2-Propenoic acid, 2-fluoro-, methyl ester

Endpoint summary

Description of key information

The substance was tested in a reliable in vitro skin corrosion assay and then in a reliable in vitro skin irritation assay. It gave negative results in both in vitro studies. The substance was also tested in a reliable in vitro eye irritation assay (BCOP) where it gave inconclusive results. Because the results were inconclusive an additional eye irritation study was performed in vivo. This study was reliable and gave negative results.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9/6/15 to 15/6/15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recently conducted study performed to OECD guideline and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiSkin Reconstructure Human Epidermis Model Kit

Strain:

not specified

Details on test animals or test system and environmental conditions:

Test System: EPISKIN Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date Received: 9th June 2015
EpiSkin Tissues (0.38cm^2) lot number: 15-EKIN-023
Maintenance Medium lot number: 15-MAIN3-023
Assay Medium lot number: 15-ESSC-023

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

not required

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours

Number of animals:

Not applicable

Irritation / corrosion parameter:

other: other: Relative mean tissue viability

Value:

102.8

Remarks on result:

other:

Remarks:

Basis: other: Prediction. Time point: 42 hours. Reversibility: no data. (migrated information)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance was non-irritant to the skin in an in vitro assay

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.

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

Results

The relative mean viability of the test item treated tissues was 102.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD and CLP: Not classified for Irritation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29/4/15 to 1/5/15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Species:

other: EpiDermn Reconstructed Human Epidermis Model Kit

Strain:

not specified

Details on test animals or test system and environmental conditions:

Test System: EpiDerm Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date Received: 28 April 2015
EpiDerm Tissues (0.5 cm^2) lot number: 21663/D
Assay Medium lot number: 0923152SC

Upon receipt of the EpiDerm tissues, the sealed 24-well plate was placed into a refrigerator.

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

not required

Duration of treatment / exposure:

3 minutes and 60 minutes

Observation period:

None

Number of animals:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

113.6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

21.3

Negative controls validity:

valid

Positive controls validity:

valid

TABLE 1 - Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Time	Mean OD562	Percentage Viability
Negative Control	3 minutes	1.852	100
Negative Control	60 minutes	2.335	100
Positive Control	3 minutes	0.191	10.3
Positive Control	60 minutes	0.066	2.8
Test Item	3 minutes	2.103	113.6
Test Item	60 minutes	0.497	21.3

The mean viability of the negative control tissues is set at 100%

The mean OD₅₆₂ values is based on the mean of EpiDermTM tissues tested in duplicate

Viability is expressed as a percentage of the 3 and 60 minute negative control tissues

Interpretation of results:

GHS criteria not met

Conclusions:

The substance was non-corrosive to the skin in an in vitro assay

Executive summary:

In an in vitro skin corrosion study human skin tissue (eipdermis keratinocytes) was exposed to undiluted substance for 3-minutes or 1-hour. There was 113.6% and 21.3% tissue viability following the 3-minutes and 1-hour exposure points, respectively. Resultantly, the test material is considered to be non-corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18/5/15 to 4/6/15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd.
- Age at study initiation: 12 to 20 weeks
- Weight at study initiation: 2.6 to 2.8 kg
- Housing: Individual suspended cages
- Diet (e.g. ad libitum): ad libitum, 2930C Teklad Global Rabbit diet
- Water (e.g. ad libitum): ad libitum, mains drinking water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours darkness, 12 hours light

Vehicle:

unchanged (no vehicle)

Controls:

other: Tested animals were also used as control - test item was adminstered to only one eye, with the other left untreated for control purposes.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml

Duration of treatment / exposure:

Eight hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.17

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

No corneal or iridial effects were noted in any animal during the study. Moderate conjunctival irritation was seen in two animals at one hour and minimal conjunctival irritation was seen in one animal at one hour. Minimal conjuctival irritation was seen at 24 hours and 48 hours. No effects were seen at 72 hours in the surviving animals.

Other effects:

The first treated animal was found dead three days after test substance application and the third animal was found dead two days after application.

Individual scores for Ocular Irritation

Rabbit name and sex	75066 Male				75073 Male				75083 Male
IPR	0				0				0
Time after treatment	1 hour	24 hours	48 hours	72 hours	1 hour	24 hours	48 hours	72 hours	1 hour	24 hours	48 hours
Cornea
Degree of Opacity	0	0	0	0	0	0	0	0	0	0	-
Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	-
Iris	0	0	0	0	0	0	0	0	0	0	-
Conjunctivae
Redness	1	1	1	0	1	1	1	0	2	1	-
Chemosis	1	1	0	0	1	0	0	0	1	1	-
Discharge	2	0	0	0	1	0	0	0	1	0	-

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance is not classified as corrosive or irritant based on the results of this study. It is not clear whether the mortalities noted on the study were test substance related.

Executive summary:

A study has been undertaken according to OECD method 405 and EU method B5 to determine the eye irritancy of 2 -Propenoic acid, 2 -fluoro-, methyl ester. The study shows that the test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008. It is not clear whether the observed mortalities within the study are test substance related.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8/5/15 to 8/5/15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recently conducted study performed to OECD guideline and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Species:

other: Cow

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Local abattoir
- Age at study initiation: 12 to 60 months

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

120 minutes

Number of animals or in vitro replicates:

9 corneas used

Irritation parameter:

in vitro irritation score

Value:

ca. 10.3 - < 10.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Individual and Mean Corneal Opacity and Permeability Measurements

			Opacity				Permeability
Treatment	Cornea Number	Pre-treatment	Post-treatment	Post-incubation	Post-incubation - Pre-treatment	Corrected Value		Corrected Value	In Vitro Irritancy Score
Negative Control	1	4	5	5	1		0.001
Negative Control	5	4	4	5	1		0.002
Negative Control	12	4	5	6	2		0.278
Negative Control					1.3		0.094		2.7
Positive Control	3	5	35	32	27	25.7	1.004	0.910
Positive Control	16	2	31	28	26	24.7	0.894	0.800
Positive Control	19	4	35	28	24	22.7	0.697	0.603
Positive Control						24.3		0.771	35.9
Test Item	14	4	17	16	12	10.7	0.020	0.000
Test Item	18	6	21	17	11	9.7	0.025	0.000
Test Item	20	4	14	16	12	10.7	0.009	0.000
Test Item						10.3		0.000	10.3

Corneal Epithelium Condition Post-reatment and Post-incubation

		Observation
Treatment	Cornea Number	Post-treatment	Post-incubation
Negative Control	1	Clear	Clear
Negative Control	5	Clear	Clear
Negative Control	12	Clear	Clear
Positive Control	3	Cloudy	Cloudy
Positive Control	16	Cloudy	Cloudy
Positive Control	19	Cloudy	Cloudy
Test Item	14	Partly cloudy	Partly cloudy
Test Item	18	Partly cloudy	Partly cloudy
Test Item	20	Partly cloudy	Partly cloudy

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item had an in vitro irritancy score of 10.3. This is in the range of 3 to 55, and within this range no predicition of eye irritation can be made.

Executive summary:

SUMMARY

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Interpretation

The test item is classified according to the prediction model below:

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Scor
Test Item	10.3
Negative Control	2.7
Positive Control	35.9

Conclusion

No prediction of eye irritation can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance gave negative results in skin corrosion and skin irritantancy assays conducted in vitro. It gave an inconclusive result in an in vitro eye irritation study and only minimal irritation effects in an in vivo eye study. Therefore classification for corrosion/irritancy effects is not justified.