Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start and completion dates: 19 January 2021 to 5 February 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the testing of Chemicals No. 431 In Vitro Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (adopted: 18 June 2019)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, of 18 December 2006, of the European Parliament and of the Council on the Registration, Evaluation, Authorisation a

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethane-1,2-diol

EC Number:

203-473-3

EC Name:

Ethane-1,2-diol

Cas Number:

107-21-1

Molecular formula:

C2H6O2

IUPAC Name:

ethane-1,2-diol

Constituent 2

Reference substance name:

Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

EC Number:

854-559-8

Cas Number:

2382963-35-9

Molecular formula:

Not applicable for a UVCB substance

IUPAC Name:

Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) Sponsor: Suzuki Techno Commercial Corporation (CoA)
- Lot/batch number of test material: 021015
- Purity: 50% (Aliphatic Carboxylic acids(UB-20) ammonium salts.50wt% and ethylene glycol:50wt%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Assumed stable for the duration of the study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Assumed stable for the duration of the study
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ Human Skin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Source strain:

other: Human skin

Justification for test system used:

The EpiDerm™ Human Skin Model in accordance with OECD 431 (26 June 2020) is an accepted guideline to permit test item classification of corrosivity potential. Increased cytotoxicity being indicative of corrosion potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 2 February 2021
EpiDermTM Tissues (0.63cm2) lot number: 34121
Assay Medium lot number: 012821LHC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

Duplicate tissues were treated with the negative control (sterile distilled water), positive control (Potassium hydroxide) and test item (UB20-LDB(50%)). Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the control items and test item were rinsed from the tissues before each tissue was taken for MTT loading. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction.
At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 50 µL UB20-LDB(50%) in duplicate wells
- Concentration (if solution): Test item used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume): 50 µL Sterile distilled water in duplicate wells
- Lot/batch no. 18L10BA1A

POSITIVE CONTROL
- Amount(s) applied (volume): 50 µL Potassium hydroxide in duplicate wells
- Concentration (if solution): 8.0 N
- Purity: 7.98 mol/L (complying with specification 7.92-8.08 mol/L)
- Lot/batch no. H3410

Duration of treatment / exposure:

Exposure periods of 3 minutes and 60 minutes at 37°C, 5% CO2.

Duration of post-treatment incubation (if applicable):

After the respective exposure periods, plates were rinsed for ca. 40 seconds, blotted and transferred to a 24-well plate for MTT-loading, which were then incubated at 37°C, 5% CO2) for 3-hrs.

Number of replicates:

Duplicate tissues were treated with the negative control, test item and positive control for exposure period of 3 minutes and 60 minutes.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative Mean Viability (%)

Run / experiment:

1 - Test Item - 3 minute exposure

Value:

94.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

1: ≥ 50% after 3 min exposure AND < 15% after 60 min exposure 2: ≥ 25% after 3 min exposure H314: Combination of sub-categories 1B-and-1C

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative Mean Viability (%)

Run / experiment:

1 - Test Item - 60 minute exposure

Value:

9.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

1: ≥ 50% after 3 min exposure AND < 15% after 60 min exposure 2: ≥ 25% after 3 min exposure H314: Combination of sub-categories 1B-and-1C

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This indicated the test item did not reduce MTT.
- Colour interference with MTT: The test item solution was pale yellow in colour. This colour was attributed to the intrinsic colour of the test item. No colour correction tissues were therefore used.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study was conducted in accordance with
current guidelines (OECD 431 and Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008 (REACH)) and to GLP. It is therefore considered that the test facility has technically proficient and appropriately trained personnel to undertake this in vitro test and maintain raw data in accordance with GLP requirements.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD570 of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
- Acceptance criteria met for positive control: Yes, the positive control is acceptable if mean relative tissue viability of the 60 minute exposure is < 15%.
- Acceptance criteria met for variability between replicate measurements: Yes, in the range between 20 and 100% viability, the Coefficient of Variation between tissue replicates was ≤ 30%.

Any other information on results incl. tables

Tissue	Exposure Period	Mean ^OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.746	1.755	0.013	0.7	100^*
		1.764
	60 Minutes	1.807	1.707	0.142	8.3
		1.606
Positive Control	3 Minutes	0.061	0.062	0.001	^na	3.5
		0.062
	60 Minutes	0.054	0.056	0.003	na	3.3
		0.058
Test Item	3 Minutes	1.623	1.665	0.059	3.6	94.9
		1.707
	60 Minutes	0.122	0.157	0.049	31.5 (na)	9.2
		0.192

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C.

Executive summary:

To evaluate the corrosivity potential of the test item, UB20-LDB(50%), an in vitro EpiDerm™ skin corrosion test (EpiDerm™ Human Skin Model) was performed with treatment periods of 3 and 60 minutes. This study was performed to OECD 431, Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008 (REACH) and to GLP. In this test corrosion is directly related to cytotoxicity in the EpiDerm™ tissue with results used to make a prediction of corrosivity potential. 

Checks to confirm the test item did not reduce MTT or its colour interfere with MTT assesment were performed. Both had no impact on MTT assessment. 

In the main test, duplicate tissues were treated with the negative control (sterile distilled saline), positive control (8N Potassium Hydroxide) and test item (UB20-LDB(50%)) for exposure periods of 3 and 60 minutes.  Post-exposure, control and test item tissues were rinsed before each tissue was taken for MTT‑loading.  After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a 96‑well plate.  The optical density (OD) was measured at 570 nm (OD₅₇₀).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.5	94.9
60 minute	100*	3.3	9.2

Based on the above results, the criteria required for acceptance of results were satisfied. The classification of corrosivity potential is based on relative viabilities for both exposure times.

Under the experimental conditions, the test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C.