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

Endpoint summary

Description of key information

Skin: Not classified as corrosive, EpiSkin, OECD 431, Hargitai 2015
Skin: Classified as irritant Category 2, EpiSkin, OECD 439, Hargitai 2015
Eye: Not classified as irritant, ICE, OECD 438, Váliczkó 2015

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:

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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.

Irritation / corrosion parameter:

other: other: Mean cell viability

Value:

12

Remarks on result:

other:

Remarks:

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

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.

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

 

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-02 until 2015-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with OECD guideline 438 and in cimpliance with GLP. The validity criteria have been fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Isolated chicken eye

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

EYES
- Source: Commercial abattoir

EXPERIMENTAL DATES: 2 December 2013

Vehicle:

unchanged (no vehicle)

Controls:

other: Yes (0.9% sodium chloride solution)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

240 minutes. The negative and positive control eyes and all test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

7 isolated eyes (3 test item, 3 positive control, 1 negative control)

Details on study design:

METHOD
- After the zero reference measurements, the eye was held in a horizontal position and 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 μL benzalkonium chloride 5 % (w/v). The negative control eye was treated with 30 μL of sodium chloride (0.9% solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (pitting or loosening of the epithelium) evaluated.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the 10 second exposure period, the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature.

SCORING SYSTEM:
- Corneal thickness or swelling. Measurements made with an optical pachymeter on a slit-lamp microscope and expressed as a percentage calculated from corneal thickness measurements. Corneal swelling was classified by the following scale: Mean cornea swelling (%) of 0-5, >5-12, >12-18 (>75 mins after treatment), >12-18 (<75 mins after treatment), >18-26, >26-32 (>75 mins after treatment), >26-32 (<75 mins after treatment), >32 classified as ICE Class I, II, II, III, III, III, IV or IV, respectively. i.e. No swelling = Class I, slight swelling = Class II, moderate swelling = Class III, severe swelling = Class IV.
- Corneal opacity. Corneal opacity was scored (on a 0-4 scale) using the area of the cornea that was most densely opacified and the mean ΔCOmax (mean value of the highest opacity scores) calculated. Mean maximum opacity scores of 0-0.5, 0.6-1.5, 1.6-2.5 and 2.6-4.0 were classified as ICE Class I, II, III and IV, respectively. i.e. No opacity = Class I, Slight opacity = Class II, Moderate opacity = Class III, Severe or total opacity = Class 4.
- Fluorescein retention: Fluorescein retention change was calculated and scored using a 0-3 scale. Mean fluorescein retention scores of 0-0.5, 0.6-1.5, 1.6-2.5 and 2.6-3.0 were classified as ICE Class I, II, III and IV, respectively. i.e. No fluorescein retention = Class I, slight fluorescein retention = Class II, Moderate fluorescein retention = Class III, Severe fluorescein retention = Class IV.

TOOL USED TO ASSESS SCORE: Hand-slit lamp / fluorescein

Irritation parameter:

other: Mean maximum corneal swelling

Basis:

mean

Time point:

other: up to 75 minutes

Score:

0

Remarks on result:

other: ICE Class I

Irritation parameter:

other: Mean maximum corneal swelling

Basis:

mean

Time point:

other: up to 240 minutes

Score:

0

Remarks on result:

other: ICE Class I

Irritation parameter:

other: Mean maximum corneal opacity change

Basis:

mean

Score:

0.67

Remarks on result:

other: ICE Class II

Irritation parameter:

other: Mean fluorescein retention change

Basis:

mean

Score:

0

Remarks on result:

other: ICE Class I

Irritation parameter:

other: Overall ICE Class

Remarks on result:

other: 2xI, 1xII

Irritant / corrosive response data:

No corneal swelling or fluorescein retention was observed during the four hour observation period. Corneal opacity (severity 0.5 or 1) was noted in all eyes. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid.

The test item is non-irritating, GHS Classification: Non-classified.

The positive control benzalkonium chloride 5% (w/v) was classed as severely irritating, GHS Classification: Category 1.

The negative control Saline (NaCl 0.9% w/v) was classed as non-irritating, GHS Classification: Non-classified.

Table 1: Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change – Run 1

Observation	Test item		Positive control		Negative control
	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0%	I	0%	I	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I	-5.3%	II	0%	I
Mean maximum corneal opacity change	0.67	II	4.00	IV	0	I
Mean fluorescein retention change	0	I	2.83	IV	0	I
Other Observations	None		Loosening of epithelium observed in all eyes (3/3) 75 mins after post-treatment rinse.		None
Overall ICE Class	2xI 1xII		1xII 2xIV		3xI

 

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information: non-irritant

Conclusions:

Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating, GHS Classification: Non-classified.

Executive summary:

An in vitro eye irritation study was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to OECD 438 (2013). After the zero reference measurements, the eye was held in a horizontal position and 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 μL benzalkonium chloride 5 % (w/v). The negative control eye was treated with 30 μL of sodium chloride (0.9% solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (pitting or loosening of the epithelium) evaluated.

No corneal swelling or fluorescein retention was observed during the four hour observation period. Corneal opacity (severity 0.5 or 1) was noted in all eyes. No other corneal effect was observed. The negative and positive control group results demonstrated that the study was valid.

Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating, GHS Classification: Non-classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritation / Corrosion

Two studies are available, both performed in compliance with GLP, using recent guideline methods which are validated for regulatory use:

In order to assess the in vitro skin corrosion potential of the substance, EpiSkin disks (Hargitai 2015) were treated with the test item, negative controls (0.9 % sodium chloride) and positive controls (glacial acetic acid) and incubated for four hours at room temperature (two replicates each). The mean cell viability was 92 % compared to the negative control (100 %) and the positive control (0.7 %). All validity criteria were within acceptable limits and therefore the study can be considered as valid. The substance is not corrosive to the in vitro skin model EpiSkin.

In order to assess the in vitro skin irritation potential of the substance, EpiSkin disks (Hargitai 2015) were treated with the test item, negative controls (PBS) and positive controls (5% SDS) for 15 min (three replicates each). The mean cell viability was 12 ± 2.31% compared to the negative control (100 ± 3.79%) and the positive control (12 ± 2.36%). All validity criteria were within acceptable limits and therefore the study can be considered as valid. The substance is considered irritant to the in vitro skin model EpiSkin.

Based on the available in vitro data which is considered to be adequate, reliable and conclusive for skin irritation/corrosion, the substance is considered to be an irritant. The results is carried forward for the purposes of risk assessment, and classification.

Eye Irritation

In order to assess thein vitroeye irritation potential of the substance, OECD Guideline 438 Isolated Chicken Eye Test Method was carried out by Váliczkó (2015). Simultaneously were performed three tests with the substance, three positive controls (benzalkonium chloride 5 % (w/v)) and one negative control (0.9 % sodium chloride) on isolated eyes. No corneal swelling or fluorescein retention was observed during the four hour observation period. Corneal opacity (severity 0.5 or 1) was noted in all eyes. No other corneal effect was observed. The negative and positive control group results demonstrated that the study was valid.

Based on the available in vitro data which is considered to be adequate, reliable and conclusive for eye irritation, the substance is considered to not be an irritant. The result is carried forward for the purposes of risk assessment, and classification.

Justification for selection of skin irritation / corrosion endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Justification for selection of eye irritation endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin Irritation / Corrosion

Two EpiSkin studies were conducted following OECD guidelines and in compliance with GLP Directive 2004/10/EC. Both results are valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

According to the criteria set in OECD Guideline 439, section 36 - Interpretation of Results and Prediction Model, the test chemical is considered to be irritant to skin under UN GHS Category 2. The result is conclusive.

Eye Irritation

Results from a validated study for eye irritation on Isolated Chicken Eye, conducted according to OECD Guideline demonstrated the substance to be non-irritating to the eye. The conclusion is considered valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

According to the criteria set in OECD Guideline 438, Section 51 - Decision Criteria, Table 6: Overall in vitro classifications, the test chemical is considered not to be classified under UN GHS.The result is conclusive.