N-(1,3-dimethylbutylidene)-5-[(1,3-dimethylbutylidene)amino]-1,3,3-trimethylcyclohexanemethylamine

Endpoint summary

Description of key information

OECD 431: corrosive: UN GHS H314 Combination of sub-categories 1B and 1C.

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:

12.02.2020-29.04.2020

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)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: CAS 71077-09-3
Batch: 12846
Lot Number: 0809103346
Purity: 97.32%
Physical state/Appearance: Pale yellow viscous liquid
Expiry Date: 30 November 2020
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EpiDermTM test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek (Bratislava, Slovakia).
- Tissue batch number(s): Lot 30853, Batch no.: 00267
- Production date: March 25, 2020

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml MTT solution (0.3 mg/ml in PBS)
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 and 60 minutes exposure period

Number of replicates:

3

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): 100% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 and 60 minutes exposure period

Number of animals:

not used, in vitro study

Details on study design:

The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.
After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.

The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = ( mean OD570 of test item / mean OD570 of negative control ) x 100

The test item was shown to directly reduce MTT and freeze-killed tissues were employed, the results of the MTT assay were therefore corrected as follows:
True viability=mean OD tvt−(OD tkt−OD ukt)
OD=optical density at 570 nm
tvt=treated viable tissues
tkt=treated killed tissues
ukt=untreated killed tissues

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

- Acceptance criteria met for positive control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-Minute positive control is < 15%.

- Acceptance criteria met for variability between replicate measurements:Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

94.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

5.3 % viability

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

4.3 % viability

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The MTT solution containing the test item turned purple. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using non-viable, freeze-killed, tissues was performed. The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
The mean OD570 for the negative control treated tissues was 1.766 for the 3-Minute exposure period and 1.792 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 4.3% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. 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 each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C. The criteria required for acceptance of results in the test were satisfied

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. 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 each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C. The criteria required for acceptance of results in the test were satisfied