Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July-August 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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Name: Cyclohexylamine Benzoate
Chemical Name: Benzoic acid, compound with cyclohexylamine (1:1)
CAS No.: 3129-92-8
Batch No.: L15CHAB26
Aggregate State at RT: solid (powder)
Colour: off-white
Chemical Formula: C7H6O2.C6H13N
Active Components: Cyclohexylamine: ~50%
Benzoic Acid: ~50%
Purity: Based on GC, LC and NMR: 97%
CoA of original supplier: 99.74%
Storage Conditions: room temperature
Expiry Date: not applicable; chemical was retested in March 2018
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell)
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
25 mg (39 mg/cm2) of the test item were applied directly atop the EpiDerm tissue
Duration of treatment / exposure:
After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min.
Duration of post-treatment incubation (if applicable):
The plates were post-incubated at 37 °C, 5.0% CO2, humidified to 95%, for 24 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2h.
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
87.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
78.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
80.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test met the acceptance criteria:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
-standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item, cyclohexylamine benzoate, 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 Cyclohexylamine Benzoate was analysed.

The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used 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.

The test item was applied topically. Cytotoxicity was 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.

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of the test item per 300 µl aqua dest. and/ per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (82.0%) after 60 min treatment and 42 h post-incubation.