Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 944-073-5 | CAS number: -
Table 1: Optical density and viability
NC - negative control
PC - positive control
C4 - test substance
SD - standard deviation
SDS - sodium dodecyl sulphate
The test substance, Acid Brown 235, was assayed for in vitro skin irritation in the human epidermal model EpiDerm.The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and protocol for: In Vitro EpiDerm Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.
In preliminary experiment neither colour interference with the endpoint nor direct MTT reduction were found.
After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.
After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.
Under the experimental design average viability of treated tissues was 94.0%, i.e. viability was >50 %.
The effect of the test substance was negative in EpiDerm model (tissues were not damaged).
According to the classification criteria, thetest substance, Acid Brown 235, is considered to have no category in regard to skin irritation.
Table 1: Opacity values
Opacity after treatment
Mean Opacity (corrected)
(20% Imidazole in 0.9% NaCl)
(52.00 – 1.00)
(Acid Brown 235)
The test substance, Acid Brown 235, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 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, Adopted 26th July 2013.
The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used. Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.
The In Vitro Irritancy Score (IVIS) for test substance, Acid Brown 235, could not be calculated. The basic values for the IVIS calculation (opacity values) could not be measured because of colouring of corneas by the test substance which were applied. For this reason the classification was not performed.
Table 1: Optical density results
NC - negative control
The test substance, Acid Brown 235, was assayed for the in vitro eye irritation in human epidermal model EpiOcular. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).
After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Two tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which was undergo the entire testing procedure excepting of incubation with MTT medium.
After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.
Tests for colour interference and direct reduction were performed as a part of another study. Direct reduction or colour interference were not found.
Under the experimental conditions, average viability of treated tissues was 4.6 % i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcular model (tissues were damaged).
According to the classification criteria, the test substance, Acid Brown 235, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
The test substance, Acid Brown 235, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).
The test was performed according to EU Method B.5 (Acute Eye Irritation/Corrosion), Commission Regulation (EU) 2017/735, published in O.J.L. 112, 2017.
Before in vivo testing, the sequential testing strategy as it is recommended in supplement to TG 405 (2012) was respected.
The test was performed initially using one animal. As no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals.
The following changes were observed on eye at 1 hour after application: conjunctivae – diffuse, crimson colour, individual vessels not easily discernible, and chemosis – obvious swelling with partial reversion of lids was observed in all rabbits.
Some hyperaemic blood vessels of conjunctivae were observed in one rabbit at 24 hours after application. No alterations were observed in the other rabbits at 24 hours after application. No alterations were observed in all rabbits at 48 and 72 hours after application.
No clinical signs of systemic intoxication were detected.
Evaluation of results after single application demonstrated that the test substance, Acid Brown 235, is not irritating to the eye of rabbit. The initial irritation (1 hour after exposure) is caused by mechanical irritation effect of test substance petty particles.
Based on the test results and according to the CLP criteria for classification and labelling requirements for dangerous substances and preparations, the test substance, Acid Brown 235, did not fall into any of quoted categories of skin or eye irritation/corrosion, and has no obligatory labelling requirement in this respect.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
Welcome to the ECHA website. This site is not fully supported in Internet Explorer 7 (and earlier versions). Please upgrade your Internet Explorer to a newer version.
Close Do not show this message again