Fenamiphos

Skin irritation / corrosion

Administrative data

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

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:

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

Version / remarks:

6 July 2012

Deviations:

no

Test guideline 2

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 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

In vitro test system

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

Results and discussion

In vitro

Results

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%).

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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”.