Propanamide, N-(aminothioxomethyl)-2-methyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.01.2015 - 19.01.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restrictions: OECD Guideline Study No. 439 ("In vitro Skin Irritation: Reconstructed Human Epidermis Test Method") under GLP.

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:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-Isobutyryl-thiourea

Cas Number:

6965-58-8

Molecular formula:

C5H10N2OS

IUPAC Name:

N-Isobutyryl-thiourea

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): N-Isobutyryl-thiourea
- Physical state: solid
- Storage condition of test material: at room temperature

Test animals

Species:

other: in vitro test

Strain:

other: human skin model EPISKIN-SM TM

Test system

Controls:

not required

Number of animals:

not applicable (in vitro test)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 107.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

ca. 101.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

ca. 97.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

1. Pre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. The mixture of 10 mg of the test item per 90 μL aqua dest. showed no colouring detectable by unaided eye-assessment.

2. Experiment

Table 1: Result of the test with N-Isobutyryl-thiourea

Name	Negative Control (NC)			Positive Control (PC)			Test Item
Tissue	1	2	3	1	2	3	1	2	3
absolute OD550	0.937 0.911	0.923 0.960	0.934 0.937	0.116 0.119	0.129 0.125	0.115 0.110	1.021 0.986	0.968 0.928	0.939 0.881
OD550(blank-corrected)	0.893 0.867	0.880 0.916	0.890 0.893	0.072 0.076	0.085 0.081	0.071 0.066	0.977 0.942	0.925 0.884	0.895 0.837
mean OD550of the duplicates (blank-corrected)	0.880	0.898	0.892	0.074	0.083	0.068	0.960	0.904	0.866
total mean OD550of 3 replicate tissues (blank corrected)	0.890*			0.075			0.910
SD OD550	0.009			0.007			0.047
relative tissue viabilities [%]	98.9	100.9	100.2	8.3	9.3	7.7	107.9	101.6	97.3
mean relative tissue viability [%]	100.0			8.4**			102.3
SD tissue viability [%]***	1.0			0.8			5.3
CV [% viability]	1.0			9.9			5.2

* Corrected mean OD550 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the three positive control tissues is ≤ 40%

*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18 %.

3. Qualitiy Criteria

Table 2: Quality Criteria

	Value	Cut off	pass / fail
Mean OD550 Blank	0.044	< 0.1	pass
Mean Absolute OD550 NC	0.934	0.6 ≤ NC ≤ 1.5	pass
Mean Relative Viability [%] PC	8.4	≤ 40 %	pass
SD of Viability [%]	0.8 – 5.3	< 18 %	pass

4. Historical Data

Table 3: Historical Data

	OD550blank	Absolute OD550NC	Relative Viability [%] PC	SD of Viability [%]
Mean	0.043	0.869	12.50	8.01
SD	0.002	0.122	8.67	8.63
n	44	44	44	176

5. Discussion and Conclusion

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM TM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study N-Isobutyryl-thiourea was applied topically to the EPISKIN-SM TM tissue for 15 min. followed by a 42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (102.3%) after 15 min. treatment and 42 h post incubation. The controls confirmed the validity of the study. The mean OD550 of the six blank values was < 0.1. The mean absolute OD550 of the three negative control tissues was ≥ 0.6 and ≤ 1.5. The mean relative tissue viability (% negative control) of the positive control was 40 % (8.4 %). The maximum standard deviation of the viability of replicate tissues of all dose groups was < 18 % (0.8 - 5.3 %)

Under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

N-Isobutyryl-thiourea was not irritating in an in vitro skin irritation test (reconstructed human epidermis test method) according to OECD 439 and EU-Method B.46.

Executive summary:

In the present study the skin irritant potential of N-Isobutyryl-thiourea was analysed. The EPISKINStandard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post incubation period and compared to those of the concurrent negative controls.

Under the given conditions N-Isobutyrylthiourea showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.