Benzoic acid, compound with cyclohexylamine (1:1)

Endpoint summary

Description of key information

Cyclohexylamine Benzoate showed an average relative viability of 91.5% (3 minute test) and 70.3% (1-hour test) in an in vitro EPIDERM™ skin corrosion test (SCT)  performed to according to OECD TG 431. As the mean tissue viability (%) was ≥ 50% in the 3 min test and and ≥ 15% in the 1 hour test, cyclohexylamine benzoate was considered to be non-corrosive to skin, and this is not classifed for skin corrosive effects.

In an in vitro skin irritation study (EpiDerm) 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"

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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)

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.

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

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%.

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.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April-June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Composition: Cyclohexyamine Benzoate - 99.7%, CAS #3129-92-8
Physical Description: Off-white crystalline powder
pH: ~7 (5% aq solution)
Stability: Test substance was expected to be stable for the duration of testing.
Expiration Date: Not applicable

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

The test system is three dimensional Reconstructed Human Epithelium (RHE) tissue, comprised of non-transformed human-derived epidermal keratinocytes cultured to form a complex model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. Cell viability was measured by dehydrogenase conversion of MTT [(3-4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, thiazol blue], present in cell mitochondria, into a blue formazan salt that was quantitatively measured after extraction.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

50 µL distilled water (negative control) was added to the first negative control tissue insert. After approximately 1 minute, the procedure was repeated with the second tissue.
This process was repeated for the test substance (approximately 25 mg of the test substance wetted with 25 µL of distilled water) and positive control substance (50 µL) at 1 minute intervals.

Duration of treatment / exposure:

Testing was performed on twelve viable tissue inserts, half of which were randomly assigned to either the 3-minute or 1-hour tests.

Duration of post-treatment incubation (if applicable):

Plates were incubated (at ~37°C and 5% CO2) for 1 hour following the time the first tissue was exposed.

Number of replicates:

2 per exposure period (3minutes, 1 hour)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Distilled water (NC) 3 minute test

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Distilled water (NC) 1 hour test

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

8N KOH (PC) 3 minute test

Value:

11.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

8N KOH (PC) 1 hour test

Value:

1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Cyclohexylamine Benzoate 3 minute test

Value:

91.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Cyclohexylamine Benzoate 1 hour test

Value:

70.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For each test substance, negative control, and the positive control, the mean relative viability of each pair of tissues was calculated and used for classification, as follows:
Prediction from Mean Tissue Viability (expressed as % of negative control)
3 min < 50% corrosive
3 min ≤ 50% and 1 hour < 15% corrosive
3 min ≥ 50% and 1 hour ≥ 15% non-corrosive

Interpretation of results:

GHS criteria not met

Conclusions:

Cyclohexylamine Benzoate showed an average relative viability of 91.5% (3 minute test) and 70.3% (1-hour test) in an in vitro EPIDERM™ skin corrosion test (SCT) performed to according to OECD TG 431.
As the mean tissue viability (%) was ≥ 50% in the 3 min test and and ≥ 15% in the 1 hour test, cyclohexylamine benzoate was considered to be non-corrosive to skin, and this is not classifed for skin corrosive effects.

Executive summary:

The in vitro EPIDERM™ skin corrosion test (SCT) was performed using three-dimensional Reconstructed Human Epithelium (RHE) obtained from MatTek Corporation (Ashland, MA).

The assay measured the destruction of the skin tissue (cytotoxicity), one component predictive of skin corrosion. The reduction of the viability of tissues exposed to chemicals in comparison to negative control treated tissue was used to predict the skin corrosivity potential.

Testing was performed on twelve viable tissue inserts, half of which were randomly assigned to either the 3-minute or 1-hour tests. Three groups containing two matrices each; one test substance group, as well as a positive control group (8N KOH) and negative control group (distilled water) were

delineated for each timed test.

The Epiderm™ Tissue inserts were conditioned in DMEM-based assay media under standard CO2 incubation conditions (~37ºC, 5% CO2). One hour after pre-incubation, the DMEM-based assay medium was refreshed and the tissues were exposed to the test and control substances. Twenty-five

milligrams of the test substance wetted with 25 µL of distilled water or 50 µL of each of the negative and positive controls were added to the designated tissue matrices. The tissues were exposed to the substances for either 3 minutes or 1 hour, depending on the assigned test. After the exposure period,

the tissues were rinsed thoroughly with Dulbecco’s Phosphate Buffered Saline (DPBS) and any excess liquid was removed.

Tissues were transferred to wells containing 300µL of MTT medium and incubated for an additional 3 hours at ~37ºC (5% CO2). The tissues were then gently rinsed with DPBS and transferred to a fresh 24-well plate for MTT extraction with isopropanol for 2 hours at room temperature. Inserts were then drained into the well of the plate and duplicate aliquots transferred to a 96-well plate for optical

density measurement.

Cyclohexylamine Benzoate showed an average relative viability of 91.5% (3 minute test) and 70.3% (1-hour test).

The positive control (8N KOH) and negative control (Distilled water) groups demonstrated appropriate effects in order to validate the study.

As per the criteria outlined in the referenced guidelines, the results indicate that Cyclohexylamine Benzoate does not cause skin corrosion.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)