Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP test performed according to OECD guideline.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: crystalline
Details on test material:
- Name of test material : 3-NITRO-1 ,2,4-TRIAZOL-5-0NE (NTO)
- Substance type: energetic explosive
- Physical state: light green to white crystalline solid with no odor
- Purity ca.99%
Specific details on test material used for the study:
Lot number: DDP12K0091-0002
Expiry: 31 December 2022
Purity: 100%

Test animals


Test system

Type of coverage:
other: EPISKIN™ human epidermis skin constructs
Preparation of test site:
other: incubation of at least 24 hours in maintenance medium
unchanged (no vehicle)
The tissues were wetted with 5 μL of distilled water prior to application of the test substance
Amount / concentration applied:
- Amount(s) applied :10 +/- 2mg of the test substance NTO

Duration of treatment / exposure:
15 minutes treatment
Details on study design:
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 +/- 0.5 minutes with the test substance, negative or positive control at room temperature. One additional tissue was dosed with the test substance, NTO, and another tissue dosed with DPBS for the colour controls. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30seconds and then re-spread with a curved flat spatula after 7 minutes application time.
After 15 +/-0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbecco’s Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 +/- 1 hour at 37 +/- 2oC in a humidified atmosphere of 5% CO2 in air. After 42 +/- 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours +/- 5 minutes at 37 +/- 2oC in a humidified atmosphere of 5% CO2 in air. The colour controls were transferred to wells containing 2 mL of assay medium without MTT. At the end of 3 hours +/- 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted for four hours, protected from light. During the four hour extraction, extracts were transported at ambient temperature within the range 19.1 to 20oC, from ERC, Eye, to HRC, Huntingdon. After two hours extraction, at HRC, the extracts were vortexed. After formazan extraction, duplicate 200 uL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.
The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: % tissue viability
ca. 100
Remarks on result:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: no data. Remarks: Conclusion: non-irritant to the skin. (migrated information)

In vivo

Other effects:
There was no change in the colour of the water control after the 15 minute shaking period. The test substance, NTO/water solution was colourless prior to incubation but was pale yellow in colour after incubation. As the test substance had shown colouring of the water, (potential interference with assessment of optical density) colour control tissues were run in parallel with the main tissues but not exposed to MTT.

Any other information on results incl. tables

There was no change in the test substance, NTO/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 +/- 2oC in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

Negative control: the mean absorbance of the triplicate negative control values was 0.916 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 7.6 which was below the maximum value of 18.

Positive control: The percentage mean viability of the positive control was 14.6 ± 4.2 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

EPISKIN™ results: The percentage value for the OD of test substance NTO, linked to colour was 2.074% of the negative control with MTT (Table 1). As this value was below 5%, correction of the test substance OD values was not required.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: OECD GHS
The test substance, NTO, elicited a mean tissue viability of 100.3 ± 2.8% using the EPISKIN™ human epidermis skin constructs and therefore was predicted as non-irritant to the skin.