Dimethyl itaconate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 12 October 2015, Experimental completion date: 19 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP; Relevant and adequate for the endpoint

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:

Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012 ( (EpiSkin™ model))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 26 July 2013 (EpiSkin™ model)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl itaconate

EC Number:

210-519-6

EC Name:

Dimethyl itaconate

Cas Number:

617-52-7

Molecular formula:

C7H10O4

IUPAC Name:

1,4-dimethyl 2-methylidenebutanedioate

Details on test material:

- Name of test material (as cited in study report): DMI
- Substance type: Organic monoconstituent chemical
- Physical state: Colourless liquid (determined by the test facility WIL Research Europe), considered being supercooled
- Analytical purity: 99.6 %
- Isomers composition: Non-chiral molecule
- Lot/batch No.: Acros 2012 – lot A 0313227
- Expiration date of the lot/batch: 12 June 2016
- Storage condition of test material: Room temperature

Test animals

Species:

human

Strain:

other: not applicable, in vitro test using Reconstructed Human Epidermis (EpiSkin™ model)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Not applicable, as a reconstructed human epidermis model was used in vitro.
- EPISKIN Standard Model™ (EPISKIN-SMTM, 0.38 cm², Batch no.: 15-EKIN-041). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: SkinEthic Laboratories, Lyon, France
- Preincubation (tissues): On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 27 hours at 37 °C

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment:
Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door.
Based on laboratory historical data these deviations are considered not to affect the study integrity.
- Temperature (°C): 37.0±1.0 °C (actual range 36.5 - 37.3 °C; Temperature was continuously monitored throughout the experiment.
- Humidity (%): 80 - 100 % (actual range 70 - 91 %; Humidity was continuously monitored throughout the experiment.
- Air: The test atmosphere contained 5.0 ± 0.5 % carbon dioxide; The CO2 percentage was monitored once on each working day.
- Photoperiod: The test was conducted in the dark.

Test system

Type of coverage:

open

Preparation of test site:

other: None, not required for use of the EPISKIN Standard Model. The liquid test item was applied directly on top of the tissue.

Vehicle:

unchanged (no vehicle)

Controls:

other: negative (Phosphate Buffered Saline, PBS) and positive (Sodium Dodecyl Sulphate, CAS 151-21-3) controls with 3 tissues each

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 25 µL of the liquid test item was applied (on 0.38 cm²)
- Concentration: The liquid test item was applied undiluted

VEHICLE
No vehicle was used

Duration of treatment / exposure:

15 min of treatment at room temperature, then rinsing, followed by 42 h of post-incubation period at 37 °C

Observation period:

After a 42 hour post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed. The incubation for the cell viability measurement was made during 3 h.

Number of animals:

The test was performed on a total of 3 tissues (i.e. three replicates of treatment, negative and positive control)

Details on study design:

TEST SITE
- Area of exposure: In the in vitro study 0.38 cm² of the skin tissue (EPISKIN Standard Model™) was exposed in well plates
- % coverage: The skin tissue (EPISKIN Standard Model™) was completely covered by the liquid test item, no wrap was therefore used

REMOVAL OF TEST SUBSTANCE
- Washing: After exposure the skin tissue was thoroughly rinsed to remove the test item and transferred to fresh medium.
- Time after start of exposure: 15 min of treatment at room temperature, then rinsing

SCORING SYSTEM: Cell viability (cytotoxicity) was measured after rinsing, followed by 42 h of post-incubation period at 37 °C. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan (CAS 504-65-4) production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, CAS 298-93-1) at the end of the treatment by OD570 (optical density at 570 nm) determination. Non-irritancy has to be considered according to the test guideline if the treatments show > 50 % of the mean viability of the negative controls.

Results and discussion

In vivo

Irritant / corrosive response data:

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that it did not interact with the MTT endpoint.
The mean absorption (Optical Density) at 570 nm (OD570) measured after treatment and in controls are presented in Table 1. Table 2 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 12 %. The absolute mean OD570 of the negative control tissues, which was within the laboratory historical control data range (Table 3). The standard deviation value of the percentage viability of three tissues treated identically with the positive control was less than 14 %. Thus the test system functioned properly.
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 14 %. Since the mean relative tissue viability was below 50 % it is considered to be irritant or corrosive.

Any other information on results incl. tables

Table 1: Mean absorption

Sample	Optical density at 570 nm [1]
	A	B	C	Mean±SD
Negative control	0.917	0.742	0.977	0.878±0.122
Test item	0.127	0.096	0.145	0.123±0.025
Positive control	0.077	0.134	0.110	0.107±0.028

OD = Optical Density

SD = Standard deviation

The triplicate exposures are indicated by A, B and C.

In the above table the values are corrected for background absorption (0.0415). Isopropanol was used to measure the background absorption.

Table 2: Mean tissue viability

Sample	Mean tissue viability [% of control]
Negative control	100
Test item	14
Positive control	12

In Tables 2 and 3 Skin irritation is expressed as the remaining cell viability after exposure to the test item.

Table 3: Historical Control Data for in vitro Skin Irritation Studies in the Test Laboratory

	Optical density at 570 nm [1]		Viability [%]
	Negative control	Positive control	Positive control
Range	0.576 – 1.352	0.020 – 0.408	2.1 – 43.0
Mean	1.03	0.14	14.8
SD	0.15	0.11	10.7
n	91	91	108

SD = Standard deviation

n = number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of March 2012 to March 2015.

Applicant's summary and conclusion

Interpretation of results:

other: positive answer, indicating either corrosive (UN-GHS Cat. 1) or irritant (UN-GHS Cat. 2) properties

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

In a valid in vitro Reconstructed Human Epidermis test using the EpiSkin™ assay the test item gave a positive answer, which indicates that it is either corrosive (US-GHS Cat. 1) or irritant (US-GHS Cat. 2).

Executive summary:

The in vitro irritation potential of the test item Dimethyl itaconate (CAS 617-52-7, purity 99.6 %) to Reconstructed Human Epidermis tissues (RHE, EpiSkin™ model) was investigated in a GLP-compliant study according to the OECD TG 439 (2013) and EU B.46 (2012) protocols. The experiment can be considered valid, relevant and adequate for final conclusion on the presence or absence of corrosive (UN-GHS category 1) and/or irritant (UN-GHS category 2) properties. Therefore it can be deemed conclusive for presence of absence of non-irritancy and was rated „reliable without restrictions“, i.e. “Klimisch 1” according to the scale of Klimisch et al. (1997).

The test was performed in three replicates employing tissues treated with undiluted test item, Phosphate Buffered Saline (PBS) serving as negative control and 5 % Sodium Dodecyl Sulphate (SDS, CAS 151-21-3) serving as positive control. The equal volume of twenty five μL was added into 12-well plates on top of the skin tissues. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with PBS to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C. After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL Methylthiazoletetrazolium (MTT, CAS 298-93-1)-medium (0.3 mg/mL). The tissues were incubated for 3 hours at 37 °C. After incubation the tissues were placed on blotting paper for drying. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (CAS 67-63-0). Tubes were stored refrigerated and protected from light for 72 hours. The amount of extracted formazan (CAS 504-65-4) was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that it did not interact with the MTT endpoint.

The positive control had a mean cell viability of 12 % and the absolute mean Optical Density at 570 nm (OD570) of the negative control tissues (set to 100 %), which was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the positive control was less than 14 %. Thus the test system functioned properly.

The relative mean tissue viability of the test item treatments compared to the negative control tissues was 14 %. Since the mean relative tissue viability was not equal to or above 50 % it cannot be regarded as an a non-irritant in agreement with the test guidelines.

In result and according to CLP (considered up to the 7th ATP of Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU) this study indicates that the test item is either corrosive (Cat. 1) or irritant (Cat. 2). To finally conclude on classification a further study designed to distinguish between corrosive and irritant effects (e.g. OECD TG 431) is required.

• Klimisch HJ, Andreae M, Tillmann U (1997). A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data. DOI 10.1006/rtph.1996.1076 PMID 9056496 Regul Toxicol Pharmacol 25:1-5.