4-chlorobenzonitrile

Endpoint summary

Description of key information

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.
Based on structural and physico chemical similarities (see justification for Read-Across) the result of a study according to OECD Guideline 405 (Acute Eye Irritation / Corrosion) with 2-Chlorobenzonitrile was read-acrossed to 4-Chlorobenzonitrile.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Cell type:

non-transformed keratinocytes

Species:

other: in vitro test

Strain:

other: human skin model Epiderm TM

Controls:

not required

Number of animals:

not applicable (in vitro test)

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for Read-Across:

According to REACH Annex VI and the Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint specific guidance), the registrant should gather and evaluate all available information before considering further testing. These include physic-chemical properties and grouping of chemicals.

One very similar substance for which the eye irritation/corrosion potential was tested in an in vivo test on rabbits is 2-Chlorobenzonitrile. This substance is as well as 4 -Chlorobenzonitrile a chloro substituted Benzonitrile and both substances only differ in the position of the chloro substituent on the benzol ring. This means that there is no additional functional group which could have an influence on the eye irritation/corrosion potential. Also the physical chemical properties are very similar. For example both substances are hardly soluble in water and have similar water octanol partition coefficients (2.2 and 2.1).

Because of this reasons a similar eye irritation/corrosion potential of 4-Chlorobenzonitrile and 2-Chlorobenzonitrile can be assumed.

 

Furthermore the standard information required (column 1 of REACH Annex VII) for eye irritation can be satisfied by following four steps: (1) assessment of the available human and animal data, (2) assessment of the acid or alkaline reserve, (3) in vitro eye irritation study.

Column 2 of REACH Annex VII lists specific adaptation that specify when step 3 in vitro eye irritation testing is not necessary. The first is when the available information already indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes.

 

The study with 2-Chlorobenzonitrile resulted in a the classification “H19: Causes serious eye irritation”. This classification was read-accrossed to substance 4-Chlorobenzonitrile because of an structural similarity and similar physic chemical properties it can be assumed that also 4-Chlorobenzonitrile causes serious eye irritation. According to Column 2 of REACH Annex VII an in vitro eye irritation test with 4-Chlorobenzonitrile was not necessary because the substance could be classified with “H319: Causes serious eye irritation” without any further testing.

Justification for selection of skin irritation / corrosion endpoint:
best study available

Justification for selection of eye irritation endpoint:
best study available

Effects on eye irritation: irritating

Justification for classification or non-classification

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. According to this result the substance does not need to be classified as irritating to skin.

In order to evaluate the endpoint eye irritation/corrosion the result of an OECD 405 study was read-acrossed from 2 -Chlorobenzonitrile to 4 -Chlorobenzonitrile based on structural and physico chemical similarities of both substances (see "Justification for Read-Across"). According to this results 4 -Chlorobenzonitrile was classified with "H319: Causes serious eye irritation".