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Description of key information

In vitro skin and eye studies were performed in accordance with GLP and the recommended guidelines.

The results of the skin corrosivity and irritation studies showed that 4-aminobenzoyl-b-alanine is not corrosive or a skin irritant.

The results of an in vitro eye study were unable to predict the classification of 4-aminobenzoyl-b-alanine with regards to irritancy. However, it can be concluded that 4-aminobenzoyl-b-alanine is not corrosive to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
29 July 2016
Deviations:
not specified
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
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)
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

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
Species:
other: Not applicable
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
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

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%

Interpretation of results:
GHS criteria not met
Remarks:
non-corrosive
Conclusions:
The test item is classified as “non-corrosive“.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22nd May 2018 to 17th July 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)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6th July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: L'Oreal Standard Operating Procedure:"EpiSkin Test Method";-ECVAM Skin Irritation Validation Study- Validation of the EpiSkin Test Method for the Prediction of Acute Skin Irritation of Chemicals
Version / remarks:
Version 1.8, Fen-2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM DB-ALM Protocol No. 131 "EpiSkin Skin Irritation test
Version / remarks:
9th June 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PharmaZell, Lot No. 12/18
- Expiration date of the lot/batch: retest date Sept 2019
- 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: not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not assessed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
None



Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Details on animal used as source of test system:
Not applicable
Details on test system:
SKIN DISC PREPARATION
- Procedure used: Upon receipt of the EPISKIN-SM, the transport and the temperature indicator were inspected. The tissues were transferred into 12-well plates containing 2 mL pre-warmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for at least 24 h

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: not specified
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] MTT
- Spectrophotometer: Yes (plate)
- Wavelength: 570 nm
- Filter: Yes
- Filter bandwidth: maximum ± 30 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: three replicates.

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): 10 ± 2 mg + 5 µL aqua dest.

VEHICLE
- Amount(s) applied (volume or weight with unit): None

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 0 µL DPBS for negative control


POSITIVE CONTROL (SDS)
- Concentration (if solution): 10 µL 5% SDS solution
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h.
Number of replicates:
3
Species:
other: not applicable
Type of coverage:
other: Not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
100.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
104.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
109.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: none
- Colour interference with MTT: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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:
with reference to section 7.2 of SOP of L’Oréal (IN VITRO SKIN IRRITATION TEST: HUMAN EPIDERMIS MODEL EPISKIN™, Feb 2009, based on a 95% confidence interval, application of 2xSD to the historical mean shows that the negative control mean values in the study are not below the lower boundary of this confidence interval

Name

Negative Control

Positive Control

Test Item

Tissue 

1

2

3

1

2

3

1

2

3

Absolute OD570

0.704

0.681

0.737

0.103

0.095

0.088

0.732

0.753

0.749

0.715

0.750

0.738

0.114

0.099

0.091

0.720

0.745

0.819

OD570(Blank Corrected)

0.660

0.637

0.694

0.059

0.052

0.044

0.689

0.710

0.705

0.671

0.707

0.695

0.071

0.056

0.048

0.677

0.702

0.775

Mean OD570 of the Duplicates (Blank corrected)

0.666

0.672

0.694

0.065

0.054

0.046

0.683

0.706

0.740

Total Mean OD570 of 3 Replicate Tissues (Blank Corrected)

0.677*

0.055

0.710

SD OD570

0.015

0.009

0.029

Relative Tissue Viability [%]

98.3

99.2

102.5

9.6

7.9

6.8

100.8

104.2

109.3

Mean Relative Tissue Viability [%]

100.0

8.1**

104.8

SD Tissue Viability [%]***

2.2

1.4

4.3

CV [% Viabilities]

2.2

17.2

4.1

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

**Mean relative tissue viability of the three positive control tissues is ≤ 40%.

***Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Interpretation of results:
GHS criteria not met
Remarks:
Non-irritant
Conclusions:
The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-July 2018 - 04-July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch no: 12/18
Details on test animals or tissues and environmental conditions:
- Tissue source: The assay used isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
750 mg
Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1°C
Number of animals or in vitro replicates:
3 corneas for the test item
3 corneas as negative controls
3 corneas as positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The assay uses isolated corneas obtained äs a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
3 corneas as negative controls treated with physiological saline 0.9% NaCI

POSITIVE CONTROL USED
3 corneas as positive controls treated with imidazole 20% in physioloaical saline 0.9% NaCI

APPLICATION DOSE AND EXPOSURE TIME
750 mg of the test item (open-chamber method) or 750 µL of the control substance

TREATMENT METHOD: Closed chamber

POST EXPOSURE PERIOD: 4 hours ± 5 minutes incubation at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Washed at least 3 times with MEM (containing phenol red).

POST-INCUBATION PERIOD: 90 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry optical density at 490 nm

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) using the following formula: IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: The IVIS cut-off values for identifying test substances as inducing serious eye damage UN GHS Category 1) and test substances not requiring classification for eye Irritation or serious eye damage (UN GHS No Category) was used (see table 1 in materials and methods)
Irritation parameter:
in vitro irritation score
Value:
4.17
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Opacity results

Cornea No.

Test item

Initial opacity

Final opacity

Change of opacity value

Corrected opacity value

1

Negative control

0.69

1.02

0.34

 

2

1.06

0.95

-0.10

 

3

1.50

1.33

-0.17

 

MV

1.08

1.10

0.02

 

4

Positive control

2.03

71.26

69.23

69.21

5

1.96

82.72

80.76

80.74

6

           2.42

65.05

62.62

62.60

MV

2.13

73.01

70.87

70.85

7

Test item

-2.07

0.92

2.99

2.97

8

-1.48

2.57

4.05

4.03

9

1.57

7.13

5.46

5.44

MV

-0.63

3.54

4.17

4.15

MV = mean value

 

Table 2: Permeability results:

Cornea No.

Test item

OD490

Corrected OD490 value

1

Negative control

0.004

 

2

0.001

 

3

0.010

 

MV

0.005

 

4

Positive control

1.132

1.127

5

0.912

0.907

6

1.374

1.369

MV

1.139

1.134

7

Test item

0.013

0.008

8

0.004

-0.001

9

0.002

-0.003

MV

0.006

0.001

MV = mean value

 

Table 3: In vitro irritation score

Cornea No.

Test item

Corrected opacity value

Corrected OD490 value

IVIS

1

Negative control

0.34

0.004

 

2

-0.10

0.001

 

3

-0.17

0.010

 

MV

0.02

0.005

0.10

4

Positive control

69.21

1.127

 

5

80.74

0.907

 

6

62.60

1.369

 

MV

70.85

1.134

87.87

7

Test item

2.97

0.008

 

8

4.03

-0.001

 

9

5.44

-0.003

 

MV

4.15

0.001

4.17

MV = mean value

 

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction can be made regarding the classification of the test substance 4-Aminobenzoyl-b-alanine according to the evaluation criteria. However, it can be concluded that 4-aminobenzoyl-b-alanine is not corrosive to eyes.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The in vitro skin corrosion and irritation studies performed in accordance with OECD Guidelines 431 and 439, respectively, show that 4-aminobenzoyl-b-alanine is not corrosive or irritating to skin. The in vitro eye irritancy screening study performed in accordance with OECD Guideline 437 could not make a prediction regarding the classification of 4-aminobenzoyl-b-alanine for eye irritancy but it can be concluded that it is not corrosive to eyes.

Therefore, in accordance with Regulation (EC) No. 1272/2008, 4-aminobenzoyl-b-alanine does not require classification as corrosive or as a skin irritant.