Dioctyl maleate, branched

Endpoint summary

Description of key information

The key studies were conducted to recognised national testing guidelines.

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:

key study

Study period:

25 October 2017 - 26 October 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: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Article: Bernel Ester DCM
CAS Number: 85566-63-8
Storage: 15 to 25˚C, protected from light
Purity: 92.78%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

Justification for test system used:

This study was conducted to determine whether the test article causes corrosion in the in vitro skin model EpiDermTM.
The test article was applied topically to a three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined using the MTT reduction assay) below defined thresholds at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System
Specifications
Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

Identification
The test system was appropriately labelled with the study number, date of treatment, duration of treatment and negative/positive/test article.

General Model Conditions
Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Functional Model Conditions
The magnitude of viability is quantified using MTT. The optical density (OD) of the extracted (solubilised) dye from the negative control tissue is at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture for the duration of the test exposure period. The stratum corneum is sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (eg 1% Triton X-100). This property has been estimated by the exposure time required to reduce cell viability by 50% (ET50). For the EpiDermTM model the lower acceptance limit is 4.08 hours and the upper acceptance limit is 8.7 hours. The tissue has been shown to demonstrate reproducibility over time, and it has been shown to be capable of predicting the corrosive potential of the reference chemicals when using the testing protocol selected.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 minute and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The OD values for the negative controls met the acceptance criteria.
Skin viability after a 3 minute or 60 minute exposure to the positive control article was 2% and 4%, respectively, demonstrating appropriate performance of the assay.

3 minute exposure period

Test Substance	OD570			Mean	Tissue Mean	Tissue Mean - FK	%Survival	%CV
Negative	0.968	0.981	0.969	0.973	1.029		100	6.1
Negative	1.066	1.105	1.082	1.084
Test article	1.021	1.031	1.017	1.023	1.009		98	1.8
Test article	0.982	1.005	0.997	0.995
Positive	0.192	0.191	0.189	0.191	0.175	0.018	2	9.6
Positive	0.16	0.162	0.158	0.16
Positive FK	0.131	0.132	0.13	0.131	0.158
Positive FK	0.183	0.186	0.184	0.184

60 minute exposure period

Test Substance	OD570			Mean	Tissue Mean	Tissue Mean - FK	%Survival	%CV
Negative	1.174	1.19	1.168	1.177	1.166		100	1.7
Negative	1.156	1.133	1.172	1.154
Test article	1.102	1.047	1.103	1.084	1.082		93	2
Test article	1.08	1.091	1.068	1.079
Positive	0.308	0.296	0.31	0.305	0.259	0.05	4	20
Positive	0.202	0.236	0.202	0.213
Positive FK	0.211	0.25	0.217	0.226	0.21
Positive FK	0.193	0.191	0.197	0.194

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, Bernal Ester DCM, was considered to be non-corrosive according to the UN GHS classification system.

Executive summary:

This study was conducted to determine whether the test article, Bernel Ester DCM, causes corrosion in the in vitro skin model EpiDerm^TM.

Duplicate EpiDerm^TM inserts were treated with test article, purified water (negative control) and 8N potassium hydroxide (positive control) for 3 minutes and 60 minutes. At the end of the treatment period, the tissues were washed with phosphate buffered saline (PBS) and cell viability was assessed using the MTT assay. The skin corrosivity potential was classified according to the remaining cell viability obtained after test material treatment with either of the two treatment times.

Skin viability after a 3 minute or 60 minute exposure to the test article was 98% and 93%, respectively.

The OD values for the negative controls met the acceptance criteria.

Skin viability after a 3 minute or 60 minute exposure to the positive control article was 2% and 4%, respectively, demonstrating appropriate performance of the assay.

The test article, Bernal Ester DCM, was considered to be non-corrosive according to the UN GHS classification system.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2017 to 1 December 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)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Article: Bernel Ester DCM
CAS Number: 85566-63-8
Storage: 15 to 25˚C, protected from light
Purity: 92.78%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM SIT (EPI-200) three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

Vehicle:

unchanged (no vehicle)

Details on test system:

Assessment of MTT Interacting Substances
In order to assess the potential non-specific reduction of the test article, a dose of 30 µL of test article was added to 1 mL of MTT and colour change assessed after 60 minutes. The solution did not turn blue or purple therefore the test article did not interact with MTT.

Assessment of Staining Potential for Non-coloured Substances
In order to assess the potential of staining, 30 µL of the test article was added to 0.3 mL deionised water and 0.3 mL isopropanol and incubated for 60 minutes at 37±1ºC, 5±1% CO2. At the end of the incubation, the mixtures were shaken. Neither of the solutions became coloured, therefore it was deemed that the test article did not have the potential to stain the tissue.

Assessment of Mesh Compatibility
To prevent capillary effects drawing the liquid test article to the edge of the insert a nylon mesh was recommended. An assessment of the interaction of the mesh and test article was therefore assessed. A nylon mesh was placed onto a glass microscope slide and 30 µL of the test article was applied. After exposure for 60 minutes, the mesh was examined microscopically for any signs of interaction (e.g. test article dissolves the mesh / changes in appearance etc.). There were no adverse effects to the mesh disc.

Test Article Preparation
The test article was administered without dilution.

Application of Test and Control Substances
EpiDermTM SIT (EPI-200) tissues were kept in their packaging at room temperature in a microbiological safety cabinet until the next step. The tissues were set up the day prior to treatment by placing each tissue onto 0.9 mL maintenance medium (supplied with the EpiDermTM SIT (EPI-200) tissues) in 6-well plates and incubating at 37°C. Immediately prior to treatment initiation, the media under the tissues was replaced with 0.9 mL of assay medium (supplied with the EpiDermTM SIT (EPI-200) tissues). Three tissues per test article, negative control and positive control were treated by application of 30 µL of the negative control, 30 µL of the positive control and 30 µL of liquid test material onto the surface of the tissues, with mesh discs used.
The treated tissues were placed into an incubator at 37±1ºC, 5±1% CO2 for 35 minutes. The plates were removed from the incubator and placed into a sterile hood until the 60 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 42.5 hours.

Cell Viability Measurements
Upon completion of the 42 hour recovery period, each tissue was rinsed with PBS before being placed on 0.3 mL of 1 mg/mL MTT in PBS and incubated for three hours at 37°C.
After incubation, any resultant colour was extracted. Each tissue was flooded with 2 mL isopropanol, the plate was sealed and then shaken at 150 rpm for 2 hours. Upon completion of the extraction each tissue was pierced using a hypodermic needle and the extract drained and placed into a 96 well microtitre plate. The optical density of each resultant extract was determined spectrophotometrically at 570 nm, using extraction solvent as a blank and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42.5 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment

Value:

98.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The group mean viability for the test article was 98.7%.
The group mean viability for the positive control was 4.1%.
The assay acceptance criteria were met.

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, Bernel Ester DCM, was not considered to be irritant in the in vitro skin model EpiDermTM SIT (EPI-200).

Executive summary:

This study was conducted to determine whether the test article causes irritation in the in vitro skin model EpiDermTM SIT (EPI-200).

EpiDermTM SIT (EPI-200) inserts were treated with Bernel Ester DCM, negative control (phosphate buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for 60 minutes. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin irritation potential was classified according to the remaining cell viability obtained after test article treatment.

The group mean viability for the test article was 98.7% and for the positive control was 4.1%, when compared to the negative control.

The test article, Bernel Ester DCM, was not considered to be irritant in the in vitro skin model EpiDermTM SIT (EPI-200).

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:

25 October 2017 to 05 February 2018

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 437 (adopted 26 July 2013) and Method B47 of Council Regulation (EC) No 440/2008.

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)

Version / remarks:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 437 (adopted 26 July 2013) and Method B47 of Council Regulation (EC) No 440/2008.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test article, a clear colourless liquid, was identified as Bernel Ester DCM and was received at Covance as follows:
Test Article: Bernel Ester DCM
CAS Number: 85566-63-8
Storage: 15-25oC, protected from light
Purity: 92.78%, assumed 100% for testing

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Attachments.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System and Study Design

Specification
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.

Assessment on Arrival
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Excision and Preparation of Corneas
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1 °C for at least 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl or sufficient to completely cover the cornea

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Opacity: 2 hours
Permeability: 1 hour 25 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Treatment of Corneas
A volume of 750 µL of the test article (or enough test article to completely cover the cornea) was applied to each of three corneas followed by a ten minute incubation at 32 °C. After this incubation, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red before being incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer.
A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.

Terminal Procedures
At the end of the assay all corneas were preserved in 10% Neutral Buffered Formalin. However, the Sponsor confirmed that microscopic examination was not required. The corneas were discarded on finalisation of the study report.

Data Evaluation
The In Vitro Irritation Score (IVIS) was determined as follows:
IVIS = mean opacity value + (15 x mean permeability value).
The opacity and mean permeability (OD490) values are corrected for background opacity and the negative control OD490 values.

Decision Criteria
The test article is concluded as inducing serious eye damage if the IVIS is >55.
The test article is concluded as not requiring classification for eye irritation if the IVIS is ≤3.
No prediction can be made if the IVIS is >3 but ≤55.

Assay Acceptance Criteria
The assay was considered valid if the following criteria are met:
The positive control yields an IVIS within 2 standard deviations of the historical control mean.
The negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean value

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

opacity reading of 0.0

Positive controls validity:

valid

Remarks:

opacity reading of 58.7

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean value

Value:

0.002

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

The mean group corrected optical density for the negative control was 0.000

Positive controls validity:

valid

Remarks:

The mean group corrected optical density for the positive control was 0.277.

Remarks on result:

no indication of irritation

Corneal Opacity

Results are presented in Table 8.1.

The mean corrected opacity reading for the test article was -1.0.

The mean corrected opacity reading for the negative control was 0.0.

The mean corrected opacity reading for the positive control was 58.7.

The corneas treated with the test article were noted to be clear and unchanged following treatment. The corneas treated with the positive control were noted to be cloudy and wrinkled following treatment.

Corneal Permeability

Results are presented in Table 8.2.

The mean group corrected optical density for the test article was 0.002.

The mean group corrected optical density for the negative control was 0.000.

The mean group corrected optical density for the positive control was 0.277.

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that Bernel Ester DCM, produced an IVIS score of -0.97 and therefore does not require classification for eye irritation.
The assay was considered valid as the assay acceptance criteria were met.

Executive summary:

This study was conducted to determine whether the test article, Bernel Ester DCM, causes serious eye damage or does not require classifying for eye irritation, using the bovine corneal opacity and permeability (BCOP) assay.

A volume of 750 µL of the undiluted test articlewas applied to each of three corneas followed by a 10 minute incubation at 32±1°C. After this incubation, each cornea was washed with media containing phenol red followed by media without phenol red. The corneas were then incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied.

For the permeability endpoint, sodium fluorescein solution was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour 25 minutes at 32±1°C. Following this period, the media in the posterior chamber was removed and three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD₄₉₀).

A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas. These corneas were subject to the procedures detailed above.

The test article, Bernel Ester DCM, produced an IVIS score of -0.97 and therefore does not require classification for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance did not meet the criteria for consideration as an irritant to the skin or the eye in accordance with the Classification, Labelling, and Packaging (CLP) regulation.