Trimethylamine, N-oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 November 2016 to 30 August 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

Instead of wetting and mixing the dry test item directly on the insert, it was decided to apply a slurry. The amount of applicated test item was in each case >36mg per insert. No details, on test system, on MTT direct or colour interference were given.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylamine, N-oxide

EC Number:

214-675-6

EC Name:

Trimethylamine, N-oxide

Cas Number:

1184-78-7

Molecular formula:

C3H9NO

IUPAC Name:

N,N-dimethylmethanamine oxide

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human epidermal keratinocytes

Cell source:

other: not applicable

Source strain:

other: not applicable

Justification for test system used:

epiCS Epidermal Skin Test (from CellSystems Biotechnology Vertrieb GmbH, Langeler Ring 5, 53842 Troisdorf Germany) consists of normal human epidermal keratinocytes and shows significant similarity to the skin found on thorax, abdomen and extremities of adult humans. The cellular structure of epiCS resembles that of natural epidermis showing a base membrane, proliferating keratinocytes and stratum corneum with an intact barrier function.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS Epidermal Skin Test (from CellSystems Biotechnology Vertrieb)
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date:not specified
- Delivery date:not specified
- Date of initiation of testing: 15 November 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time:3 hours
- Spectrophotometer: 96-well Plate reader spectrophotometer/fluorimeter/bioluminometer (eg. TECAN infinite M200)
- Wavelength: 570 nm
- Filter: no filter was used
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: viable skin inserts
- Barrier function: intact barrier function
- Morphology: normal human epidermal keratinocytes, and stratum corneum
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3 inserts were used per condition

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): not specified.
- N. of replicates : not specified
- Method of calculation used: not specified

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 : preparation of the inserts and main test (including application of the test item and MTT assay)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: no modification was performed

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 72 mg of prepared slurry (at least 36 mg of the test item)
- Concentration (if solution): not applicable

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

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

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

3 inserts were used per condition, duplicate were used per insert

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Test item - 3 minutes exposure period

Value:

97.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - test item - 60 minutes exposure period

Value:

92.9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Mean OD of the tissue replicates treated with the negative control (H2O) should exhibit (undiluted) OD ≥ 0.8 to ≤ 2.8 for every exposure time.
- Mean viability of the tissue replicates exposed for 1 hour with the positive control (8N
KOH). expressed as % of the negative control. should be < 20%.
- In the range 20 – 100% viability. and for ODs ≥ 0.3. difference of viability between the tissue replicates should not exceed 30%.

All acceptability criteria were met.

Any other information on results incl. tables

Table1 :Mean values of the absorbance values (λ=570nm)

 

OD 570 nm	Test Item   3minutes	Test Item   60 minutes	Negative Control 3 minutes	Negative Control 60 minutes
Mean Value 3 Inserts	0.7284	0.6192	0.7464	0.6669
SD	0.0095	0.0937	0.0344	0.0797

 

 

Table2 :Viability (based on MTT-Assay)

TestItem         Test Item

                                          3minutes        60minutes   

Viability (Vitality)

                [%]                             97.6                92.9

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive

Conclusions:

Based on the results of this experimental study, the test item TMAO did not induced decrease of viability (97.6 and 92.9% viability after, respectively, 3 and 60 minutes of exposure period). Hence, the test item was not considered as corrosive substance.

Executive summary:

This GLP compliant study was performed in order to assess the potential corrosive property of the test substance TMAO trimethylamine-N- oxyde. An in vitro test was performed according to OECD 431 Guideline, using epiCSepidermal skin test.

Inserts were incubated with test item for a period of 3 and 60 minutes at 37°C. At least 72 mg of the prepared slurry (at least 36 mg of the test item) were applied topically to the centre of the skin. For each incubation period 3 inserts were used. Sterile water and KOH were used as, respectively negative and positive control. At the end of the exposure period, the viability of each insert was assessed using a MTT assay.

Based on the results of this experimental study, the test item TMAO did not induced decrease of viability (97.6 and 92.9% viability after, respectively, 3 and 60 minutes of exposure period). Hence, the test item was not considered as corrosive substance.