Benzoic acid, compound with cyclohexylamine (1:1)

Skin irritation / corrosion

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

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, compound with cyclohexylamine (1:1)

EC Number:

221-516-4

EC Name:

Benzoic acid, compound with cyclohexylamine (1:1)

Cas Number:

3129-92-8

Molecular formula:

C7H6O2.C6H13N

IUPAC Name:

cyclohexanamine benzoate

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

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

87.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

78.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

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.