1-(2,4,6-trichlorophenyl)acetone O-methyloxime

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-18 2013-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD guideline 439 and in compliance with GLP. All validity criteria were within acceptable limits.

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: COMMISSION REGULATION (EU) No 640/2012 B.46. (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,4,6-trichlorophenyl)acetone O-methyloxime

EC Number:

807-534-0

Cas Number:

1228284-89-6

Molecular formula:

C10H10Cl3NO

IUPAC Name:

1-(2,4,6-trichlorophenyl)acetone O-methyloxime

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Purity test date: 25 September 2013
- Expiration date of the lot/batch: End September 2015
- Stability under test conditions: Stable until end September 2015
- Storage condition of test material: Room temperature (<30°C), protected from light and humidity

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

HUMAN SKIN: EPISKIN® (SM) (Manufacturer: SkinEthic, France, Batch No.: 13-EKIN-045, Expiry Date: 23 December 2013) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. 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. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Test system

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

15 mins

Details on study design:

Disks of EPISKIN (3 units/chemical) were treated with the test item and incubated for 15 mins at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Mean cell viability

Value:

12

Remarks on result:

other:

Remarks:

Basis: mean. Remarks: irritant. (migrated information)

In vivo

Irritant / corrosive response data:

The optical density value for the test item treated skin showed a viability of 12±2.31%. In addition, after MTT reduction, the treated discs became white around the edges.

The mean OD value of the three negative control tissues was 0.740. The positive control result showed a mean of 12% viability. Each standard deviation value (SD) of the % viability was below 18. Control OD values were below historically established boundaries. All validity criteria were within acceptable limits and, therefore, the study can be considered as valid.

As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation. Optical density (measured at 540 nm) of this tissue was determined as 0.018, Non Specific Colour % was calculated as 2.4%. Therefore additional data calculation was not necessary.

No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated % viability of the cells

Substance	Optical density		Viability (%)
Negative control (PBS)	1	0.770	104
	2	0.728	98
	3	0.721	97
	mean ± SD	0.740	100±3.79
Positive control (5% SDS)	1	0.100	14
	2	0.095	13
	3	0.070	9.5
	mean ± SD	0.088	12±2.36
Test item	1	0.107	14
	2	0.071	9.6
	3	0.093	13
	mean ± SD	0.090	12±2.31

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In this in vitro EPISKIN®(SM) model test, the results indicate that the test item is Irritant (UN GHS: Category 2).

Executive summary:

The reconstructed human epidermis model EPISKIN® (SM) is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

Disks of EPISKIN (3 units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Following exposure with the test item, the mean cell viability was 12% compared to the negative control and, therefore, irritant. In addition, after MTT reduction, the treated discs became white around the edges.

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In this in vitro EPISKIN®(SM) model test, the results indicate that the test item is Irritant (UN GHS: Category 2).