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skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-11-24 - 2021-03-01
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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

Chemical structure
Reference substance name:
By-product from Guanidinoacetic acid manufacturing
EC Number:
Molecular formula:
- Hydantoic Acid Sodium salt: C3H5N2O3Na - Cyanoguanidine: C2H4N4 - Sodium acetate: C3H3O2Na - Glycine: C2H5NO2 - N,N'-Guanidinodiacetic acid: C5H9N3O4 - N,N-Guanidinodiacetic acid: C5H9N3O4 - Guanidinoacetic acid:C3H7N3O2 - Urea: CH4N2O - Water: H2O - Biguanidinoacetic acid: C4H10N6O2 - Sodium formate: CHO2Na - Glycylglycine: C4H8N2O3
By-product from Guanidinoacetic acid manufacturing
Test material form:
Specific details on test material used for the study:
201009MA2 solid

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of
normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of
the species of interest and closely mimics the biochemical and physiological properties of the upper
parts of the human skin, i.e. the epidermis.
other: Dulbecco’s phosphate buffered saline (DPBS)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg + 25 µL DPBS
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
ca. 59.2
Vehicle controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In this study under the given conditions the test item showed no irritant effects. The relative mean
tissue viability after 60 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”.
Executive summary:

In the present study the skin irritant potential of By-product from Guanidinoacetic acid manufacturing
was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human
epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) 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 60 min exposure and 42 h post-incubation period
and compared to those of the concurrent negative controls.
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed
per 1 mL MTT medium and incubated for 60 min at 37  1 °C in the incubator. The mixture did not turn
blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were
not necessary.
To check the colouring potential of the test item 25 mg of the test item was mixed per 300 μL aqua
dest. and/or per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for
60 min. The mixture showed no relevant colouring detected by unaided eye-assessment. Thus, the
additional test with viable tissues and the quantitative corrections were not necessary.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after
mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of
possible false-negative results were necessary.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was
> 50% (59.2%) after 60 min treatment and 42 h post-incubation.