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Description of key information

Based on both studies available (OECD 439 and 437), 3.3.5-trimethylcyclohexyl acrylate is considered to be not irritating on skin and eyes.

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:
23 November 2015 -- 22 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EU Method B.46 (In vitro dermal irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 439 (In vitro dermal irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Details on animal used as source of test system:
EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
Vehicle:
unchanged (no vehicle)
Details on test system:
REMOVAL OF TEST SUBSTANCE
- Rinsing: at the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

POSITIVE CONTROL
Name: Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution.

NEGATIVE CONTROL
Name: Dulbecco’s Phosphate-Buffered Saline (D-PBS).

SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank

Interpretation: see below
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Amount per tissue: 10 µL
Duration of treatment / exposure:
Exposure period of 15 minutes, followed by rinsing
MTT-loading after a 42h-incubation period. Observation of MTT-> formazan transformation by viable cells.
Number of replicates:
Triplicate tissues for each tested substance (test item, negative control, positive control).
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
treated
Value:
86
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 86% with a Standard Deviation of 22%. Despite a Standard Deviation higher than 18% was noted, all viability data of test item-treated tissues were above the cut-off of 50%, and the
determination of IL-1a concentrations was used to consolidate the trend for a non-irritant response.
Therefore, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the Limit Of Quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration
from the three test item-treated tissues was not calculated. The IL-1a concentration values of the two other test item-treated tissues were found < 60 pg/mL (39.1 and 6.30 pg/mL).
Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.
Interpretation of results:
GHS criteria not met
Conclusions:
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 France 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 +37°C, 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 addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

 

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 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 86% with a Standard Deviation of 22%. Despite a Standard Deviation higher than 18% was noted, all viability data of test item-treated tissues were above the cut-off of 50%, and the determination of IL-1a concentrations was used to consolidate the trend for a non-irritant response.

Therefore, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the Limit Of Quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration from the three test item-treated tissues was not calculated. The IL-1a concentration values of the two other test item-treated tissues were found < 60 pg/mL (39.1 and 6.30 pg/mL).

Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

Conclusion

The test item is considered to be non-irritant to skin.

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:
24 November 2015 - 10 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EU method B.47 (In vitro eye irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guideline 437 (In vitro eye irritation)
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 freshly slaughtered cattle at the abattoir 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 to 3 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 corneas were used within a maximum of 24 hours.

(Pre)Incubation T°C: 32°C
Dates of experimental phase: from 01 December 2015 to 10 December 2015
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro negative and positive controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL


Duration of treatment / exposure:
Exposure period of 10 minutes (± 30 seconds) followed by rinsing
Observation period (in vivo):
Opacity measurement:
- before treatment
- after 2-hour incubation in 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:
Not applicable
Triplicate corneas for each timepoint and 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 7 times with pre-warmed cMEM.

NEGATIVE CONTROL:
As the test item was tested undiluted (i.e. in its original form), the following negative control was used:
- Name: 0.9% Sodium Chloride (NaCl)
- Batch No.: A4044
- Supplier: Lavoisier

Since several test items were assayed concurrently, the negative control was shared.

POSITIVE CONTROL:
As the test item is a non-surfactant containing liquid (tested using a 10-minute treatment), the positive control was 10% sodium hydroxide solution (10% NaOH).
The positive control was prepared by CiToxLAB France pharmacy on the morning of use by dissolving anhydrous pellets of sodium hydroxide (batch No. SZBE3360V supplied by Sigma-Aldrich) in water obtained by reverse osmosis using ELIX 5 (Millipore SA).

Since several test items were assayed concurrently, the positive control was shared.

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 of positive control and test item-treated corneas.
- 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 of positive control and test item-treated corneas.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below
Irritation parameter:
in vitro irritation score
Run / experiment:
treated
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All acceptance criteria were fulfilled. The study was therefore considered as valid.
The mean In Vitro Irritancy Score (IVIS) obtained with the test item was: 0.
Interpretation of results:
GHS criteria not met
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 potential irritant and corrosive properties of the test item, to the eye. 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 design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France Standard Operating Proceduresand with the OECD 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.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

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

The test item was applied undiluted in a single experiment. The test item, negative and positive controls were evaluated using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes)at +before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at +32°C.

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

Results

Macroscopic examinations

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

In Vitro Irritancy Score

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

The mean In Vitro Irritancy Score (IVIS) obtained with the test item was: 0.

As the mean IVIS was = 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

In vitro skin irritation test (Richez 2016a)

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).

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 main study was therefore considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 86% with a Standard Deviation of 22%. Despite a Standard Deviation higher than 18% was noted, all viability data of test item-treated tissues were above the cut-off of 50%, and the determination of IL-1a concentrations was used to consolidate the trend for a non-irritant response.

Therefore, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the Limit Of Quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration from the three test item-treated tissues was not calculated. The IL-1a concentration values of the two other test item-treated tissues were found < 60 pg/mL (39.1 and 6.30 pg/mL).

Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

BCOP / Eye corrosion/irritation test (Richez 2016b)

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, to the eye. 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 (OECD 437).

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

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

The mean In Vitro Irritancy Score (IVIS) obtained with the test item was: 0.  

As the mean IVIS was = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Skin irritation: No CLP classification (Regulation EC no.1272/2008) is required for 3.3.5-trimethylcyclohexyl acrylate based on the in vitro study available.

Eye irritation: No CLP classification (Regulation EC no.1272/2008) is required for 3.3.5-trimethylcyclohexyl acrylate based on the in vitro study available.

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