2-hydroxy-4-(trifluoromethyl)benzoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 17, 2017 - November 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-4-(trifluoromethyl)benzoic acid

EC Number:

700-368-9

Cas Number:

328-90-5

Molecular formula:

C8H5F3O3

IUPAC Name:

2-hydroxy-4-(trifluoromethyl)benzoic acid

Test material form:

solid: particulate/powder

Details on test material:

Beige to brownish solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra.
- Characteristics of donor animals (e.g. age, sex, weight): Age between 1 to 5 years (age of animals was determined based on the teeth count and horn ring count).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported under cold condition in Hank's Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 µg/mL].
- Time interval prior to initiating testing: The eyes were used within 24 h from the slaughtering.
- indication of any existing defects or lesions in ocular tissue samples: No. Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.
- Indication of any antibiotics used: Antibiotic used during transport in buffered Hanks medium [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Test system

Vehicle:

other: Corn oil

Remarks:

(Sigma Aldrich; Batch MKBZ9899V)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL applied on each cornea.
- Concentration (if solution): 20% (w/v) in corn oil.

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL.

Duration of treatment / exposure:

4 hours (± 5 minutes) at 32 ± 1 ºC.

Duration of post- treatment incubation (in vitro):

90 min (± 5 min) at 32 ± 1 ºC.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : Corneas, free from defects were dissected to a 2 to 3 mm rim and were transferred to container containing Hank's Balanced Salt Solution containing antibiotics (e.g., penicillin at 100 IU/mL and streptomycin at 100 µg/mL).

QUALITY CHECK OF THE ISOLATED CORNEAS : Selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on the top of the cornea and fixed in place with screws. Both chambers will then be filled to excess with prewarmed phenol red free Eagle's Minimum Essential Medium (EMEM) (posterior chamber first to return the cornea to its natural concave position), ensuring that no bubbles were present within the holders. The device was then equilibrated at 32 ± 1 ºC for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, the medium was removed from both the chambers and fresh pre-warmed phenol red free EMEM is added to both chambers and baseline opacity readings were taken for each cornea. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes, normal saline.

SOLVENT CONTROL USED (if applicable) : Yes, corn oil.

POSITIVE CONTROL USED : Yes, Imidazole 20% (w/v) concentration in normal saline

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of 20% (w/v) in corn oil for 4 hours exposure.

TREATMENT METHOD: [closed chamber]

POST-INCUBATION PERIOD: yes, holders are incubated in a horizontal position for 90 ± 5 min at 32 ± 1ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red). Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Media in the anterior and the posterior chamber was removed and fresh EMEM (without phenol red) was filled.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer BASF-OP3.0 was used. Opacity value calculation for selecting the cornea for the experiment:
Mean Opacity value =
Step 1. Baseline opacity reading (with medium without cornea)/post incubation opacity reading (with medium and cornea)
Step 2. [Step 1 value of each cornea – b]/a
Where b = 0.9894 and a = 0.0251 (a and b values are empirical derived for the instrument)

Mean Opacity value =
Step 1. [Baseline opacity reading (with medium without cornea)/post incubation opacity reading (with medium and cornea)] – post treatment opacity reading (Calculate for each cornea)
Step 2. Calculate the mean of control group
Step 3. {[Step 1 value of each cornea (except control) – Step 2 value] – b}/a
Step 4. Calculate Mean of Step 3 value
Where b = 0.9894 and a = 0.0251 (a and b values are empirical derived for the instrument)

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader BioTek Instruments Inc. USA (OD490) .
Mean Permeability value =
Step 1. OD490 value of all the individual cornea – Blank OD490 value
Step 2. Calculate the mean of control group
Step 3. Step 1 value of each cornea (except control) – Step 2 value
Step 4. Calculate Mean of Step 3 value
Any dilution made to bring OD490 into linear range was multiplied by the dilution factor.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) was calculated for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The in vitro irritancy score was calculated for each individual treatment.

DECISION CRITERIA: as indicated in the TG.
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

159.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

151.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

174.19

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

161.84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

153.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

145.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

161.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

153.63

Vehicle controls validity:

valid

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:
Yes, a proficiency study (study no. 530-1-01-10123) was performed from November 06, 2014 to February 06, 2015, which demonstrates the laboratory's proficiency to conduct Bovine corneal Opacity and Permeability test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes, the mean In-Vitro Irritancy Score (IVIS) of imidazole (20% w/v) (positive control) treated corneas were found to be 130.83 which is within the range of two standard deviation of the mean of the historical control data, confirming the reliability of the test procedure.

Any other information on results incl. tables

TABLE 1: In Vitro Irritation Score

 

Group : Normal Saline, 0.75 mL
Cornea Holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	OD490 Value	Corr. OD490 Value	IVIS
2	1095	1032	2.85	999	4.25	1.40	0.125	0.084	2.66
7	1090	1012	3.49	977	5.03	1.54	0.114	0.073	2.64
8	1092	1035	2.62	987	4.66	2.04	0.123	0.082	3.27
Mean						1.66	-	0.080	2.86
SD						0.34	-	0.006	0.36

 

Group : Imidazole at 20% (w/v) in normal saline, 0.75 mL
Cornea Holder N°	Io  (LUX)		I (Initial) (LUX)		Initial Opacity Value	I (Post Treatment) (LUX)		Post Treatment Opacity	Corr. Opacity	Final Opacity		OD490	Corr. OD490	Final OD490		IVIS Score
3	1117		1066		2.33	277		121.24	118.91	117.25		1.564	1.523	1.443		138.90
9	1109		1034		3.31	289		113.46	110.15	108.49		2.870	2.829	2.749		149.73
15	1066		1002		2.97	309		98.03	95.06	93.40		0.819	0.778	0.698		103.87
Mean										106.38		-	1.710	1.630		130.83
SD										12.06		-	1.038	1.038		23.97
Group : Corn Oil, 0.75 mL
Cornea Holder N°		Io  (LUX)		I (Initial) (LUX)		Initial Opacity Value	I (Post Treatment) (LUX)		Post Treatment Opacity Value		Corr. Opacity Value		OD490 Value		Corr. OD490 Value		IVIS
4		1070		1024		2.21	989		3.69		1.48		0.14		0.099		2.97
11		1064		997		3.10	969		4.33		1.23		0.142		0.101		2.75
17		1066		1011		2.59	972		4.28		1.69		0.126		0.085		2.97
Mean											1.47		-		0.095		2.90
SD											0.23		-		0.009		0.13

Group : 4 - Trifluoromethylsalicylic acid (ATFMS) (suspension) at 20% (w/v) in Corn Oil, 0.75 mL
Cornea Holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	Final Opacity Value	OD490 Value	Corr. OD490 Value	Final OD490 Value	IVIS
5	1147	1071	3.25	231	158.41	155.16	153.69	0.542	0.501	0.406	159.78
6	1106	1045	2.75	233	149.70	146.95	145.48	0.540	0.499	0.404	151.54
12	1080	1008	3.27	209	166.46	163.19	161.72	0.967	0.926	0.831	174.19
Mean							153.63	-	0.642	0.547	161.84
SD							8.12	-	0.246	0.246	11.46

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX 

Reading with Medium and Cornea, OD₄₉₀= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD₄₉₀value = 0.041.

 

InitialOpacity Value = [((Io⁄I)-b)/a)], Where I = Initial LUX, Io = Baseline Reading Note: a (0.0251) and b (0.9894) are constant. Example:Initial Opacity Value = [((1095/1032)-0.9894)/0.0251] =2.85

Post TreatmentOpacity Value = [((Io⁄I)-b)/a)], Where, I = Post Treatment LUX, Io = Baseline Reading. Note: a (0.0251) and b (0.9894) are constant. Example:Initial Opacity Value = [((1095/999)-0.9894)/0.0251] =4.25

Corr. Opacity Value = Post Treatment Opacity Value - Initial Opacity Value Example:Corr. Opacity Value = 4.25 – 2.85 =1.40

Final Opacity Value = Corr. Opacity Value – Mean Corr. Opacity Value of Control (Group I) Example:Final Opacity Value= 118.91 – 1.66 =117.25

Corr. OD490Value = OD490Value – Blank OD490Value, Where Blank Value for this trial was 0.044 Example:Corr. OD490Value = 0.125 -0.041 =0.084

Final OD490Value = Corr. OD490Value –Mean Corr.OD490Valueof Control (Group I) Example:Corr. OD490Value 1.523 -0.080 =1.443

IVIS (Control) = Corr. Opacity Value + (15 x Corrected OD490) Example:IVIS = 1.40 + [15 x 0.084] =2.66

IVIS (Treatment) = Final Opacity Value + (15 x Final OD490) Example:IVIS = 117.25 + (15 x 1.443) =138.90

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the tested condition, the mean IVIS value of the test item was found to be 161.84. According to decision criteria for classification, test item has to be classified as eye irritant, category 1.

Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in accordance with OECD TG 437 (GLP study) in order to determine the potential severe eye damaging effects of the test item. The corneas were obtained from freshly slaughtered cattles. An experiment was performed using three corneas for each treated series using the closed chamber method during 4 h ± 5 minutes. One set of corneas were treated with 750 μL of the prepared test item 20% (w/v) concentration in corn oil. One set of corneas served as positive control (750 μL of 20% (w/v) concentration in normal saline) and two set of corneas served as negative control and was treated with 750 μL of normal saline and corn oil, respectively. At the end of exposure period, the test item, positive and negative controls were removed from the anterior chamber and the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red). Post opacity reading permeability was measured by applying1 mL of fluorescein sodium solution (5 mg/mL) on to the anterior surface of the cornea and was incubated for approximately 90 min at 32 ºC. At the end of the incubation period OD was measured at 490 nm for the fluid collected from the posterior chamber. The mean in-vitro irritancy score (IVIS) of normal saline, corn oil and 750 µL 20% (w/v) imidazole in normal saline (positive control) treated corneas were found to be 2.86, 2.90 and 130.83, respectively. The IVIS score for the corneas treated with 750 μL of test item at 20% (w/v) concentration in corn oil were found to be 161.8. Based on the results of this study, an indication of the classification for test item is Category 1.