Butyl anthranilate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

other information

Study period:

02 August 2016 until 15 August 2016

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Butyl anthranilate

EC Number:

231-816-7

EC Name:

Butyl anthranilate

Cas Number:

7756-96-9

Molecular formula:

C11H15NO2

IUPAC Name:

butyl 2-aminobenzoate

Test material form:

liquid

Specific details on test material used for the study:

Identification: Anthranilsäurebutylester
Batch: 1/12
CAS No.: 7756-96-9
EINECS: 231-816-7
Purity: 99.9%
Appearance: Bright yellow clear liquid
Expiry Date: 02 May 2018
Storage Conditions: At room temperature
Stability in Solvent: Not indicated by the Sponsor
Certificate of Analysis: AZ 1014/Toxd1, dated 2 May 2016

In vitro test system

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL (47 µL/cm exp. 2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue

Duration of treatment / exposure:

60 minutes.

Number of replicates:

3 tissues

Test animals

Species:

other: reconstituted human epidermis model

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
30 µL (47 µL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue for a complete treatment time of 60 minutes.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) were used as negative control per tissue.


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 5% SLS solution in deionised water (MatTek)

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Results and discussion

In vivo

Irritant / corrosive response data:

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 8.8% after exposure of the skin tissues to the test item. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Other effects:

No

Any other information on results incl. tables

Results after treatment with Anthranilsäurebutylester and the controls (60 minutes exposure time):

Dose	Tissue	Absorbance	Absorbance	Absorbance	Mean	Mean
Group	No.	570 nm	570 nm	570 nm	Absorbance	Absorbance
		Well 1	Well 2	Well 3	of	of three well
					3 Wells	blank
	-	-	-	-	-	corrected
Blank	-	0.037	0.038	0.038	0.038	0.000
Negative Control	1	1.770	1.757	1.747	1.758	1.721
Negative Control	2	1.852	1.733	1.745	1.777	1.739
Negative Control	3	1.748	1.700	1.675	1.708	1.670
Positive Control	1	0.119	0.116	0.116	0.117	0.079
Positive Control	2	0.107	0.111	0.107	0.108	0.071
Positive Control	3	0.103	0.104	0.104	0.104	0.066
Test Item	1	0.231	0.225	0.224	0.227	0.189
Test Item	2	0.295	0.290	0.287	0.291	0.253
Test Item	3	0.260	0.261	0.264	0.262	0.224

Dose	Tissue	Mean	Rel. Absorbance*	Relative	Mean Rel.
Group	No.	Absorbance	[%]	Standard	Absorbance
		of 3 tissues	Tissue	Deviation	[% of
		After blank	1, 2 + 3**	[%]	Negative
		correction*			Control]***
Blank	-	-	-	-	-
Negative Control	1	-	100.6	-	-
Negative Control	2	1.710	101.7	2.1	100.0
Negative Control	3	-	97.7	-	-
Positive Control	1	-	4.6	-	-
Positive Control	2	0.072	4.1	9.4	4.2
Positive Control	3	-	3.9	-	-
Test Item	1	-	11.1	-	-
Test Item	2	0.222	14.8	14.3	13.0
	3	-	13.1	-	-

 

*   Mean of three replicate wells after blank correction

**  relative absorbance per tissue [rounded values]:

*** relative absorbance per treatment group [rounded values]:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 13.0% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Anthranilsäurebutylester is irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Anthranilsäurebutylester by means of the Human Skin Model Test.

The test item passed the MTT- and the colour interference pre-tests.

Each 30 µL of the test item, of the negative control (DPBS), and of the positive control (5% SLS) were applied to each tissue and spread to match the surface of triplicate tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following approximately 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD at or higher 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.2% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 15% (threshold of the "OECD Guideline for the Testing of Chemicals 439 :In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative controlthe mean relative absorbance value was reduced to 13.0% after exposure of the skin tissues to the test item. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.