Amines, N-coco alkyltrimethylenedi-, acetates

Endpoint summary

Description of key information

Skin corrosion in vitro: The test item was considered to be non-corrosive to skin because tissue viability viability relative to the negative control was determined to be 131.2 % after 4 hours using the EPISKIN reconstructed human epidermis model (OECD 431).

 

Skin irritation in vitro: The test item was determined to be irritating to skin because viability of test item-treated tissues was reduced to 8 % relative to the negative control (OECD 439; interim results).

Eye irritation in vitro: It is considered applicable to classify the substance as H318: Eye Dam 1 under the terms of Regulation (EC) No. 1272/2008 because the test item has been determined to be irritating to skin.

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:

29 June 2011 and 02 July 2011

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

other: adult

Vehicle:

unchanged (no vehicle)

Details on test system:

PURPOSE OF THE TEST
- The purpose of this test was to evaluate the corrosivity potential of the test item using the EPISKIN in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.
- The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item-treated cultures relative to the negative control.

EPISKIN MODEL KIT 0.38 cm2
- Supplier: SkinEthic Laboratories, Nice, France
- Date received: 29 June 2011

MTT DYE METABOLISM, CELL VIABILITY ASSAY
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
- As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below.
- Test item (50 µL) was added to 2.2 mL of a freshly prepared 0.3 mg/mL MTT solution. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control.
- If the MTT solution containing the test item turned blue relative to the control, the test item was presumed to have reduced the MTT.
- The test item was shown to directly reduce MTT. There was a possibility that, if the substance could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could give rise to a false negative result. Determination of skin corrosion potential was therefore performed in parallel on viable and water-killed tissues. This step was a functional check, which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues.
- Water-killed tissues were prepared by placing untreated EPISKIN tissues in a 12-well plate containing 2.2 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 hours ± 1 hour. At the end of the incubation, the water was discarded. One killed, the tissues were stored in a freezer (-14 to -30 °C) for up to six months. Before use, each tissue was thawed by placing in 2.2 mL of maintenance medium for approximately one hour at room temperature.
- In addition to the normal test procedure, the MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
- Maintenance medium (2.2 mL), warmed to approximately 37 °C, was pipetted into two wells of the first column of a pre-labelled 12-well plate.
- Each epidermis unit was transferred into the maintenance medium-filled wells (2 units per plate).
- A different 12-well plate was used for each test item, control and time point.
- Tissues were incubated at 37 °C, 5% CO2 in air overnight.
- After 24 hours, the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

APPLICATION OF TEST ITEM AND RINSING (DAY 2)
- Assay medium (2.2 mL), warmed to approximately 37 °C, was pipetted into two wells of the second and third columns of the 12-well plate. Tissues were then transferred into the second column.
- Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes.
- Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes.
- Test item (50 µL) was applied topically and uniform coverage of the tissues was ensured.
- Duplicate tissues treated with 0.9% sodium chloride (50 µL) served as negative controls.
- Duplicate tissues treated with glacial acetic acid (50 µL) served as positive controls.
- Treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.
- At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 s using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
- MTT solution 0.3 mg/mL freshly prepared in assay medium (2.2 mL) was pipetted into two wells of the fourth column of each 12-well plate. Tissues were transferred into the MTT-filled wells. The tissues were incubated for 3 hours ± 5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light.
- At the end of the 3-hour incubation period, each tissue was placed onto absorbent paper to dry.
- A total biopsy of the epidermis was taken using the EPISKIN biopsy punch.
- The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 850 µL of acidified isopropanol.
- Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals from the MTT-loaded tissues.

ABSORBANCE / OPTICAL DENSITY MEASUREMENTS (DAY 3)
- At the end of the formazan extraction period, each tube was mixed thoroughly on a vortex mixer to produce a homogeneous coloured solution.
- For each tissue, duplicate samples (200 µL) were transferred to the appropriate wells of a pre-labelled 96-well plate. Acidified isopropanol alone (200 µL) was added to the two wells designated as blanks. Opitcal density was measured (quantitative viability analysis) at 540 nm without a reference filter using an Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240 minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% sodium chloride solution.
- The relative mean viabilities were calculated using the equation relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100.

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) Negative control: The mean optical density for the negative control-treated tissues is ≥ 0.115 and ≤ 0.400.
(ii) Positive control: Mean tissue viability for the positive control-treated tissues is 0 to 20% relative to the control-treated tissues after a 240-minute exposure period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3, 60 and 240 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

120

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: relative mean viability of test item-treated tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

69.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: relative mean viability of test item-treated tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240-minute exposure

Value:

131.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: relative mean viability of test item-treated tissues

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The test item was found to be able to directly reduce MTT. However, results showed no degree of interference due to direct reduction of MTT.
- It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD540 values and viabilities for the negative control, positive control and test item are given in Table 1 (attached).

QUALITY CRITERIA
- The mean tissue viability for the positive control-treated tissues was 8.0% relative to the negative control-treated tissues after an exposure period of 240 minutes. The positive control acceptance criterion was therefore satisfied.
- The mean OD540 for the negative control-treated tissues was 0.125. The negative control acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

Relative mean viabilities of test item-treated tissues were determined to be 120.0% after 3 minutes exposure, 69.6% after 60 minutes exposure and 131.2% after 240 minutes exposure. The test item was considered to be non-corrosive to skin.

Executive summary:

The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (13 April 2004) and Method B.40 of Commission Regulation (EC) No 440/2008 of 30 May 2008. The purpose of the test was to evaluate the corrosivity potential of the test item using the EPISKIN in vitro Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

 

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading, a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from the MTT-loaded tissues.

 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540nm (OD540). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Relative mean viabilities of test item-treated tissues were determined to be 120.0% after 3 minutes exposure, 69.6% after 60 minutes exposure and 131.2% after 240 minutes exposure. The test item was considered to be non-corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Irritation / corrosion parameter:

% tissue viability

Value:

8

Remarks on result:

other: interim result

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item was determined to be irritating to skin because viability of test item-treated tissues was reduced to 8 % relative to the negative control.

Executive summary:

The test item was determined to be irritating to skin because viability of test item-treated tissues was reduced to 8 % relative to the negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion in vitro

 

The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (13 April 2004) and Method B.40 of Commission Regulation (EC) No 440/2008 of 30 May 2008. The purpose of the test was to evaluate the corrosivity potential of the test item using the EPISKIN in vitro Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

 

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading, a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from the MTT-loaded tissues.

 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540nm (OD540). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Relative mean viabilities of test item-treated tissues were determined to be 120.0% after 3 minutes exposure, 69.6% after 60 minutes exposure and 131.2% after 240 minutes exposure. The test item was considered to be non-corrosive to skin.

 

Skin irritation in vivo: Testing on vertebrate animals is not required because results of a validated in vitro test permitted unambiguous classification.

Skin irritation in vitro: Investigation of skin irritation in vitro is ongoing.

 

Eye irritation in vivo: Testing involving vertebrate animals is not required because the substance has been shown to be irritating to skin and it is considered applicable to assume that it will also cause serious eye damage.

Justification for classification or non-classification

Skin corrosion in vitro: The test item was considered to be non-corrosive to skin because relative mean tissue viability was determined by OECD 431 (EPISKIN Reconstructed Human Epidermis Model) to be ≥ 35 % after 4 hours. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Skin irritation: The test item was considered to be irritating to skin because viability of test item-treated tissues was reduced to 8 % relative to the negative control (OECD 439).In accordance with Regulation (EC) No. 1272/2008, classification as H315: Skin Irrit 2 is therefore applicable.

Eye irritation: The substance has been demonstrated to be irritating to skin and it is considered applicable to also classify as H318: Eye Dam 1 under the terms of Regulation (EC) No. 1272/2008.