3-[(4-aminobenzoyl)amino]propanoic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th April 2018 to 23rd May 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July 2016

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

May 30, 2008

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for : In Vitro EpiDermTM Skin Corrosion Test (EIP-200-SCT)

Version / remarks:

07/11/2014

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-[(4-aminobenzoyl)amino]propanoic acid

EC Number:

616-017-7

Cas Number:

7377-08-4

Molecular formula:

C10 H12 N2 O3

IUPAC Name:

3-[(4-aminobenzoyl)amino]propanoic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PharmaZell, batch no. 12/18
- Expiration date of the lot/batch: not provided
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble. Assumed stable within the short time-frame of the test
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: notne
- Final preparation of a solid: none

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek

Details on test system:

SKIN DISC PREPARATION
- Procedure used: skin supplied in ready-to-use kits. The tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± 1°C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C for both the 3 minute and 60 minute tests

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20 times for both 3 minute and 60 minute tests
- Observable damage in the tissue due to washing: non specified
- Modifications to validated SOP: none stated

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] : MTT
- Spectrophotometer: plate
- Wavelength: 570 nm
- Filter: not stated
- Filter bandwidth: not stated

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL H2O

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N KOH

Duration of treatment / exposure:

3 mins and 60 minutes

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2

Test animals

Species:

other: Not applicable

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of replicates 1+2 in the 3 minute test

Value:

98.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of replicates 1+2 in the 60 minute test

Value:

96.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: test item did not cause direct MTT reduction
- Colour interference with MTT: none


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline:
OD570 for negative control (3 minute test) - mean 1.895, with SD 0.313 (10 samples)
OD570 for negative control (60 minute test) - mean 1.867, with SD 0.261 (11 samples)
Historical control data were generated from 2015-2016

Any other information on results incl. tables

Pre-experiments

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

The mixture of 25 mg test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

Experiment

3 minute experiment

Name	Negative Control		Test item		Positive control
Tissue	1	2	1	2	1	2
Absolute OD570	1.736	1.769	1.697	1.722	0.080	0.083
	1.713	1.715	1.726	1.735	0.079	0.081
	1.770	1.762	1.724	1.725	0.084	0.088
OD570(Blank Corrected)	1.691	1.724	1.652	1.678	0.035	0.039
	1.669	1.670	1.682	1.691	0.035	0.036
	1.725	1.718	1.680	1.680	0.040	0.044
Mean OD570of 3 aliquots (Blank corrected)	1.695	1.704	1.671	1.683	0.037	0.040
SD OD570of 3 aliquots	0.028	0.030	0.016	0.007	0.003	0.004
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.700*		1.677		0.038
SD OD570 of 2 Replicate Tissues	0.006		0.008		0.002
Mean Relative Tissue Viability [%]	100.0		98.7		2.2
Coefficient Of Variation [%]***	0.4		0.5		5.1

*corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

60 minute experiment

Name	Negative Control		Test item		Positive control
Tissue	1	2	1	2	1	2
Absolute OD570	1.786	1.922	1.867	1.713	0.121	0.145
	1.780	1.887	1.854	1.716	0.120	0.139
	1.770	1.923	1.827	1.693	0.126	0.146
OD570(Blank Corrected)	1.741	1.877	1.822	1.668	0.076	0.100
	1.734	1.842	1.809	1.671	0.075	0.093
	1.725	1.878	1.782	1.648	0.080	0.100
Mean OD570of 3 aliquots (Blank corrected)	1.733	1.865	1.804	1.662	0.077	0.098
SD OD570of 3 aliquots	0.008	0.021	0.021	0.012	0.003	0.004
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.799*		1.733		0.087
SD OD570 of 2 Replicate Tissues	0.093		0.101		0.015
Mean Relative Tissue Viability [%]	100.0		96.3		4.9**
Coefficient Of Variation [%]***	5.2		5.8		16.9

*corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

**mean relative tissue viability of the 60 min positive control < 15%,

***coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

non-corrosive

Conclusions:

The test item is classified as “non-corrosive“.