Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion:

- not corrosive in-vitro (OECD TG 431, GLP)

- not irritant in-vitro (OECD TG 439, GLP)

Eye irritation:

- not irritant in-vitro (OECD TG 437, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 December 2017 to 29 January 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
30 mg of test item was used instead of 25 mg and the post incubation period was 24h instead of 42h,as required in the current OECD method. No details were given on MTT interference or colou interference assessment, no certificate on tissue was given
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
supplied by the sponsor, lot No. 160405
- Expiration date of the lot/batch:
30June 2018
- Purity test date:not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: prepared as slurry with sterile water

FORM AS APPLIED IN THE TEST (if different from that of starting material)
the test item was applied with 25 µL of DPBS
Test system:
human skin model
Source species:
human
Cell type:
other: EpiDermTM
Cell source:
other: not applicable
Source strain:
other: not applicable
Justification for test system used:
The test system is given by the sponsor
Vehicle:
other: DPBS
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpidermTM SIT
- Tissue batch number(s): not detailed
- Production date: not detailed
- Shipping date: not detailed
- Delivery date: not detailed
- Date of initiation of testing: 12 December 2017

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: washing was performed 15 times with sterile PBS
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP:not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: 96 well plate reader spectrophotometer/fluorimeter/bioluminometer (TECAN infinite 2000)
- Wavelength:570nm
- Filter: no filter was used
- Filter bandwidth:no filter was used
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No deviation from test system or model condition was observed. No historical data was mentionned

NUMBER OF REPLICATE TISSUES: triplicates were used

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

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Only MTT assay was described in the report

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if tthe viability after 60 minutes and 24 hours post incubation period is equal to or less than 50
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes and 24 hours post incubation period is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 29 , 30.2, 31.2 mg test item powder with 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes (including 35 minutes at 37°C)
Duration of post-treatment incubation (if applicable):
24 hours
Number of replicates:
triplicates were used
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item
Value:
107.98
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control
Value:
100
Negative controls validity:
not valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: not specified
- Colour interference with MTT: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: positive control indicated proficiency of the test system to measured tissue viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: conform
- Acceptance criteria met for positive control: conform
- Acceptance criteria met for variability between replicate measurements: conform
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental condition of the study, the test item TMAO did not induced decrease of tissue viability after exposure. Hence, according to CLP criteria, the TMAO did not required skin irritation classification.
Executive summary:

The aim of this GLP compliant study was the detection of the skin irritation potency of TMAO tested on Human skin model EpiDermTM SIT according to OECD 439 method.

30 mg of the test item was applied on the tissue (EpiDermTM) used in triplicate for 60 minutes (including 35 minutes period at 37°C).5% SDS was used as positive condition and PBS as negative condition. Thereafter, the test item was removed from the tissue by rincing step. The cells were incubated for 24 hours. MTT assay was performed at the end of the post incubation period. The tissue viability was measured at 570 nm using a spectrophotometer.

The viability of the tissue exposed to test item was measured at 107,98%

Under the experimental condition of the study, the test item TMAO did not induced decrease of tissue viability after exposure. Hence, according to CLP criteria, the TMAO did not required skin irritation classification.

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
Qualifier:
according to
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. certificate)
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
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
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.

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

 

 

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.

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:
From 20 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted October 09, 2017
Deviations:
yes
Remarks:
One of the negative control eye was excluded from analysis because opacity value was above historical data. This does not affect the study outcome.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch No. 160405
- Expiration date of the lot/batch: 31 January 2019
- Purity test date: only purity (>98%) was given, no certificate was added.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature desiccated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: soluble in physiological saline
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 20% w/v solution was performed from TMAO anhydrous in physiologicalsaline
- Preliminary purification step (if any): not applicabme
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: prepared in solution

FORM AS APPLIED IN THE TEST (if different from that of starting material) in solution
Species:
cattle
Strain:
other: bovine. not more details
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- Number of animals: 9 eyes were used
- Characteristics of donor animals (e.g. age, sex, weight): young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: not detailed
- indication of any existing defects or lesions in ocular tissue samples: no lesions
- Indication of any antibiotics used: not specified
Vehicle:
physiological saline
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µL
- Concentration (if solution): 20% (w/v) in physiological saline
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
triplicates were used per condition
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.


QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
Triplicates were used

NEGATIVE CONTROL USED
Physiological saline

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
Imidazole 20% (w/v) in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750µl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32±1°C.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. 90 minutes (for application of sodium fluorescein)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to: Opacity = ((Io/I) - 0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)


DECISION CRITERIA: ≤ 3 No Category

> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
3.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
164
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
TMAO anhydrous
Value:
-0.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Negative control
Value:
2.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control
Value:
131
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
TMAO anhydrous
Value:
-0.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Negative control
Value:
0.055
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Positive control
Value:
2.161
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
TMAO anhydrous
Value:
0.007
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: the positive control condition showed proficiency of the test system to measure effect

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual in vitro irritancy scores for the negative controls ranged from 1.8 to 5.3. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. Since the mean of the two remaining eyes completely met the criteria and the test item results were not influenced by this result, this does not affect the study outcome
- Acceptance criteria met for positive control: yes

Table1          
Summary of Opacity, Permeability and In Vitro Scores

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

2.7

0.055

3.6

Positive control

131

2.161

164

TMAO anhydrous

-0.8

0.007

-0.7

1     Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2     In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


 

 

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the experimental condition of the study, IVIS score of the test item TMAO was below 3, no classification is required for eye irritation or serious eye damage according to CLP criteria.
Executive summary:

The objective of this GLP compliant study was to evaluate the eye hazard potential of TMAO anhydrous as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test according to OECD TG 437 method).

This studydescribes the potency of chemicals to induce serious eye damage using isolated bovine corneas.  The eye damage of TMAO anhydrous was tested through topical application for approximately 240 minutes.  

Batch 160405 of TMAO anhydrous was a white powder.  The test item was applied as a 20% (w/v) solution (750 µL) directly on top of the corneas.  

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.  The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 164 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

TMAO anhydrous did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.2 after 4 hours of treatment.  

In conclusion, since TMAO anhydrous induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.  

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion:

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

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.

Skin irritation:

The skin irritation potency of TMAO was tested on Human skin model EpiDermTM SIT according to OECD 439 method.

30 mg of the test item was applied on the tissue (EpiDermTM) used in triplicate for 60 minutes (including 35 minutes period at 37°C). 5% SDS was used as positive condition and PBS as negative condition. Thereafter, the test item was removed from the tissue by rincing step. The cells were incubated for 24 hours. MTT assay was performed at the end of the post incubation period. The tissue viability was measured at 570 nm using a spectrophotometer.

The viability of the tissue exposed to test item was measured at 107,98%

Under the experimental condition of the study, the test item TMAO did not induced decrease of tissue viability after exposure. Hence, according to CLP criteria, the TMAO did not required skin irritation classification.

Eye irritation:

The eye hazard potential of TMAO anhydrous was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test according to OECD TG 437 method).

The eye damage of TMAO anhydrous was tested through topical application for approximately 240 minutes.  

The test item was applied as a 20% (w/v) solution (750 µL) directly on top of the corneas.  

TMAO anhydrous did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.2 after 4 hours of treatment.  

In conclusion, since TMAO anhydrous induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.  

Justification for classification or non-classification

Klimisch-1-rated in-vitro studies on skin and eye irritation/corrosion properties are available with the substance TMAO itself, revealing no adverse effect. These results justify an absence of classification for skin and eye irritation/corrosion according to the CLP criteria is thus required.

No data is available by inhalation.