(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14th June 2018 - 17th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

EC Number:

617-116-8

Cas Number:

80573-04-2

Molecular formula:

C17 H15 N3 O6

IUPAC Name:

(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

Test material form:

solid

Specific details on test material used for the study:

Batch: 0400

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue lot number: 28623

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were gently rinsed about 20 times with PBS (phosphate buffered saline). Excess PBS was removed by gently shaking the insert and blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of pre-warmed MTT solution was used
- Incubation time: 3 hours
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Concentration (if solution): 25 mg + 25 μL H2O

NEGATIVE CONTROL
- Concentration (if solution): 50 μL distilled water

POSITIVE CONTROL
- Concentration (if solution): 50 μL 8 N KOH

Duration of treatment / exposure:

3 min and 60 min exposure time

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes treatment

Value:

112.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes treatment

Value:

84.8

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:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: The test material showed no non-specific MTT- reducing.
- Colour interference with MTT: The test item showed water-colouring potential in Aqua dest. and in isopropanol in the range of 570 ± 30 nm. Therefore, additional viable tissue controls were treated with the test item to determine the non-specific colour (NSCliving) and the results were corrected to the true MTT metabolic conversion (TODTT).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - mean absolute OD570 nm for 3 min experiment: 1.651; mean absolute OD570 nm for 60 min experiment: 1.896
- Acceptance criteria met for positive control: Yes - mean relative tissue viability for 60 min experiment: 6.6%
- Acceptance criteria met for variability between replicate measurements: Yes - CV: 4.4 - 12.8%

Any other information on results incl. tables

Table 1: Results of 3 min experiment

Name	Negative control		Test item		Positive control
Tissue	1	2	1	2	1	2
Absolute OD570	1.585	1.687	1.926	1.819	0.192	0.161
	1.596	1.716	1.920	1.782	0.195	0.161
	1.617	1.705	1.896	1.805	0.195	0.168
OD570 - Blank Corrected	1.540	1.642	1.882	1.774	0.148	0.116
	1.551	1.671	1.875	1.737	0.150	0.116
	1.572	1.660	1.851	1.760	0.150	0.123
Mean OD570 of 3 Aliquots (Blank Corrected)	1.554	1.658	1.869	1.757	0.149	0.118
SD OD570 of 3 Aliquots	0.016	0.015	0.016	0.019	0.002	0.004
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.606*		1.813		0.134
TODTT			1.813
SD OD570 of 2 Replicate Tissues	0.073		0.080		0.022
Mean Relative Tissue Viability [%]	100.0		112.9		8.3
Mean Relative Tissue Viability [%] - NSCliving Corrected	-		112.9		-
Coefficient Of Variation [%]***	4.6		4.4		16.3

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is

≤ 30%.

Table 2: Results of 60 min experiment

Name	Negative control		Test item		Positive control
Tissue	1	2	1	2	1	2
Absolute OD570	2.007	1.778	1.747	1.476	0.187	0.148
	1.996	1.803	1.760	1.475	0.187	0.151
	1.987	1.803	1.763	1.468	0.186	0.148
OD570- Blank Corrected	1.962	1.733	1.702	1.431	0.142	0.104
	1.951	1.758	1.715	1.430	0.142	0.106
	1.942	1.759	1.719	1.423	0.141	0.104
Mean OD570of 3 Aliquots (Blank Corrected)	1.952	1.750	1.712	1.428	0.142	0.105
SD OD570of 3 Aliquots	0.010	0.015	0.009	0.004	0.001	0.002
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.851		1.570		0.123
TODTT	-		1.570		-
SD OD570of 2 Replicate Tissues	0.143		0.201		0.026
Mean Relative Tissue Viability [%]	100.0		84.8		6.6**
Mean Relative Tissue Viability [%] - NSClivingCorrected	-		84.6		-
Coefficient Of Variation [%]***	7.7		12.8		21.2

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%,

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is

≤ 30%

Table 3: Result of the NSC_living control at 3 min

NSCliving	TVT		Negative control
Tissue	1	2	1	2
absolute OD570 - values	0.047	0.047	1.585	1.687
	0.049	0.049	1.596	1.716
	0.048	0.048	1.617	1.705
absolute OD570- Blank corrected values	0.002	0.002	1.540	1.642
	0.005	0.004	1.551	1.671
	0.003	0.003	1.572	1.660
mean OD570 (mean of 3 aliquots)	0.003	0.003	1.554	1.658
total mean OD570 (mean of replicate tissues)	0.003		1.606
SD OD570 (of the 2 replicate tissues)	0.000		0.073
NSCliving [%]	0.2		-
Relative Tissue Viability [%]	-		96.8	103.2
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		4.6
CV [% Viabilities]	-		4.6

Table 4: Result of the NSC_living control at 60 min

NSCliving	TVT		Negative control
Tissue	1	2	1	2
absolute OD570 - values	0.048	0.048	2.007	1.778
	0.053	0.050	1.996	1.803
	0.048	0.048	1.987	1.803
absolute OD570- Blank corrected values	0.003	0.003	1.962	1.733
	0.008	0.005	1.951	1.758
	0.003	0.003	1.942	1.759
mean OD570 (mean of 3 aliquots)	0.005	0.003	1.9552	1.750
total mean OD570 (mean of replicate tissues)	0.004		1.851
SD OD570 (of the 2 replicate tissues)	0.001		0.143
NSCliving[%]	0.2		-
Relative Tissue Viability [%]	-		105.4	94.6
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		7.7
CV [% Viabilities]	-		7.7

The test item showed no non-specific MTT-reducing but water-colouring potential in Aqua dest. And in isopropanol in the range of 570 ± 30 nm . Therefore, additional viable tissue controls were treated with the test item to determine the non-specific colour (NSC_living) and the results were corrected to the true MTT metabolic conversion (TOD_TT).

  

As NSC_livingwas ≤ 5% relative to the negative control of living epidermis, no correction of the results was necessary.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥50% (112.9%) after 3 min treatment and ≥15% (84.8%) after 60 min treatment.

The controls confirmed the validity of the study. The mean OD_570nmof the two negative control tissues was ≥0.8 and ≤2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (1.896%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤30% (4.4% - 12.8%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no corrosive effects and is therefore classified as 'non-corrosive'.