Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Aug - 13 Oct 2017

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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted Jul 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Deutschland

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

EC Number:

947-727-8

Cas Number:

568591-00-4

Molecular formula:

C9H13N3

IUPAC Name:

Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200, MatTek)
- Tissue batch number: 25834, 25848

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min exposure), 37 ± 1 °C (60 ± 5 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed using a wash bottle containing phosphate buffered saline (PBS) to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
LOT NUMBER: 25834
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.866 ± 0.143 (acceptance criteria: 1.0 - 3.0).
- Barrier function: According to the Supplier`s Data Sheet, the ET50 value was determined to be 6.02 h (Lower acceptance limit ET50 = 4.77 h, Upper acceptance limit ET50 = 8.72 h).
- Morphology: Tissue was inspected visually.
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.

LOT NUMBER: 25848
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.257 ± 0.111 (acceptance criteria: 1.0 - 3.0).
- Barrier function: According to the Supplier`s Data Sheet, the ET50 value was determined to be 7.34 h (Lower acceptance limit ET50 = 4.77 h, Upper acceptance limit ET50 = 8.72 h).
- Morphology: Tissue was inspected visually.
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.

NUMBER OF REPLICATE TISSUES: 2 replicates for each treatment condition (3 min and 60 min experiment)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance directly reduced MTT and showed colouring as compared to the solvent, an additional test was performed.

For quantitative correction of results, the part of absorption due to the non specific reduction of MTT (NSMTT) was determined by using killed tissues.
Therefore two tissues per treatment period were treated with the test substance (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100

NSMTT was ≤ 30% relative to the negative control of living epidermis (5.38% in the 3 min experiment and 4.66% in the 60 min experiment). The true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula:
TODTT = ODTM – (ODKT – ODKU)

- Colour interference with MTT: The test substance mixed with aqua dest. or isopropanol showed colouring and absorbed light in the range of 570 ± 30 nm. Therefore, the tissue staining potential of the test substance was checked by treatment of two additional viable tissues per treatment period with test substance (TVT). For quantitative correction of results, the part of absorption due to non-specific colour (NSCliving) was determined by using MTT-free assay medium for staining according to the following formula:
NSCliving [%] = [ODTVT/ODNK]*100.

NSCliving was ≤ 5% (2.37% on the 3 min experiment and 0.37% in the 60 min experiment) relative to the negative control of living epidermis. Therefore, no correction of the results was necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue 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
- Amount(s) applied: 50 µL undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume): 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

4 tissues per dose group, in duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean relative tissue viability non specific reduction of MTT corrected

Run / experiment:

3 min experiment

Value:

98.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean relative tissue viability non specific reduction of MTT corrected

Run / experiment:

60 min experiment

Value:

65.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct-MTT reduction:
The test substance showed non specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test substance to determine the non-specific reduction of MTT (NSMTT) and the resuts were corrected for the true MTT metabolic conversion (TODTT).

NSCkilled was 1.18% for the 3 min experiment and 0.27% for the 60 min experiment relative to the negative control of living epidermis. Since theNSCliving control was ≤ 5% and no correction was performed, NSCkilled correction to prevent possible double correction was not performed.

- Colour interference with MTT:
The test substance showed water-colouring potential, when mixed with isopropanol in the range of 570 ± 30 nm. Therefore additinal viable controls were treated with test substance to determine the non-specific colour (NSCliving). Since the NSCliving was ≤ 5% (2.37% on the 3 min experiment and 0.37% in the 60 min experiment) relative to the negative control of living epidermis, no correction of the results was necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time thereby confirming the acceptable quality of the tissues.

- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (13.3%) and for the 60 min exposure period (6.3%) thus confirming the validity of the test system and the specific batch of tissue models (acceptance criteria: mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%)

- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (in the range 20 - 100% viability) between tissue replicates was ≤ 30% (3.3% -18.8%), thus the validity of the test system and the specific batch of the tissue models is confirmed.

Any other information on results incl. tables

Table 2: Result of the 3 min experiment

Name	Negative Control		Positive Control		Test Substance
Tissue	1	2	1	2	1	2
OD570values (blank-corrected)	1.689	1.719	0.171	0.279	1.800	1.800
	1.698	1.802	0.169	0.291	1.681	1.866
	1.751	1.742	0.180	0.297	1.721	1.898
Mean OD570of 3 Aliquots	1.713	1.754	0.173	0.289	1.734	1.855
SD OD570of 3 Aliquots	0.034	0.045	0.024	0.025	0.059	0.051
Total Mean OD570of 2 Replicate tissues	1.733*		0.231		1.794
SD OD570of 2 Replicate tissues	0.030		0.082		0.085
Mean tissue viability [%]	100.0		13.3		103.5
Mean tissue viability [%] - NSMTT	-		-		98.1
Coefficient of Variation [%]**	1.7		35.4		4.8

* 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 tissues treated identically is ≤ 30%

NSMTT = non-specific reduction of MTT

OD = optical density

SD = standard deviation

Table 3: Evaluation of the NSMTT Results 3 min

NSMTT	Tissue untreated		Tissue treated with test substance
Tissue	1	2	1	2
OD570values (blank-corrected)	0.028	0.012	0.110	0.113
	0.032	0.009	0.116	0.115
Mean OD570of 2 Aliquots	0.030	0.010	0.113	0.114
Total Mean OD570of 2 Replicate tissues	0.020		0.113
SD OD570of 2 Replicate tissues	0.014		0.001
NSMTT [%]	5.38

NSMTT = non-specific reduction of MTT

OD = optical density

SD = standard deviation

Table 4: Result of the 60 min experiment

Name	Negative Control		Positive Control		Test Substance
Tissue	1	2	1	2	1	2
OD570values (blank-corrected)	1.733	1.653	0.120	0.090	1.361	1.084
	1.840	1.798	0.132	0.100	1.429	1.071
	1.809	1.704	0.1.29	0.093	1.425	1.072
Mean OD570 of 3 Aliquots	1.794	1.718	0.127	0.094	1.405	1.076
SD OD570 of 3 Aliquots	0.055	0.070	0.025	0.024	0.042	0.025
Total Mean OD570 of 2 Replicate tissues	1.756*		0.110		1.240
SD OD570 of 2 Replicate tissues	0.054		0.023		0.233
Mean tissue viability [%]	100.0		6.3***		70.6
Mean tissue viability [%] - NSMTT	-		-		65.9
Coefficient of Variation [%]**	3.1		21.1		18.8

* 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 tissues treated identically is ≤ 30%

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

NSMTT = non-specific reduction of MTT

OD = optical density

SD = standard deviation

Table 5: Evaluation of the NSMTT Results 60 min

NSMTT	Tissue untreated		Tissue treated with test substance
Tissue	1	2	1	2
OD570 values (blank-corrected)	0.005	0.003	0.080	0.090
	0.005	0.002	0.088	0.089
Mean OD570 of 2 Aliquots	0.005	0.003	0.084	0.090
Total Mean OD570 of 2 Replicate tissues	0.004		0.087
SD OD570 of 2 Replicate tissues	0.002		0.004
NSMTT [%]	4.66

NSMTT = non-specific reduction of MTT

OD = optical density

SD = standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive according to OECD 431

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model, but no prediction on the skin irritation potential can be made and additional testing should be conducted for classification and labeling purposes.