Fenamiphos

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-02-12 to 2015-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products Appendix B1, NIH Publication No. 10-7553

Version / remarks:

2010

Deviations:

no

Test guideline 2

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:

adopted: 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fenamiphos

EC Number:

244-848-1

EC Name:

Fenamiphos

Cas Number:

22224-92-6

Molecular formula:

C13H22NO3PS

IUPAC Name:

{ethoxy[3-methyl-4-(methylsulfanyl)phenoxy]phosphoryl}(propan-2-yl)amine

Test material form:

solid: crystalline

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Transport conditions of ocular tissue:
On the test day, fresh eyes were collected from the slaughterhouse and were transported in Hanks’ balanced salt solution (HBSS) with Ca++ and Mg++, containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.

- Time interval prior to initiating testing: The corneas were incubated for one hour at 32 ± 1 °C in a water bath using deionized water prior to testing.

- Indication of any existing defects or lesions in ocular tissue samples:
The eyes were carefully examined for defects and any defective eyes were discarded.

- Indication of any antibiotics used: used HBSS contains Pen/Strep

- Selection and preparation of corneas:
The tissue surrounding the eyeball was carefully pulled away and the cornea was
excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri
dish containing HBSS. Before the corneas were mounted in corneal holders (MC2,
Clermont, France) with the endothelial side against the O-ring of the posterior chamber,
they had been visually examined for defects and any defective cornea had been
discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first.

- Quality check of the isolated corneas: visual examination for defects

Test system

Vehicle:

physiological saline

Remarks:

0.9 % sodium chloride

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

20 %

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3 replicates

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes, concurrent vehicle

SOLVENT CONTROL USED: physiological saline 0.9% NaCl

POSITIVE CONTROL USED: yes, imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME:
750 μL of the test item preparation or the control substance was introduced into the
anterior chamber (closed-chamber method) and incubated for 4 hours ± 5 minutes.

TREATMENT METHOD: closed chamber


POST-INCUBATION PERIOD: yes, 90 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.

- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The IVIS cut-off values for identifying test substances as inducing serious eye damage
(UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the following:

1. IVIS ≤ 3: No Category
2. IVIS > 3; ≤ 55: No prediction can be made
3. IVIS > 55: Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

37.33

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

ACCEPTABILITY
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore, this assay is considered acceptable.

Any other information on results incl. tables

Table 1: In Vitro Irritation Score

Cornea No.	Test item	Corrected opacity	Corrected OD490 value	IVIS
1		6.00	0.206
2	Negative control	6.00	0.200
3		6.00	0.283
MV		6.00	0.230	9.45
4		185.00	4.020
5	Positive control	192.00	2.375
6		174.00	2.850
MV		183.67	3.082	229.90
7		40.00	0.144
8	Test item	44.00	0.325
9		19.00	0.130
MV		34.33	0.200	37.33

MV = mean value

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made regarding the eye irritation potential of the test item according to the evaluation criteria.

Executive summary:

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay (BCOP). The test item was suspended with physiological saline 0.9% sodium chloride to gain a 20% concentration. The suspension was prepared just before application on the test system. Homogeneity of the suspension was checked just before application on the test system. The test item or the control substance were introduced into the anterior chamber. After 4 hours ± 5 minutes of incubation, either the test substance or the control substance was removed and the epithelium washed at least three times with minimum essential medium (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed. After the opacity measurement, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. Sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a UV/VIS spectrophotometer.

Mean in vitro irritation score (IVIS) was 37.33. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

No prediction can be made regarding the eye irritation potential of the test substance according to the evaluation criteria.