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

Endpoint summary

Description of key information

OECD Guideline in vitro/ in chemico studies have been performed to address each of the key events of skin sensitisation (molecular interaction with skin proteins, inflammatory response in keratinocytes and activation of dendritic cells). The results of these tests are considered valid and all tests concluded that 4-aminobenzoyl-b-alanine is not a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th June 2018 to 19th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

January 12, 2013

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Reduction in animal testing by use of a validated non-vertebrate test, in accordance with the requirements to consider non-vertebrate testing for this endpoint, in accordance with the REACH regulation.

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 specified
- Purity test date: not specfied, but CoA states retest data in March 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: assumed to be stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed non-reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.04 was used to correct for the purity of the test item
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: None; stock solution was used directly with peptide solutions

FORM AS APPLIED IN THE TEST (if different from that of starting material) : a solution of the solid was used in the test, and mixed at specified ratios with the peptide solutions

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

Mean Cysteine peptide depletion = 71.18% (SD= 0.06%, CV = 0.08%)
Mean Lysine peptide depletion = 58.47% (SD = 2.23%, CV = 3.81%)
Prediction model 1, mean of peptides depletions = 64.83%

Key result

Run / experiment:

other: 1

Parameter:

other: % Cysteine Peptide depletion

Value:

0.41

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: % Lysine peptide depletion

Value:

1.07

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: Mean of both % peptide depletions

Value:

0.74

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: not stated.

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

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.7290	0.1451	71.19	71.18	0.06	0.08
	4.7400	0.1454	71.12
	4.7210	0.1449	71.24
Test Item	16.6830	0.5110	0.00	0.41	0.57	140.65
	16.3680	0.5014	1.06
	16.5170	0.5060	0.16

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	6.2940	0.2177	56.28	58.47	2.23	3.81
	5.9870	0.2071	58.41
	5.6530	0.1955	60.73
Test Item	14.5170	0.5025	0.22	1.07	0.80	74.95
	14.3800	0.4977	1.16
	14.2860	0.4945	1.81

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July 2018 - 04 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E: In vitro skin sensitisation assays addressing the key event on activtation of dendritic cells on the adverse outcome pathway for skin sensitisation

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Batch no: 12/18

Key result

Run / experiment:

other: CD86 upregulation (%)

Parameter:

other: dendritic cell activation

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD54 upregulation (%)

Parameter:

other: dendritic cell activation

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Evaluation of results:

For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day of on the same day provided that for each run: independent fresh stock solutoins and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.

Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal or greater than 150% at any tested dose at a cell viability of ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a call viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥50% in at least two independent runs.

A negative test result of a test item is only accepted if the cell viability of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accpeted when the test item is tested at the highest soluble concentration (5000 µg/ml for 0.9% NaCl solution; 1000 µg/ml for DMSO or a different organic solvent) even if cell viability is >90%.

Table 1: Results for CD54 and CD86 expression experiment 1

Sample	Conc (µg/ml)	Cell viability (%)			Mean fluorescence intensity			Corrected mean fluorescence intensity		Relative fluorescence intensity (RFI)		Ratio isotype IgG1 to (%)
	-	CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium control		96.6	95.7	95.3	1873	978	521	1352	457	97	91	360	188
Solvent control	0.20%	95.4	96.0	95.6	1899	1016	512	1387	504	100	100	371	198
DNCB	4.00	84.2	84.5	83.9	5529	2516	543	4986	1973	359	391	1018	463
4-Aminobenzoyl b-alanine	1000	95.7	95.2	95.1	1769	984	525	1244	459	90	91	337	187
	833.33	95.4	95.0	95.0	1864	984	553	1311	431	95	86	337	178
	694.44	95.3	95.4	94.6	1912	995	540	1372	455	99	90	354	184
	578.70	95.4	95.3	95.1	1998	994	582	1416	412	102	82	343	171
	482.25	95.8	95.7	95.3	1944	1011	554	1390	457	100	91	351	182
	401.88	95.0	95.2	95.2	2012	988	555	1457	433	105	86	363	178
	334.90	95.7	95.5	95.2	1969	1019	555	1414	464	102	92	355	184
	279.08	95.3	95.5	94.6	1803	991	568	1235	423	89	84	317	174

Table 2: Results for CD54 and CD86 expression experiment 2

Sample	Conc (µg/ml)	Cell viability (%)			Mean fluorescence intensity			Corrected mean fluorescence intensity		Relative fluorescence intensity (RFI)		Ratio isotype IgG1 to (%)
	-	CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium control		96.7	96.1	96.5	1515	1005	559	956	446	84	94	271	180
Solvent control	0.20%	97.0	96.9	96.8	1668	1002	528	1140	474	100	100	316	190
DNCB	4.00	85.1	85.3	84.1	4510	2139	580	3930	1559	345	329	778	369
4-Aminobenzoyl b-alanine	1000	95.6	95.8	95.3	1675	1076	673	1002	403	88	85	249	160
	833.33	95.1	95.8	96.0	1796	1027	569	1227	458	108	97	316	180
	694.44	96.2	96.2	96.3	1638	1040	567	1071	473	94	100	289	183
	578.70	96.7	96.6	96.8	1735	1072	582	1153	490	101	103	298	184
	482.25	97.3	97.2	96.9	1612	975	663	949	312	83	66	243	147
	401.88	96.6	96.9	96.9	1631	1056	567	1064	489	93	103	288	186
	334.90	96.0	96.3	95.7	1651	998	667	984	331	86	70	248	150
	279.08	95.8	96.5	96.5	1592	972	572	1020	400	89	84	278	170

Interpretation of results:

GHS criteria not met

Conclusions:

In the study under the given conditions the test substance did not upregulate the expression of the cell surface markers in at least two independent cell runs. Therefore it is conisdered as a non-sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th June 2018 - 29th June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Reduction in animal testing by use of a validated non-vertebrate test, in accordance with the requirements to consider non-vertebrate testing for this endpoint, in accordance with the REACH regulation.

Specific details on test material used for the study:

Batch no: 12/18

Key result

Run / experiment:

other: Experiments 1 and 2

Parameter:

other: Luciferase induction

Value:

1.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No dose response for luciferase activity induction was observed.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The test meets the acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the thresehold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64µM is between 2 and 8
-the EC1.5 value of the positive control is within two standard devations of the historical mean
-the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Table 1: Results of the Cytotoxicity Measuerment

	Concentration [µM]	Cell Viability [%}
	-	Experiment 1	Experiment 2	Mean	SD
Solvent control		100.0	100.0	100.0	0.0
Positive control	4.00	107.0	99.8	103.4	5.1
	8.00	111.9	102.8	107.3	6.5
	16.00	114.0	107.5	110.7	4.6
	32.00	116.6	106.9	111.8	6.8
	64.00	112.8	96.9	104.9	11.2
Test item	0.98	106.9	101.9	104.4	3.5
	1.95	105.5	96.3	100.9	6.5
	3.91	103.7	102.3	103.0	1.0
	7.81	98.8	99.2	99.0	0.3
	15.63	93.6	102.7	98.1	6.5
	31.25	95.1	93.6	94.4	1.1
	62.50	97.3	93.1	95.2	3.0
	125.00	88.4	89.5	89.0	0.8
	250.00	81.9	85.7	83.8	2.7
	500.00	76.7	69.2	72.9	5.3
	1000.00	66.4	52.3	59.4	10.0
	2000.00	66.3	56.8	61.6	6.8

Table 2: Induction of Luciferase activity experiment 1

Experiment 1	Concentration [µm]	Fold induction					Significance
		Rep 1	Rep 2	Rep 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00	1.25	1.27	1.23	1.25	0.02
	8.00	1.28	1.25	1.58	1.37	0.18
	16.00	1.33	1.40	1.47	1.40	0.07
	32.00	2.02	2.03	2.47	2.17	0.25	*
	64.00	4.42	5.04	6.19	5.22	0.90	*
Test item	0.98	0.98	0.74	0.93	0.88	0.13
	1.95	1.08	0.69	0.80	0.86	0.20
	3.91	0.87	0.77	0.92	0.85	0.08
	7.81	0.89	0.71	0.81	0.80	0.09
	15.63	1.07	0.86	0.86	0.93	0.12
	31.25	0.89	0.70	0.75	0.78	0.10
	62.50	0.95	0.77	0.77	0.83	0.10
	125.00	0.92	0.76	0.86	0.85	0.08
	250.00	1.01	0.81	0.82	0.88	0.11
	500.00	1.00	0.83	0.91	0.91	0.09
	1000.00	0.83	0.71	0.76	0.77	0.06
	2000.00	0.97	0.81	0.99	0.92	0.10

*= significant induction according to Students t-test p<0.05

Table 3: Induction of Luciferase Activity experiment 2

Experiment 2	Concentration [µm]	Fold induction					Significance
		Rep 1	Rep 2	Rep 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00	1.26	1.26	1.31	1.28	0.03
	8.00	1.43	1.34	1.45	1.40	0.06
	16.00	1.92	1.63	1.95	1.83	0.18	*
	32.00	2.37	2.06	2.52	2.32	0.24	*
	64.00	6.03	4.59	6.19	5.60	0.88	*
Test item	0.98	1.47	0.93	0.84	1.08	0.34
	1.95	0.99	0.91	0.83	0.91	0.08
	3.91	1.00	1.01	0.96	0.99	0.02
	7.81	1.10	0.93	1.19	1.07	0.13
	15.63	0.99	0.95	0.96	0.97	0.02
	31.25	1.10	1.03	1.01	1.05	0.04
	62.50	1.05	1.02	1.06	1.04	0.02
	125.00	1.10	1.09	1.09	1.09	0.01
	250.00	1.17	0.96	1.00	1.04	0.11
	500.00	1.09	1.08	1.07	1.08	0.01
	1000.00	1.06	1.02	1.07	1.05	0.03
	2000.00	0.97	0.93	0.99	0.96	0.03

*= significant induction according to Students t-test p<0.05

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test item is not considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

4-aminobenzoyl-b-alanine is not a skin sensitiser based on the negative results of the three recommended in vtro/ in chemico OECD Guideline skin sensitisation studies.