Carboxylic acids, C5-9

Endpoint summary

Description of key information

Two recent in vitro experimental studies carried out on the target substance according to the ECHA guidelines for skin irritation/corrosion are available (OECD 431 and OECD 435).

The results of the two studies are in contrast:

1) Corrosion category 1A for the study according to OECD 431;

2) Non-Corrosive for the study according to OECD 435.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 July 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Cell source:

other: commercial reconstructed human epidermis (RhE) model, EPISKIN™. from human derived epidermis keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

MTT method: The test system EPISKIN™is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The principle of the RhE test method is based on the premise that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS N. 298-93-1] into a blue formazan salt that is quantitatively measured after extraction from tissues. Corrosive chemicals are identified by their ability to decrease cell viability below defined threshold levels.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL per well

Duration of treatment / exposure:

Exposure times of 3, 60±5 and 240±5 minutes were allowed in a ventilated cabinet at room temperature

Duration of post-treatment incubation (if applicable):

Plates were incubated for 3 hours ± 5 minutes at 37°C, 5% CO2 and saturated humidity

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 3 minutes of exposition

Run / experiment:

Main test

Value:

ca. 15

Negative controls validity:

valid

Positive controls validity:

valid

TABLE 1 - Preliminary Test

Direct MTT reduction test (Step1) 

Test item (mg)	MTT ready to use solution(mL)	Container	Incubation condition	Colour observation
50µL	2.0	well	3h at 37°C, 100% nominal humidity, 5% CO2	Opaque yellow solution with oily yellow drops and crystalline precipitate (no interaction)

 

 Colouring potential test (Step 2) 

Test item (mg)	Water (µL)	Container	Incubation condition	Colour Observation
10 µL	90	Eppendorf tube	15’, ambient condition, in agitation	Colourless solution (no interaction)

TABLE 2 - Main Assay - TREATMENT TIME: 3 minutes

			Test Item
							ODTI		Viability(%)
			TI1A-1 TI1A-2	0.146 0.154	0.1083 0.1163		0.1123		14.5
			TI1B-1 TI1B-2	0.153 0.162	0.1153 0.1243		0.1198		15.4

 

 

Mean	0.116	Mean	0.116		15
SD	0.007
CV(%)	5.6			∆(%)	6.5

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

The test item induced cell death at all treatment times. Each mean cell viability, after the concurrent blank subtraction, was as follows:

Treatment time (minutes) Mean cell viability (%)
 3  15
 60 1
  240  2
Intra-replicate variability was acceptable with a difference of viability between the two replicates lower than 30%, for all treatment times.
Based on the results obtained, the test item MATRILOX IL001M is identified as corrosive to the skin (UN GHS Sub-category 1A).

Executive summary:

The potential of the test item MATRILOX IL001M to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. A preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. No interaction was recorded between the test item and MTT in test conditions similar to those of theMain Assay. In a second step, the test item was assayed for the ability of colouring water per se. No colouring potential was noted. Based on these results, no additional control were included in the main experiment. In theMain Assay, for each treatment time, the test item (physical state: liquid) was applied as supplied in two replicates, at the treatment level of 50 μL/epidermis unit, each measuring 0.38cm2 (treatment level: 131.6 μL/cm2). Positive and negative controls (Glacial acetic acid and Physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 μL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

In the Main Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability. The positive control caused the expected cell death (1% of cell viability, when compared to the negative control). Based on the stated criteria, the assay was regarded as valid.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

artificial membrane barrier model

Remarks:

CORROSITEX® is composed of two components, a synthetic macromolecular bio-barrier and a CDS (Chemical Detection System)

Vehicle:

unchanged (no vehicle)

Details on test system:

CORROSITEX® is composed of two components, a synthetic macromolecular bio-barrier and a CDS (Chemical Detection System)
Supplier in vitro INTERNATIONAL (17751 Sky Park East, Ste. G. Irvin - California 92614 - US), Batch CT061118

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Valeroyl chloride (classified as GHS 1B substance, Fluka, batch no. BCBK1876V)) and 10% (w/v) citric acid solution (citric acid Sigma, batch no. SZBF3230V diluted in sterile water for injection (Baxter, batch no. 19B0101)).were concurrently tested as positive and negative controls.

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

4

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

Main experiment (Samples 1-4)

Value:

> 65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The assay was considered valid if the following criteria were met:
– the positive control should give the expected penetration response time (historical mean value + 3 SD), based on ERBC historical control values (13.13 ± 6.48 minutes; n= 26).
– the negative control should not be corrosive within 60 minutes.

Preliminary test

Results of Qualification and Categorisation tests are shown in Table 1. The test item is suitable for this test system since orange colour was recorded after adding the test item to the “Qualify” tube.

The time category was 2 since Tube A content changed the colour to pink apricot. In order to confirmthis result, pH misurement was performed. The pH value of test item solution in water (10% v/v) was 3.55 (lower than 7), hence 150 µL of the test item was added to tube A. A pH value of 6.15 was obtained, confirming a time scale category 2.

Means and standard deviations of CORROSITEX® Time were calculated for replicates of the same sample. Cut-off time values for each of the three corrosive subcategories are

established as follows:

Time category	CORROSITEX® Time (minutes)
Category 2	0 to 3	>3 to 30	>30 to 60	>60
Corrosivity category	GHS 1A (Packing Group I)	GHS 1B (Packing Group II)	GHS 1C (Packing Group III)	Non corrosive

Main Assay

The results of theMain Assay are shown in the table herein below. The test item did not show the ability to penetrate the biobarrier during the whole observation interval.

Negative control was not corrosive within 60 minutes. The UN GHS prediction for the positive control was as indicated in the relevant Guideline (Corrosive Sub-Category 1B)

and the penetration response time (13.4 minutes) was coherent with the historical control range.

Results:

Test or control item	Sample Code	Chemical/Physical changes	Breakthrough observation time (min)
			Values observed	Mean ± SD
Test Item	1	No change	> 65.00	Not applicable
Test Item	2	No change	> 65.00	Not applicable
Test Item	3	No change	> 65.00	Not applicable
Test Item	4	No change	> 65.00	Not applicable
UN/GHS Prediction	Non Corrosive
Valeroyl Chloride	+	Colour change	13.4	Not applicable
10% Citric Acid	-	No change	>60.00	Not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

The skin corrosion potential of the test item MATRILOX IL001M was assessed with an in vitro membrane barrier assay, using the validated commercial kit CORROSITEX®.
The test item did not show the ability to penetrate the biobarrier during the whole observation interval. Positive and negative control results met the acceptance criteria indicating a good functioning of the test system.
According to OECD guideline for testing of chemicals No. 435, MATRILOX IL001M is classified as non-corrosive to the skin, based on UN GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Justification for type of information:

According to the avilable studies according to OECD 431 and OECD 435 and the ones available on the substance constituents, this substance is classified as corrosive to the skin

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

	Component	Typical Value (%)	Range (%)	Skin corrosion/irritation classification in the relative REACH registration dossier
1	Nonan-4-olide	6	0 - 10
	EC: 203-219-1			Not classified
	CAS: 104-61-0
2	2-pentylcyclopentan-1-one	1	0 - 5
	EC: 225-392-2			Not classified
	CAS: 4819-67-4
3	6-butyltetrahydro-2H-pyran-2-one	0,7	0 - 2
	EC: 221-974-5			Not classified
	CAS: 3301-94-8
4	Valeric acid	7	3   20
	EC: 203-677-2			Skin corrosion 1B
	CAS: 109-52-4
5	Heptanoic acid	10	3  20
	EC: 203-838-7			Skin corrosion 1B
	CAS: 111-14-8
6	Hexanoic acid	18	5 20
	EC: 205-550-7			Skin corrosion 1C
	CAS: 142-62-1
7	Octanoic acid	34	15 - 80
	EC: 204-677-5			Skin corrosion 1C
	CAS: 124-07-2
8	Nonanoic acid	24	15 - 80
	EC: 203-931-2			Skin Irritant 2
	CAS: 112-05-0

Justification for classification or non-classification

In consideration of the different results of the two carried out studies on skin corrosion, the classification of the substance was calculated according to the chemical composition, using the in vivo data available in the art for the constituents (carboxylic acids from C5 to C9, lactones).

According to the data reported in the table herein above, the classification of the substance is: Skin corrosion, category 1B.