Registration Dossier

Administrative data

Description of key information

There is one in vitro skin irritation test available and one in vivo eye irritation study both performed on Acid Yellow 23.

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:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
other: reconstructed human epidermis (RhE)
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
- Principle of the assay
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential. Evaluation is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean OD value of the negative control tissues. Skin irritation potential of the test substance is predicted if the remaining cell viability is below 50%.

- Test system
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures. Certificate of quality is given in Annex1. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

Test procedure
Direct MTT reduction - functional check in tubes
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional check for this possibility is performed as follows: the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.

- Colour interference
Test chemical has intensive yellow colour.To identify potential interference by coloured test chemical with OD570 absorbance measuring the spectral analysis of the test chemical in water (environment during exposure) and/or isopropanol (extracting solution) should be performed. If the test chemical in water and/or isopropanol absorbs light in the range of 570±30 nm, further colorant controls should be done. In this study the absorbance in isopropanol was detected. A colour may affect the results only if the test chemical is present in the tissues and tracted with MTT at the same time. Due to exclusion of colour influence two frozen tissue replicates, which undergo the entire testing procedure but are incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCkilled) control. The true tissue viability is then calculated as the percent tissue viability obtained with tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with tissues exposed to the interfering test chemical and incubated with medium without MTT.

MTT test
a) Application: The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 25 µL of PBS. The material is spread on the tissue surface. A single testing, composed of three replicate tissues, was run.
b) Procedure:On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations durable for approximately 1 and 18 hours, tissues are topically exposed to the test chemicals for 1 hour (25 minutes at room temperature and the remaining 35 minutes at 37°C, 5% CO2). Three tissues are used per the test substance, for the positive (PC) and negative (NC) controls. Tissues are then thoroughly rinsed with PBS, blotted to remove the test substances, and transferred to fresh medium. After 24±2 hours post-incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18±2 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg•mL-1). After 3 hour of incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/48/3 (VUOS-CETA, 2011).
c) OD570 measuring:OD570 is measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter is used.
d) Viability calculation: Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit.
Amount/concentration applied:
25 mg of the tested substance
Duration of treatment / exposure:
1 and 18 hours
Number of replicates:
3
Irritation / corrosion parameter:
other: irritating effects
Remarks on result:
no indication of irritation
Remarks:
See attcahed table with results

The tested substance is considered to be non irritant for the skin.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the test results, the tested item is to be considered as non-irritant to human skin..
Executive summary:

The results show no irritation on human skin therefore the tested item is to be considered as non-irritant to skin under CLP classification.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
according to
Guideline:
other: Draize test
Deviations:
yes
Remarks:
no details
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Body weights: 3 - 3.5 kg Source: commercial supplier (Summit View Farm, Hazelton, PA, USA)Acclimation in the test area for one week prior to the start of the trial.Rabbits were individually housed in stainless steel, wire mesh-floor cages in a room with a 12-hr light/dark cycle, maintained at 16-21°C and an average relative humidity of 38%. All animals were supplied with water ad lib; feed (Certified Lab Rabbit Chow HF; Purina No. 5325) was limited to 125g/day. All animals were acclimatized for at least 2 wk prior to the start of the study at which time only healthy animals of the normal weight range found to be free from ocular irritation or damage (i.e. as determined by gross ocular examination, slit-lamp biomicroscope and indirect ophthalmoscope examination) were used for administration of test and control materials. All animals were checked for viability twice daily. Currently acceptable practices of good animal husbandry were adhered to throughout the study (NIH, 1985).
Vehicle:
other: 0.5% (w/v) hydroxypropyl methylcellulose (Methocel 90 HG, premium, 15,000 cps; Dow Chemical, Midland, MI, USA) and 0.25% (w/v) laureth°10 acetate (Solulan 98; Amerchol Corp., Edison, NJ, USA), using sterile technique and materials.
Controls:
other: the right eye serves as control
Amount / concentration applied:
10 µl of a 10% solution of the test substance (test solution: 3 %)The maximum volume capacity of the rabbit conjunctival sac is 10-30 µl (Crai et al., 1973). The maximum volume capacity of the human conjunctival sac with blinking is 10 µl (Ehlers, 1976; Wright and Meger, 1962), whereas a volume of 30µlis considered to be the maximum capacity of the human conjunctival sac in the absence of blinking (Mishima et al., 1966), and therefore represents the maximum likely accidental exposure to finished product applied to the eye area.
Duration of treatment / exposure:
1 seconds
Observation period (in vivo):
up to 21 days
Number of animals or in vitro replicates:
12 (6 males / 6 females)
Details on study design:
All eyes were scored for ocular irritation pretest (8 days, 24 hr and immediately prior to the initial dose) and approximately 24 hr after each treatment, prior to the next instillation of test material; on days 1, 3, 7, 14 and 21, the eyes were also evaluated for irritation 1 hr after treatment.In addition, all readily observable ocular structures were evaluated for eye stain and particle embedment 24 hr after each treatment. The degree of ocular staining was scored (following rinsing of residual material where necessary) and noted. Ophthalmic observations were performed 7 days and 24 hr prior to the initial dose, on days 3, 7 and 14 (prior to daily dosing), and at the end of the study. Both eyes of all animals were examined by slit-lamp bimicroscopy (including examinations of fluorescein stain retention to evaluate integrity of the corneal epithelium). Indirect ophthalmoscopic examinations (using 1% tropicamide as mydriatic) were performed on both eyes.SCORING SYSTEM: The ocular reactions were scored by the criteris of Consumer Product Safety Commission (1972).
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24 h
Score:
0
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Time point:
48 h
Remarks on result:
not measured/tested
Irritation parameter:
conjunctivae score
Time point:
72 h
Remarks on result:
not measured/tested
Irritation parameter:
cornea opacity score
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritation parameter:
iris score
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritation parameter:
chemosis score
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritant / corrosive response data:
Evaluation of eye irritation: All animals were free of significant signs of ocular irritation other than slight conjunctival redness (score of 1) or discharge (score of 1 or 2) seen sporadically in the eyes of both test and control animals throughout the study.Evaluation of eye staining and embedment: All animals were free of significant signs of eye staining and partial embedment.Ophthalmoscopic examinations. Most animals were free of ocular abnormalities in the test eye. One female rabbit in Group III (treatedwith FD & C Blue No. 1) exhibited focal injection of conjunctival blood vessels in the test eye at the end of the study. In the opinion of the study ophthalmologist, this finding did not appear to be of clinical importance.
Other effects:
Body weight: All animals survived and were free of significant clinical signs throughout the study. Most animals gained weight; single animals in both control and treated groups lost weight at one or more intervals but subsequently gained weight by the end of the study. One male rabbit in Group II (treated with FD & C Yellow No. 5) and one male rabbit in Group III (treated with FD & C Blue No.l) exhibited slight net weight losses over the 21 days of study.
Interpretation of results:
GHS criteria not met
Conclusions:
The tested substance needs to be considered not irritant for rabbits eyes under Regulation 1272/2008.
Executive summary:

All animals were free of significant signs of ocular irritation other than slight conjunctival redness (score of 1) or discharge (score of 1 or 2) seen sporadically in the eyes of both test and control animals throughout the study.

The tested substance is considered to be not irritant for the rabbits eyes based on the results under Regulation 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Based on the results of the in vitro test, the tested substance is to be considered as non-irritant to the skin of rabbits.

There is anothe study available for the substance which confirm the absence of irritating effects.

Eye irritation:

The irritation index for the tested substance was found to be 0 for the cornea, for the iris and for the conjunctivae.

Therefore the tested item is to be considered as non-irritant to the eye of rabbits.

Justification for classification or non-classification

Based on CLP citeria:

Skin irritant cat 2:

1) Mean value of ≥ 2,3 - ≤ 4,0 for erythema/ eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions;

or 2) Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling;

or 3) In some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

Eye irritant Cat 2: at least in 2 of 3 tested animals, a positive response of:

1)corneal opacity ≥ 1 and/or

2) iritis ≥ 1, and/or

3) conjunctival redness ≥ 2 and/or

4) conjunctival oedema (chemosis) ≥ 2 5) calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days

Based on the test results, no irritating effects were observed both for skin and for eye.