Fenamiphos

Endpoint summary

Description of key information

Endpoints for irritation and corrosion are covered by available animal and in vitro studies.

The test substance is irritating to the eye and non-irritating to skin.

 

Eye: irritating

 

Skin: non-irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-02-16 to 2015-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: kit: EPISKIN-SM™, supplier: SkinEthic

Source strain:

not specified

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of human keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

water

Remarks:

aqua dest.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ reconstructed human epidermis model (SkinEthic)
This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number: 15-EKIN-007
- Date of initiation of testing: 2015-02-16

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: washing was done with PBS to remove any residual test item or control item. Excess PBS was removed by blotting bottom with blotting paper.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: yes
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: yes
- Barrier function: IC50 determination (SDS concentration, MTT test, n = 14); specification ≥ 1.5 mg/mL; result: 2.2 mg/mL
- Morphology: Well-differentiated epidermis consiting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3


NUMBER OF INDEPENDENT TEST EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL
The test item is considered to be irritant to skin, if the tissue viability after 15 min. of exposure and 42 h of post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin if the tissue viability after exposure and post-treatment incubation is higher than 50%.

ACCEPTABILITY CRITERIA:
The test meets acceptance criteria if:
- OD570 nm of the blank is < 0.1
- mean OD570 nm of the three negative control tissues is ≥ 0.6 and ≤ 1.5.
- mean relative tissue viability of the three positive control tissues is ≤ 40%.
- the standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg + 10 μL aqua dest

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

89.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Pre-Experiments
The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. The mixture of 10 mg of the test item per 90 µL aqua dest. showed no colouring detectable by unaided eye-assessment.


Acceptance of results
The controls confirmed the validity of the study. The mean OD570 of the six blank values was < 0.1. The mean absolute OD570 of the three negative control tissues was ≥ 0.6 and ≤ 1.5. The mean relative tissue viability (% negative control) of the positive control was ≤ 40% (18.1%). The maximum standard deviation of viability of replicate tissues of the negative and positive control was < 18% (4.9% - 6.1%).

Table 1: Result of the Test Item

Name	Negative Control			Positive control			Test item
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.148 1.141	1.135 1.153	1.056 1.051	0.185 0.210	0.200 0.205	0.324 0.301	1.134 1.197	1.045 1.082	0.789 0.784
OD570 (blank-corrected)	1.104 1.097	1.091 1.109	1.012 1.007	0.141 0.166	0.156 0.161	0.280 0.257	1.090 1.153	1.001 1.038	0.745 0.740
mean OD570 of the duplicates (blank-corrected)	1.100	1.100	1.010	0.154	0.158	0.268	1.121	1.020	0.743
total mean OD570 of 3 replicate tissues (blank-corrected)	1.070*			0.193			0.961
relative tissue viabilities [%]	102.8	102.8	94.4	14.4	14.8	25.1	104.8	95.3	69.4
mean relative tissue viability [%]	100.0			18.1**			89.8
SD tissue viability [%]***	4.9			6.1			18.3

* Corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is ≤ 40%.

*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%. The test item treated tissue is excluded from this statement.

 

Table 2: Quality criteria

	Value	Cut off	pass/fail
Mean OD570 nm Blank	0.044	< 0.1	pass
Mean Absolute OD570 nm NC	1.11	0.6 ≤ NC ≤ 1.5	pass
Mean Relative Viability [%] PC	18.1	≤ 40%	pass
SD of Viability [%]	4.9 – 18.3	< 18%	fail

 

 

Table 3: Historical data

	OD570 blank	Absolute OD570 NC	Relative Viability [%] PC	SD of Viability [%]
Mean	0.044	0.883	12.3	8.4
SD	0.001	9.1	9.1	8.6
n	54	54	54	248

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant”.

Executive summary:

In the present study the skin irritant potential of the test substance was analysed. The EPISKIN-Standard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. The test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after a 15 minutes exposure and 42 hours post incubation period and compared to those of the concurrent negative controls. In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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)

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

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

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.

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

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.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984-04-27 to 1984-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: guidelines of the United States Environmental Protection Agency

Version / remarks:

1978

Deviations:

no

GLP compliance:

no

Species:

rabbit

Strain:

other: Japanese native derived albino rabbits

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Ichikawaya Co., Ltd., Takenotsuka, Adachi, Tokyo, Japan.
- Age at study initiation: 12 weeks old
- Weight at study initiation: 2.92 to 3.01 kg
- Housing: aluminum cages ( 350 x 480 x 350 mm)
- Diet (ad libitum): pellet diet (RC-4 : Oriental Yeast Industries Co., Ltd.)
- Water (ad libitum): tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1984-04-27 To: 1984-05-21

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

group I: 24 h
group II: 20 to 30 sec

Observation period (in vivo):

10 days

Number of animals or in vitro replicates:

group I: 6 animals
group II: 3 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): for group II
- Time after start of exposure: at 20 to 30 seconds following an installation of test material in conjunctival sac

SCORING SYSTEM: according to the Draize-scoring-system

TOOL USED TO ASSESS SCORE:
An ophthalmoscope was used for examinations.
At 24 hours 0.1 ml of 2 % fluorescein sodium solution was used for detailed examination. Fluorescein sodium dropped and eyes were washed immediately by 20 mL of distilled water and observed for staining mark on cornea.

CLINICAL OBSERVATIONS
The animals were observed for any change in general condition and for mortality at appropriate intervals up to 6 hours after application and then daily at regular time in a day for 7 days or for a period in which reversible change were seen in eyes.

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 24 h

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 96 h

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

probability of weak irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

probability of weak irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 96 h

Remarks on result:

probability of weak irritation

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

probability of weak irritation

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

The following effects were seen in treated, unwashed eyes: scattered or diffused opacity in cornea, hyperemia in iris, slight flare in conjunctivae, swelling and ocular discarge. No treatment-related findings were obtained in eyes immediately washed after instillation of the test item.

Other effects:

Systemic findings: The following cholinergic symptoms indicative for organophosphate intoxication were seen in rabbits without eye washing: reduction of pupil diameter (midriasis), salivation, rhinorrhea, increased respiration, cyanosis, slight convulsions.

Time after administration	CORNEA opacity	IRIS		CONJUNCTIVAE redness                     chemosis
Animal no. 1/2/3/4/5/6
24 h	1/1/1/1/1/1		1/1/1/1/1/1	1/1/1/1/1/1	1/1/0/1/1/1
48 h	1/1/1/1/1/1		1/1/1/1/1/1	0/1/1/1/1/1	0/0/0/0/0/0
72 h	1/1/1/1/1/1		0/1/1/1/1/1	0/1/1/1/1/1	0/0/1/0/0/0
96 h	1/1/1/1/1/1		0/1/1/1/0/1	0/1/1/1/0/1	0/0/0/1/0/1
7 days	1/1/0/1/0/1		0/0/0/1/0/0	0/0/1/0/0/0	0/0/0/0/0/1
10 days	0/0/0/0/0/0		0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In this primary eye irritation study, the test item was found to be irritant to rabbit eye.

Executive summary:

This is a pre-guideline study. In a primary eye irritation study, 0.1 mL of the test item was placed into the conjunctival sac of the left eye of each animal after gently pulling away the lower lid from the eyeball. The lids were then held together for about one second in order to prevent loss of the test material. The right eye remained untreated and served as a control. The animals were divided in two groups: in Group I (6 animals) the treated eye was not washed after installation while in Group II (3 animals) both the eyes were washed out with lukewarm water at 20-30 seconds following application of the test material. At 24 hours post-instillation, 0.1 mL of 2% fluorescein sodium solution was used for examination. The animals were checked daily for systemic symptoms and mortality for 10 days. The ocular reactions were evaluated at 24, 48 and 72 hours as well as at 7 and 10 days after the instillation of the test item.

 

In the animals of Group I, the following effects were seen: scattered or diffused opacity in cornea, hyperaemia in iris, slight flare in conjunctivae, swelling and ocular discharge. Mydriasis was observed 10 min. after application. It recovered to normal on day 2 to 3. General effects observed in the animals without rinsing included salivation, rhinorrhoea, licks, increased respiration, cyanosis and slight convulsion. These changes appeared 3-4 hours after application and disappeared 6 hours after application. In the animals of Group II, where the eyes were rinsed, no ocular or clinical effects were observed.

 

The following effects were seen in treated, unwashed eyes (Group I): scattered or diffused opacity in cornea, hyperemia in iris, slight flare in conjunctivae, swelling and ocular discarge. No treatment-related findings were obtained in eyes immediately washed after instillation of the test item. The following cholinergic symptoms indicative for organophosphate intoxication were seen in rabbits without eye washing: reduction of pupil diameter (midriasis), salivation, rhinorrhea, increased respiration, cyanosis, slight convulsions. 

The following scores were obtained for Group I animals for conjuctivae redness, chemosis, iris and corneal opacity and the time point mean of 24/48/72 h: (0.33, 1, 1, 1, 1, 1); (0.33, 0.33, 0.33, 0.33, 0.33, 0.33); (0.67, 1, 1, 1, 1, 1); (1, 1, 1, 1, 1, 1).

It can be conluded that the test item is slightly irritating to the eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The information provided below was taken from the original plant protection dossier on the active substance submitted in 2017 for inclusion of the test substance to Annex I of Directive 91/414/EEC and has been previously evaluated in the Draft Assessment Report (DAR) according to the Commission Regulation (EU) No 1107/2009 (2003) (revisioned in the Renewal Assesment Report (December 2018)) and subject to peer review by EFSA and Member States (2006).

 

Skin:

Gehrke (2015): In the present study the skin irritant potential of the test substance was analysed. The EPISKIN-Standard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. The test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after a 15 minutes exposure and 42 hours post incubation period and compared to those of the concurrent negative controls. In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant”

 

Eye: The following studies were assessed in a weight of evidence approach. 

Kato (1984): In an eye irritation study (no OECD guideline) male rabbits received a single application of 0.1 mL of the test substance (purity 90.7%) into the conjunctival sac. At 24 hours 0.1 mL of 2 % fluorescein sodium solution was used for examination. Midriasis was observed 10 min. after application. It recovered to normal on day 2 to 3. General effects observed included salivation, rhinorrhoea, licks, increased respiration, cyanosis and slight convulsion. These changes appeared 3 - 4 h after application and disappeared 6 h after application. In three additional animals, where the eyes were rinsed, no effects on the eyes were observed.

 

Weidmann (2015): 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.

 

Based on the obtained experimental results outlined above, it can be concluded that the test item is slightly irritating to the eye. The CLP-classification to eye irritation category 2 based on the in vivo study (Kato, 1984) is supported by the in vitro result which does not enable any classification to category 1 or non-classification (Weidmann, 2015).

 

A summary of the results of skin and eye irritation studies is presented in Table 1.

 

Table 1: Summary of skin and eye irritation studies

Test / Reference	Key data	CLP
Skin irritation in vitro / Gehrke (2015)	non-irritant (mean relative tissue viability (% negative control)  > 50% (89.8%) after 15 minutes and 42 hours post incubation)	No Category
Eye irritation in vivo / Kato (1984)	slightly irritating	Eye irrit. 2 (H319)*
Eye irritation in vitro / Weidmann (2015)	IVIS: 37.33	No prediction can be made

*This classification is line with the harmonized classification and labelling approved by the European Union (Adaptation to Technical Progress (ATP) 2 to Regulation (EC) No 1272/2008).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

 

Skin: Based on available data on skin irritation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.

 

Eye: Based on available data on eye irritation, the test item is  classified as eye irritation category 2 according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.

 

This classification is line with the harmonized classification and labelling approved by the European Union (Adaptation to Technical Progress (ATP) 2 to Regulation (EC) No 1272/2008).