(octahydro-4,7-methano-1H-indenediyl)bis(methylene) diacrylate

Endpoint summary

Description of key information

Two in vitro tests are available to evaluation the irritancy potential of Tricyclododecane dimethanol diacrylate.
The Episkin irritation test showed no skin irritation with 109% of viability cells. And BCOP test showed no eye irritation with an IVIS of -1.
Therefore Tricyclododecane dimethanol diacrylate is considered to be not skin or eye irritating.

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:

key study

Study period:

20 December 2013 -- 03 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: not applicable

Cell type:

other: reconstructed human epidermis

Cell source:

other: EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

Amount applied per tissue: 10 ± 2 µL.

Duration of treatment / exposure:

Exposure period of 15 minutes, followed by rinsing.
MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test

Value:

109

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Following a 15 minutes exposure and a 42 hours recovery periods, the relative mean viability of the tissues treated with the test item was 109% with a standard deviation of 17% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.

Interpretation of results:

GHS criteria not met

Remarks:

Not skin irritating

Conclusions:

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstructed human epidermis model.

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB’s standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

 

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

Results

Preliminary tests

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and a 42 hours recovery periods, the relative mean viability of the tissues treated with the test item was 109% with a standard deviation of 17% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.

Conclusion

 

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

 

According to the results of this study, the classification of the test item should be:

- not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).

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:

03 January 2014 -- 13 February 2014

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:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Species: bovine cattle.
Origin: bovine eyes were obtained from EVA, Saint-Pierre-sur-Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas, and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.
The prepared corneas were stored and used within 24 hours.
(Pre)Incubation T°C: 32°C

Dates of experimental phase: from 12 February 2014 to 13 February 2014

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro negative and positive controls

Duration of treatment / exposure:

Exposure period of 10 minutes, followed by rinsing

Duration of post- treatment incubation (in vitro):

Opacity measurement:
- before treatment
- after 2-hour incubation period in a water bath at +32°C (± 1°C).
Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement

Number of animals or in vitro replicates:

Triplicate corneas for each tested substance (test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Rinsing: the anterior part of the eye was emptied and then rinsed up to six times with pre-warmed cMEM.

NEGATIVE CONTROL:
As the test item was tested in its original form, the negative control was 0.9% NaCl, batch No. 3A077 supplied by CDM Lavoisier.

POSITIVE CONTROL:
10% sodium hydroxide solution (10% NaOH).

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values in positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values in positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below

Irritation parameter:

in vitro irritation score

Run / experiment:

test

Value:

-1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Macroscopic examinations : No notable opaque spots or irregularities were observed on test item-treated corneas following the treatment.
In Vitro Irritancy Score : The In Vitro Irritancy Score (IVIS) was: -1.
All acceptance criteria were fulfilled. The study was therefore considered as valid.

Interpretation of results:

GHS criteria not met

Remarks:

Not eye irritating

Conclusions:

Under the experimental conditions of this study, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item. Indeed, the Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The study design was based on the OECD guideline No. 437 and was performed in compliance with CiToxLAB France’s standard operating procedures and with the principles of Good Laboratory Practice.

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer.

The test item was tested undiluted (i.e. in its original form) in a single experiment using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, the test and control items were removed from the front opening of the anterior chamber and the corneas were rinsed. The corneas were then incubated for 2 hours at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed for each cornea and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at +32°C.

At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities.

Results

Macroscopic examinations

No notable opaque spots or irregularities were observed on test item-treated corneas following the treatment.

 

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The In Vitro Irritancy Score (IVIS) was: -1. As the test item induced an IVIS = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). 

Conclusion

Under the experimental conditions of this study, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation study

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstructed human epidermis model.

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46).

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and a 42 hours recovery periods, the relative mean viability of the tissues treated with the test item was 109% with a standard deviation of 17% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response. Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

Eye irritation study

The objective of this study was to evaluate the eye hazard potential of the test item. Indeed, the Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The study design was based on the OECD guideline No. 437. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. Three corneas were used for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed using an opacitometer. The test item was tested undiluted (i.e.in its original form) in a single experiment using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, the test and control items were removed from the front opening of the anterior chamber and the corneas were rinsed. The corneas were then incubated for 2 hours at+32°Cbefore a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed for each cornea and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at+32°C.

At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on test item-treated corneas following the treatment.

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The In Vitro Irritancy Score (IVIS) was: -1. As the test item induced an IVIS = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). 

Justification for classification or non-classification

Based on the available data, Tricyclododecane dimethanol diacrylate is not classified as skin or eye irritating according to the Regulation EC 1272/2008.