4-chlorobenzonitrile

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.01.2014 - 28.01.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorobenzonitrile

EC Number:

210-765-4

EC Name:

4-chlorobenzonitrile

Cas Number:

623-03-0

Molecular formula:

C7H4ClN

IUPAC Name:

4-chlorobenzonitrile

Details on test material:

- Name of test material (as cited in study report): 4-Chlorobenzonitrile
- Physical state: solid
- Analytical purity: 99.7 %
- Impurities (identity and concentrations): 0.05 % 2-Chlorobenzonitrile; 0.09 % 3-Chlorobenzonitrile; 0.06 % Chlorobenzaldehyde; < 0.05 % 4-Chlorotoluene, Benzonitrile; 0.1 % another organic impurity max.
- Purity test date: 04.09.2013
- Lot/batch No.: 324601
- Expiration date of the lot/batch: 31.12.2015
- Storage condition of test material: Room Temperature (20 +/- 5 °C)

In vitro test system

Cell type:

non-transformed keratinocytes

Test animals

Species:

other: in vitro test

Strain:

other: human skin model Epiderm TM

Test system

Controls:

not required

Number of animals:

not applicable (in vitro test)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

60 min

Value:

>= 93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

other: not aplicable

Any other information on results incl. tables

1     Findings and Results

1.1     Measured Values

As a blank control, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table 1:      Absorption values of the blank isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorption	0.038	0.037	0.039	0.037	0.035	0.036	0.036	0.036	0.037

The absorption values of negative control, test item and positive control are given in the following table:

Table 2:     Absorption values of the negative control, test item and positive control (OD at 570 nm)     

Designation	Measurement	Negative Control	4-Chlorobenzonitrile	Positive Control
Tissue 1	1	2.124	2.232	0.097
	2	2.308	2.198	0.102
Tissue 2	1	2.144	1.646	0.102
	2	2.139	2.100	0.102
Tissue 3	1	2.350	2.139	0.097
	2	2.282	2.114	0.103

 

From the measured absorptions, the mean of each tissue was calculated, subtracting the mean absorption of isopropanol as given in table 3 Mean and relative standard deviation (comparison of the three tissues) were also calculated.

Table 3    Mean absorption values

Designation	Negative Control	4-Chlorobenzonitrile	Positive Control
Mean – blank (Tissue 1)	2.179	2.178	0.063
Mean – blank (Tissue 2)	2.105	1.836	0.065
Mean – blank (Tissue 3)	2.279	2.090	0.063
Mean of the three Tissues	2.188	2.035	0.064
Relative Standard Deviation of the three tissues	4.0 %	8.7 %	1.8 %

 

1.2     Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table 4     % Formazan Production

Designation	4-Chlorobenzonitrile	Positive Control
% Formazan production (Tissue 1)	99.5 %	2.9 %
% Formazan production (Tissue 2)	83.9 %	3.0 %
% Formazan production (Tissue 3)	95.5 %	2.9 %
% Formazan production Mean	93.0 %	2.9 %

1.3     Assessment and Validity

1.3.1    Irritation Potential of the Test Item

The relative absorbance values were reduced to 93.0 % after the treatment. This value is above the threshold for irritation (50 %).Therefore, the test item is considered as “not irritant”.

1.3.2    Validity and Acceptability

Validity criteria and results are stated in the following table:

Table 5  Validity

Criterion	Demanded	Found
OD of negative control	between 1.0 and 2.5	2.188
% Formazan production of positive control	£20% of negative control	2.9 %
Variation within replicates (RSD)	< 18%	4.0 % (negative control) 1.8 % (positive control) 8.7 % (test item)

 Values for negative control and for positive control were within the range of historical data of the test facility. Therefore the experiment is considered valid.

2 Discussion

The test item is considered as being not irritant.

After the treatment, the relative absorbance values were decreased to 93.0%. This value is well above the threshold for irritation (50%).

 

The optical density of the negative control was well within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control induced a decrease in the relative absorbance as compared to the negative control to 2.9 % (required:£20 %) for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item.

For these reasons, the result of the test is considered valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

4-Chlorobenzonitrile was not irritating in an in vitro skin irritation test (reconstructed human epidermis test method) according to OECD 439 and EU-Method B.46.

Executive summary:

In an in vitro skin irritation test (reconstructed human epidermis test method) according to OECD 439 and EU-Method B.46 three tissues of the human skin model EpiDerm TM were treated with 4-Chlorobenzonitrile for 60 minutes.

In average 25 mg of the solid test item (wetted with 25 µL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control, 5 % SDS-solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritation effects. Variation within tissues was acceptable (< 18 %). After the treatment with the test itrm, the relative absorbance values were reduced to 93.0 %. This value is well above the threshold for irritaion potential (50 %). Therefore 4 -Chlorobenzonitrile is considered as not irritant in the Human Skin Model Test.