Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives

Endpoint summary

Description of key information

In vitro studies of skin corrosivity and skin irritation are available for the submission substance [Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives].

In vitro studies of eye irritation (BCOP and EpiOcular assays) are available for the submission substance [Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives].

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:

experimental study

Adequacy of study:

weight of evidence

Study period:

14-18 August 2017

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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Commercial model

Justification for test system used:

Standard model; commerically available

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 26767).
The model 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

Three minutes, 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Four

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

8.1% viability

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

39

Vehicle controls validity:

not applicable

Remarks:

10% viability

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

6.4% viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 91% and 39% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 6.4%. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <=6% for the negative control, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives is not corrosive in the in vitro skin corrosion test under the experimental conditions.

Executive summary:

The study was performed to evaluate [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item was applied undiluted (50 μL) directly on top of the skin tissue. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 91% and 39%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, the test item is considered to be not corrosive. In conclusion, [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10 -13 -sec-alkyl derivatives] is not corrosive in the in vitro skin corrosion test under the experimental conditions.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19-25 September 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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-038)

This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 μL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Details on study design:

The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. The test item was viscous and difficult to remove. Skin tissues were rinsed thoroughly. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for 68 hours. Extracted formazan was determined spectrophotometrically at 570 nm in duplicate with a plate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment

Value:

9.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

5.4% viability

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.4%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <8%, indicating that the test system functioned properly.

The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because no colour changes were observed it was concluded that the test item did not interact with the MTT endpoint. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 9.6%. Since the mean relative tissue viability for the test item was below 50% it is considered to be irritant.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Since the mean relative tissue viability for the test item was below 50% after 15 minutes treatment it is considered to be irritant. In conclusion, the test item is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified in Category 2 according to the CLP Regulation.

Executive summary:

The objective of this study was to evaluate the substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The skin irritation potential of the test item was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item was applied undiluted (25 μL) directly on top of the skin tissue for 15 minutes. After a 42 -hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. The relative mean tissue viability obtained after 15 minutes treatment compared to the negative control tissues was 9.6%. Since the mean relative tissue viability for the test item was below 50% after 15 minutes treatment it is considered to be irritant. In conclusion, the test item is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified in Category 2 according to the CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21-25 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Species:

other: Bovine eyes were used as soon as possible after slaughter.

Details on test animals or tissues and environmental conditions:

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. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. 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 (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) 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 32C. The corneas were incubated for a minimum of 1 hour at 32C. 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. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined wasrecorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three

Details on study design:

The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test item 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 item over the entire cornea. Corneas were incubated in a horizontal position for 10 minutes at 32°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item 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 minutes at 32C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. The opacity of a cornea was measured by the diminution of light passing through the cornea. 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 treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control 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.

Following the final opacity measurement, permeability of the cornea to Na-fluorescein 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/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 minutes at 32°C.

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. 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 has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment

Value:

26

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean IVIS -1.6

Positive controls validity:

valid

Remarks:

Mean IVIS 26

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

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 mean in vitro irritancy score of the positive control (Ethanol) was 61 and 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.

 Summary of results

Treatment	Mean opacity	Mean permeability	Mean IVIS
PBS	-1.5	-0.00	-1.6
Ethanol	22	2.585	61
Test material	6.7	1.317	26

(IVIS) = mean opacity value + (15 x mean OD490 value)

Interpretation of results:

study cannot be used for classification

Conclusions:

Since [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] induced an IVIS > 3 ≤ 55, no prediction on the CLP classification can be made.

Executive summary:

The objective of this study was to evaluate the eye irritation potential of the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives], as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea (BCOP test). The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied (750 μL, undiluted) directly onto of the corneas. Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-secalkyl derivatives induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 26 after 10 minutes of treatment. In conclusion, since reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives induced an IVIS > 3 ≤ 55, no prediction on the CLP classification can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11-15 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives
Appearance: Brown liquid
Batch: P718261998
Storage: At room temperature
Stable under storage conditions until: 01 June 2018 (expiry date)

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23497

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Two replicates

Details on study design:

The test was performed on a total of 2 tissues per test item together with a negative control and positive control. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively. 50 μL of the undiluted test item was added into the 6-well plates on top of the tissues. After the exposure period with the test item (30 minutes at room temperature), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 minutes at 37°C. After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium. The tissues were incubated for 18010 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 ml of isopropanol in each designated well. Formazan was extracted with 2 mL isopropanol for 18 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate using a plate reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

other: reletive tissue viability

Run / experiment:

Experiment mean

Value:

7.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

40

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The positive control had a mean cell viability after 30 minutes exposure of 40%. The absolute mean OD570 of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 5%, indicating that the test system functioned properly.

Summary of results

	OD570				Relative viability
	Replicate A	Replicate B	Mean	SD	Mean	SD
Negative control	2.046	1.914	1.980	0.094	100	4.7
Test material	0.122	0.171	0.147	0.035	7.4	1.8
Positive control	0.750	0.816	0.783	0.047	40	2.4

Values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

Interpretation of results:

study cannot be used for classification

Conclusions:

Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives is potentially irritant in the EpiOcular test under the experimental conditions.

Executive summary:

The objective of this study was to evaluate the eye irritation potential of the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] using the reconstructed human EpiOcular model (OECD 492). The test material was applied undiluted (50 μL) directly onto the tissue for 30 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test material and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours prior to determination of cytotoxicity. The relative mean tissue viability obtained after 30 minutes compared to the negative control tissues was 7.4%. Since the mean relative tissue viability for the test material was ≤60%, it is considered to be potentially irritant and potentially requiring classification and labelling according to CLP in Category 2 or Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

A skin corrosion study in vitro with the submission substance shows that CLP classification for skin corrosion (Category 1) is not required. A skin irritation study in vitro with the submission substance shows that CLP classification for skin irritation (Category 2) is required.

Serious eye damage / serious eye irritation

A BCOP assay with the submission substance shows that CLP classification for serious eye damage (Category 1) is not required. An EpiOcular assay with the submission substance indicates that CLP classification for serious eye damage (Category 1) or serious eye irritation (Category 2) is required. Based on an assessment of the skin and eye irritation data, classification in Category 2 for serious eye irritation is considered appropriate.

Justification for classification or non-classification

The submission substance [Reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives] is classified for skin and eye irritation in CLP Category 2 on the basis of the in vitro data.