissue viability for the test substance was not below 50% after 3 min treatment and not below 15% after 1 h treatment, test substance is considered to be not corrosive.

Interpretation of results:

other: CLP criteria not met

Conclusions:

The test substance was not corrosive to the skin.

Executive summary:

A study was conducted to evaluate the skin corrosion potential of the test substance in an in vitro human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD Guideline 431 and EU method B.40, in compliance with GLP. Skin tissue was moistened with 25 μL of Milli-Q water and an excessive amount of the test substance was applied directly on its surface. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 89 and 91%, respectively. Because the mean relative tissue viability was not below 50% after the 3 min treatment and not below 15% after the 1 h treatment, the test substance was considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3 min exposure to the positive control was 8%. Under the study conditions, the test substance was not corrosive to skin (Eurlings IMJ, 2015a).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 28, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 437, EU Method B.47 and other internationally accepted guidelines, in compliance with GLP.

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:

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:

other: Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006 (ICCVAM).

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: NVITTOX) study plan 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F. (1992). Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449.

Deviations:

no

GLP compliance:

yes

Species:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Due to the consistency of the test substance, an excess amount was applied

Duration of treatment / exposure:

10 min

Details on study design:

Negative control: A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

Positive control: 10% (w/v) Benzalkonium Chloride (Merck KGaA, Darmstadt, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

PREPARATION OF CORNEAS
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32±1°C. The corneas were incubated for the minimum of 1 h at 32±1°C.

CORNEA SELECTION AND OPACITY READING
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
- The medium from the anterior compartment was removed and 750 μL of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or an excessive amount of the test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10±1 mins at 32±1°C. After incubation, the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120±10 mins at 32±1°C. After the completion of the incubation period, opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY MEASUREMENT
- The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.

- The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, Germany) was evaluated.
- The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90±5 mins at 32±1°C.

PERMEABILITY DETERMINATIONS
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

- The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes

Value:

ca. -1.8

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 1 Summary of opacity, permeability and in vitro scores

Treatment	Mean opacity^1	Mean permeability^1	Mean in vitro irritation score^1, 2
Negative control	-0.3	0.000	-0.3
Positive control (Benzalkonium Chloride)	103.0	2.481	140.2
Test substance	-2.0	0.011	-1.8

1 Calculated using the negative control mean opacity and mean permeability values.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:

other: CLP criteria not met

Conclusions:

The test substance is not-irritating to the eyes.

Executive summary:

The eye hazard potential of the test substance was evaluated in a bovine corneal opacity test conducted according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test consisted of the topical application of undiluted test substance on the epithelium of a bovine cornea for 10 min. The test was carried out using three replicates each for test substance, negative control and positive control. After exposure, the cornea was thoroughly rinsed and incubated for 2 h with fresh medium, followed by opacity measurement. The permeability of the cornea was then determined after a 90 min incubation period with sodium fluorescein. The test substance did not induce ocular irritation in both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of -1.8 after 10 min of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range, indicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (10% (w/v) benzalkonium chloride) was 140, within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Hence, based on the results of the study, the test substance was considered to be non-irritating to eyes (Eurlings IMJ, 2015b).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion test

A study was conducted to evaluate the skin corrosion potential of the test substance in an in vitro human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD Guideline 431 and EU method B.40, in compliance with GLP. Skin tissue was moistened with 25 μL of Milli-Q water and an excessive amount of the test substance was applied directly on its surface. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 89 and 91%, respectively. Because the mean relative tissue viability was not below 50% after the 3 min treatment and not below 15% after the 1 h treatment, the test substance was considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3 min exposure to the positive control was 8%. Under the study conditions, the test substance was not corrosive to skin (Eurlings IMJ, 2015a).

In vitro skin irritation test

A study was conducted to evaluate the skin irritation potential of test substance in an in vitro human skin model according to OECD Guideline 439 and EU method B.46, in compliance with GLP. The test consisted of topical exposure to the test substance, positive control (5% aqueous sodium dodecyl sulphate) and negative control (phosphate buffered saline) of a human reconstructed epidermis model (EPISKIN Small Model^TM, 0.38 cm², Lot no.: 15-EKIN-044) for 15±0.5 min. The skin irritation potential of the test substance was calculated according to a cell viability assay. The mean relative tissue viability for the test substance was 99%, i.e. above 50%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The positive control had a mean cell viability of 24%, thus confirming the validity of the test system. Under the study conditions, the test substance was found to be non-irritating to skin (Eurlings IMJ, 2015a).

In vitro eye irritation test (Bovine corneal opacity test)

The eye hazard potential of the test substance was evaluated in a bovine corneal opacity test conducted according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test consisted of the topical application of undiluted test substance on the epithelium of a bovine cornea for 10 min. The test was carried out using three replicates each for test substance, negative control and positive control. After exposure, the cornea was thoroughly rinsed and incubated for 2 h with fresh medium, followed by opacity measurement. The permeability of the cornea was then determined after a 90 min incubation period with sodium fluorescein. The test substance did not induce ocular irritation in both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of -1.8 after 10 min of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range, indicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (10% (w/v) benzalkonium chloride) was 140, within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Hence, based on the results of the study, the test substance was considered to be non-irritating to eyes (Eurlings IMJ, 2015b).

Justification for classification or non-classification

Skin irritation:

Based on the results of in vitro skin corrosion and skin irritation studies, the test substance does not warrant classification for skin irritation according to EU CLP (EC 1272/2008) criteria.

Eye irritation:

Based on the result of an in vitro eye irritation study, the test substance does not warrant classification for eye irritation according to EU CLP (EC 1272/2008) criteria.