4-bromo-3,3,4,4-tetrafluorobut-1-ene

Endpoint summary

Description of key information

In Vitro Skin Irritation: irritant (OECD TG 439, Reconstructed Human Epidermis Test Method)

In Vitro Skin Corrosion: non-corrosive (OECD TG 431, reconstructed human epidermis (RHE) test method).

Serious eye damage / eye irritation: No classification (OECD TG 437: Bovine Corneal Opacity and Permeability Test Method)

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:

24-28 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables"

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other:

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm™ Model (EPI-200) (MatTek Corporation, Ashland, USA) consists of normal, human derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analagous to those found in vivo. The EpiDerm™ Model incorporates several features which make it advantageous in the study of the potential dermal toxicity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction [1]. Secondly, the target cells are epithelial, derived from human skin. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of post-treatment incubation (if applicable):

42 ± 2 hours

Number of replicates:

3 tissues/treatment

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 experiment

Value:

10.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

3.18%

The figure below summarizes the results of the Skin Irritation Test (SIT) for the test article and the positive control. The assay results were accepted. The mean OD570 of the negative control, sterile Ca++ and Mg+ Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS) was 1.712. The mean viability of the positive control, 5% SDS, was 3.18%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was <18% for the positive and negative controls. The test article was not observed to directly reduce MTT in the absence of viable cells. The test article was not considered to have potential interference with the MTT measurement (was not considered a colorant).

Interpretation of results:

other: requires further testing for classification and labeling for skin irritation according to UN GHS and EU classification system (Category I/II)

Conclusions:

The mean viability of the test substance was 10.0%. Based upon the results of this assay and according to the prediction model presented in OECD TG 439, the test substance was predicted to be an irritant. Additional testing would be needed to determine if the test article was a GHS Category 1 (Severe/ Corrosive) or GHS Category 2 (Irritant) for skin irritation.

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test substance. The MTT (3‑[4,5‑dimethylthiazol‑2‑yl]‑2,5‑diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H‑dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to the test substance. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439). Under the conditions specified in this study, the mean viability of the tissues treated with the test article was 10.0%. According to the prediction model presented in OECD TG 439, the test substance was predicted to be an irritant. Additional testing would be needed to determine if the test article was a GHS Category 1 (Severe/Corrosive) or GHS Category 2 (Irritant) for skin irritation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-25 March 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)

Version / remarks:

18 June 2019

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables"

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm™ Model (EPI-200) (MatTek Corporation, Ashland, USA) that will be used in this study consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ Model incorporates several features which make it advantageous in the study of potential dermal corrosivity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test substance interaction [1]. Secondly, the target cells are epithelial, derived from human skin [2]. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly [3].

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Fifty microliters of the test substance per tissue was administered to the test system without dilution (neat). After dosing the 60 minute exposure, a sterilized adhesive seal was applied over the top of the plastic tissue insert

Duration of treatment / exposure:

3 minutes / 60 minutes

Number of replicates:

4 tissues per treatment -- 2 tissues per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 experiment 60 minute - 91.6% 3 minute- 108.9%

Value:

91.6 - 108.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

60 minute - 7.4% 3 minute - 8.4%

The figure below summarizes the results for the test substance and the positive control. The assay results were accepted. The 3- and 60-minute average negative control OD₅₅₀ value was 1.724. The positive control resulted in a corrosive classification at 3- and 60-minute exposures. The CV for the negative control was 2.7% (60 minute) and 0.3% (3 minute). According to the prediction model presented below, the test substance was considered to be non-corrosive because the viability (91.6%) was ≥15% after the 60 minute exposure, and ≥50% after the 3-minute exposure (108.9%). The test substance was not observed to reduce MTT directly in the absence of viable cells; therefore, a killed control experiment was not performed. The test substance was not considered to have probable photometric MTT interference.

Interpretation of results:

other:

Remarks:

Non-corrosive based on GHS criteria

Conclusions:

According to the prediction model presented below, the test substance was considered to be non-corrosive because the viability (91.6%) was ≥15% after the 60 minute exposure, and ≥50% after the 3-minute exposure (108.9%).

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential skin corrosivity of the test substance. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test substance exposure. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test.” Under the conditions specified in this study, the mean viabilities of the tissues treated with the test substance were 91.6% and 108.9% for the 60- and 3-minute treatments, respectively. According to the prediction model presented in OECD TG 431, the test substance was predicted to be non-corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

9 October 2017

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

other:

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

-0.1

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Yes: 52.1

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Yes: 0.7

Positive controls validity:

valid

Remarks:

Yes: 32.3

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

-0.007

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Yes: 0.011

Positive controls validity:

valid

Remarks:

Yes: 1.315

Table 1 summarizes the results of the BCOP assay. The followng prediction models were used to evaluate the ocular irritancy potential as described by Sina et al. (1995)[2] for non-regulatory purposes or as described in OECD TG437 (2017). 

 

The prediction model according to Sina et. al based on a wide range of materials (note: While this classification system provides a good initial guide to interpretation of these in vitro data, these specific ranges may not be applicable to all classes of materials or other exposure times. Whenever possible, results should be compared to "benchmark" materials tested under similar exposure conditons.): 

 

In Vitro Score	Sina et al Prediction
≤ 25	Mild irritant
> 25 ; ≤ 55	Moderate irritant
> 55	Severe irritant

 

Prediction model outlined in OECD TG 437:

In Vitro Irritation Score (IVIS)	UN GHS
≤ 3	No Category
> 3; ≤ 55	No prediction can be made*
> 55	Category 1

*- Additional testing would be required for a definitive classification of ocular irritation potential according to GHS

 

The BCOP assay was accepted when the positive control (ethanol) produced an In Vitro Score that fell within two standard deviations of the historical mean. The current acceptance range for ethanol is 37.9-63.4. 

 

[2] Sina, J.F., Galer, D.M., Sussman, R.G., Gautheron, P.D., Sargent, E.V., Leong, B., Shah, P.V., Curren, R.D., and Miller, K. (1995) A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates. Fundamental and Applied Toxicology 26:20-31.

Interpretation of results:

other: No Category

Conclusions:

According to the current OECD test guideline 437 (OECD TG 437) the test substance is predicted to not require classification or labeling for ocular irritation according to the Globally Harmonized System (GHS No Category).

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test substance, BTFB, also referred to as 4-Bromo-3,3,4,4-tetrafluorobut-1-ene (CAS 18599-22-9). Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test substance for 10 minutes + 2 hrs post exposure period. The test substance was tested neat. An In Vitro Score was determined for the test substance based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The In Vitro Score obtained for the test substance was -0.1. The test substance would be classified as a mild irritant according to the classification system established by Sina Scale (i.e. In Vitro Score ≤ 25). According to the OECD test guideline 437 (OECD TG 437) the test substance is predicted to not require classification or labeling for ocular irritation according to the Globally Harmonized System (GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A GLP compliant OECD 439, Skin Irritation Test (Sit) Using The Epiderm™ Skin Model was performed on the test substance (Reliability = 1). The test substance was administered to the test system without dilution (neat). After dosing, a sterilized adhesive seal was applied over the top of the plastic tissue insert to reduce evaporation of the test substance. The test substance was not observed to directly reduce MTT in the absence of viable cells and was not considered to have probable photometric MTT interference.  All criteria for a valid study were met.  According to the prediction model presented in OECD TG 439, the test substance was predicted to be an irritant. Additional testing was performed to determine if the test article was corrosive.

 

A GLP compliant OECD 431, In Vitro Skin Corrosion Assay Using The EpiDerm™ Skin Model (EPI-200): 3- and 60-Minute Exposure Protocol (Reliability = 1) was performed. The test substance was administered to the test system without dilution (neat). The test substance was not observed to directly reduce MTT in the absence of viable cells. All criteria for a valid study were met.  According to the prediction model presented in OECD TG 431, the test substance was considered to be non-corrosive.

 

When applying the OECD test guideline 439 and 431 classification criteria, the test substance can cause skin irritation but is not corrosive.  Therefore, the appropriate GHS category is Skin Category 2.

 

A GLP compliant OECD 437, Bovine Corneal Opacity and Permeability Test (Reliability = 1) was performed.  The potential ocular irritancy of the test substance, as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas was examined. Bovine corneas were incubated in the presence of neat test substance at 32 ± 1ºC for 10 minutes. All criteria for a valid study were met.  The test substance was determined to have an in vitro score of -0.1, indicating it is not an eye irritant.

Justification for classification or non-classification

Based on in vitro tests performed in accordance with OECD guidelines 439, 431, and 437, the test substance is irritating to the skin and is not irritating to eyes. Therefore, the substance classified for skin as Category 2 and does not need to be classified for eye irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.