N,N-dimethyl-N'-(2,2,6,6-tetramethylpiperidin-4-yl)propane-1,3-diamine

Endpoint summary

Description of key information

skin irritation/corrosion: skin corrosive
eye irritation/corrosion: eye corrosive

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

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

Qualifier:

according to guideline

Guideline:

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

other: EpiSkin™ tissue; human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis

Justification for test system used:

Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ kits
- Tissue batch number(s): Lot No.: 12-EKIN-024
- Production date: 12.06.2012
- Date of initiation of testing: 13.06.2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C,
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: n.a.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, 85737 Ismaning, Germany
- Wavelength: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT
- Barrier function: Each batch of the epidermal model used meets defined production release criteria, set by the supplier
- Morphology: no abnormalities
- Contamination: absence of any contamination verified by supplier
- Reproducibility: Associated and appropriate measures of variability between tissue replicates are defined

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
n.a.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
9 tests in total (Negative control, Test item and Positive control in triplicate at 1 timepoint)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin with UN GHS category 2 if the tissue viability after exposure and post-treatment incubation was ≤ 50%
- The test substance is considered to have no category if the tissue viability after exposure and post-treatment incubation was > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of undiluted test item
- Concentration (if solution): n.a.

VEHICLE
n.a.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): n.a.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% in deionised water

Duration of treatment / exposure:

15 min.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Amount / concentration applied:

10 µL of the liquid test item were applied to each tissue, spread to match the tissue size.

Duration of treatment / exposure:

15 min

Details on study design:

This in vitro study was performed to assess the irritation potential of TAT (Triacetontriamine) by means of the Human Skin Model Test. An EpiSkin kit was used.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

46.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control

Value:

24.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

After treatment with the test item TAT (Triacetontriamine) the mean relative absorbance value was reduced to 46.3%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In conclusion, the test item TAT (Triacetontriamine) is irritant to skin under the experimental conditions in this study.

Executive summary:

This in vitro study was performed to assess the irritation potential of TAT (Triacetonetriamine) by means of the Human Skin Model Test according to OECD TG 439.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. 10 μL of the liquid test item were applied to each tissue, spread to match the tissue size. 10 μL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

After treatment with the test item TAT (Triacetonetriamine) the mean relative absorbance value decreased to 46.3%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item TAT (Triacetonetriamine) is irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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

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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Freshly isolated bovine cornea

Details on test animals or tissues and environmental conditions:

. SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim 64625 Bensheim, Germany
- Number of animals: 9 corneae
- Characteristics of donor animals (e.g. age, sex, weight): 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day.
- Time interval prior to initiating testing: next day
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: streptomycin / penicillin

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 ml (undiluted)

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

The experiment was performed to determine an irritation effect of the test item on the corneal opacity according to OECD 437.
The positive control 2-Ethoxyethanol was tested neat. Saline was used as negative control item.

Negative control: Number of cornea 3
Positive control: Number of cornea 3
Test item: Number of cornea 3

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item TAT (Triacetonetriamine), the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was measured a second time (t130).
After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

78.84

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive Control

Value:

72.17

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

1.8

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is corrosive to the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of TAT (Triacetonetriamine) by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item TAT (Triacetonetriamine), the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the undiluted test item TAT (Triacetonetriamine) caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 78.84. According to OECD 437 the test item is classified as corrosive / severe irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

A reconstructed human epidermis model test according to OECD 439 was performed to assess the skin irritation potential of triacetonetriamine. After treatment with the test item the mean relative absorbance value decreased to 46.3%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

A Human Skin Model Test with EpiSkin™ tissues according to OECD 431 was performed to assess the corrosive potential of triacetonetriamine. Relative absorbance values of 113.6% (after 3 minutes treatment), 52.0% (after 60 minutes treatment) and 42.9% (after 240 minutes treatment). Since the 240 minutes relative absorbance value was not reduced below 35% compared to the negative control value, the test item was not considered to be corrosive.

Taking into account that triacetonetriamine is corrosive to the eye as shown in an eye corrosion study in vitro, the pH measured is 12.32 (100 g/L) up to 13.28 (500 g/L) and from structural related amine compounds skin corrosive properties are known, it is concluded that the substance should be considered as skin corrosive although experimentally not confirmed.

Eye irritation/corrosion:

The eye irritation potential of triacetonetriamine was assessed by means of the Human Cornea Model Test using tissues of human cornea model EpiOcular™. Irritating effects were observed following incubation with the test item from 3 minutes up to the highest tested exposure period of 60 minutes.

The corneal irritation and damage potential of triacetonetriamine was assessed by means of the BCOP assay according to OECD 437. Relative to the negative control, the undiluted test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 78.84. This value is above the trigger value of 55.1, thus, according to OECD 437 the substance is classified as corrosive / severe irritant to the eye.

Justification for selection of skin irritation / corrosion endpoint:
There are in vitro studies available for skin irritation and skin corrosion. According to the study results the test item would be classified as skin irritant. However, the overall conclusion for this endpoint is that the substance has skin corrosive properties. For more details refer to the justification for classification or non-classification.

Justification for selection of eye irritation endpoint:
There are in vitro studies available for eye irritation and eye corrosion. Based on the study results it is concluded that the substance has eye corrosive properties.

Justification for classification or non-classification

Skin irritation/corrosion:

For triacetonetriamine there are in vitro studies available for skin irritation and skin corrosion. Triacetonetriamine is corrosive to the eye as shown in an eye corrosion study in vitro, the pH measured is 12.32 (100 g/L) up to 13.28 (500 g/L) and from structural related amine compounds skin corrosive properties are known. Taking into account the information stated above it has to be expected that the skin corrosion potential of triacetonetriamine may be underestimated based on available in vitro data. For animal welfare reason no in vivo studies were performed. Thus, it is concluded that the substance should be considered as skin corrosive although experimentally not confirmed. Triacetonetriamine is classified as Skin Corrosive Category 1C.

Eye irritation/corrosion:

For triacetonetriamine there are in vitro studies available for eye irritation and eye corrosion. In a BCOP assay according to OECD 437 it was demonstrated that the substance has eye corrosive properties. Furthermore, in the water solubility study the pH measured was 12.32 (100 g/L) up to 13.28 (500 g/L). Thus, the substance is classified as Eye Corrosive Category 1.