Methyl 2,3-dibromopropionate

Endpoint summary

Description of key information

Skin corrosion:

The study was performed in accordance with OECD Guideline 431. The test substance was corrosive on a human three dimensional epidermal model.

Eye irritation:

The study was performed in accordance with OECD Guideline 437. The test substance did not induce ocular irritation using isolated bovine corneas.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-02-06 to 2019-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 29944 Kit I and J

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): 37.2 – 37.5 °C (actual range)

REMOVAL OF TEST MATERIAL AND CONTROLS : washed with phosphate buffered saline to remove residual test item, rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL diluted (1:5)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0.8 - 2.8

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 20 - 100%

NUMBER OF REPLICATE TISSUES: 2 tissues for 3-minute exposure and two for a 1-hour exposure, 2 tissues for the negative and positive controls for both the 3-minute and 1-hour time point

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%.

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): 98% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Milli-Q water

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

Duration of treatment / exposure:

3-minute and 1-hour

Number of replicates:

2 tissues for 3-minute exposure and two for a 1-hour exposure, 2 tissues for the negative and positive controls for both the 3-minute and 1-hour time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute treatment

Value:

29

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean Tissue Viability

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour treatment

Value:

2.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean Tissue Viability

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT: the test item did not interfere with the MTT endpoint

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8)
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 13%.
- Acceptance criteria met for variability between replicate measurements: ≤14% for the negative control; 36% for the positive control; Since both
individual tissues were clearly positive it was concluded that the test system functioned properly.
For the test item, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 79%. Since both individual tissues were in the same category and clearly positive, it was concluded that this did not influence the outcome of the test.

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In conclusion, the test substance was corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

This study was to evaluate the test substance for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD 431. 

The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour. The test item was applied undiluted (50 μL) directly on top of the skin tissue.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 29% and 2.4%, respectively. Because the mean relative tissue viability for the test substance was below 15% after the 1-hour treatment it is considered to be corrosive.

In conclusion, the test substance was corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

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

Study period:

2018-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue:
Storage: The isolated corneas were stored in a petri dish with cMEM (Earle's Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum).
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

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): 750 μL
- Concentration (if solution): 98%

Duration of treatment / exposure:

10 ± 1 minutes

Number of animals or in vitro replicates:

Three corneas for each treatment group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Preparation of Corneas: The isolated corneas were mounted in a corneal holder (one cornea per holder) 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.
- Cornea Selection: 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. Corneas that had an initial opacity reading higher than 7 were not used.

QUALITY CHECK OF THE ISOLATED 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.

NUMBER OF REPLICATES : Three corneas for each treatment group

NEGATIVE CONTROL USED : physiological saline

POSITIVE CONTROL USED : Ethanol

APPLICATION DOSE AND EXPOSURE TIME : 750 μL of either the negative control, positive control (Ethanol) or test item; 10 ± 1 minutes

REMOVAL OF TEST SUBSTANCE: After the incubation the control or test item was washed with MEM with phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Value:

>= -0.7 - <= 1.1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas were clear after the 10 minutes of treatment with test item. No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean 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.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (Ethanol) was 47 and was within two standard deviations of the current historical positive control mean.

Interpretation of results:

other: unclassfied

Conclusions:

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

Executive summary:

The study was performed to evaluate the eye hazard potential of the test substance in accordance with OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage.

The test item was applied as it is (750 μL) directly on top of the corneas for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test item and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The test conditions were adequate and that the test system functioned properly.

The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 10 minutes of treatment.

In conclusion, since the test substance 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:

This study was to evaluate the test substance for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD 431. 

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 29% and 2.4%, respectively. Because the mean relative tissue viability for the test substance was below 15% after the 1-hour treatment it is considered to be corrosive.

In conclusion, the test substance was corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Eye irritation:

The study was performed to evaluate the eye hazard potential of the test substance in accordance with OECD Guideline 437.

The test item was applied as it is (750 μL) directly on top of the corneas for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test item and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 10 minutes of treatment.

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

Justification for classification or non-classification

Skin corrosion / irritation:

The test item is corrosive after the 1-hour treatment in the in vitro skin corrosion test.

According to Regulation (EC) No 1272/2008, section 3.2.2.1, this substance should be classified as category 1 for this endpoint.

 

Serious eye damage/eye irritation:

No irritation, IVIS≤3

According to Regulation (EC) No 1272/2008, section 3.3.2.1, this substance is not classified for this endpoint.