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Administrative data

Description of key information

The in vitro skin irritation showed that the test substance should be considered as a skin irritant. The Isolated Chicken Eye test did not produce irreversible effects on the eye.

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:
29 February - 5 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted by GLP accredited laboratory. Method according to OECD guideline.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
human
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
10µl undiluted test material. The same amount is applied to the positive and negative control.
Duration of treatment / exposure:
15 minutes at room temperature.
Details on study design:
Prior to the analysis, the test substance was checked for possible direct reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; (CAS number 298-93-1) (MTT).

10 µl of the test substance was added to 2.2 ml MTT solution and incubated for 3h at 37°C, 5% CO2. A negative control of distilled water was included.
Three tissues per test were treated with the test material. Three tissues were treated with the negative control and three tissues were exposed to the positive control (5% aqueous solution of sodium dodecylsulphate-SDS CAS 151-21-3). The tissue samples were exposed to the test substance / controls for 15 minutes. After the exposure, the tissues were rinsed with phosphate buffered saline (PBS) and incubated for 42h at 37°C, 5% CO2.
 
After the incubation, the tissues were subsequently incubated for another 3h in the presence of 2.2ml MTT. The formed formazan was extracted with 0.5 ml isopropanol for 2h under agitation in the dark. The amount of Formazan was determined spectrophotometrically (Optical Density - OD) at 570 nm in duplicate. The cell viability was measured against the mean of the negative control.
Irritation / corrosion parameter:
other:
Value:
7.5
Remarks on result:
other:
Remarks:
Basis: mean. Max. score: 100.0. (migrated information)
Irritant / corrosive response data:
The test substance was visually checked for possible direct MTT reduction causing a colour change. No interaction with MTT was observed.
Details of the data and the mean of the spectroscopic measurements can be found in Table 1. Table 2 presents the mean relative tissue viability obtained after 15 minutes treatment with the test substance being the mean of the absorption of the test substance divided by the negative control mean for each application. Since the mean relative tissue viability of the test substance was below 50% after an exposure duration of 15 minutes, the test substance is considered to be irritant.

Table 1  Absorptions (OD570) in the in vitro skin irritation test

15-minute exposure
1 2 3 Mean
Negative control 0.548 0.529 0.507 0.528
Test substance 0.029 0.050 0.040 0.040
Positive control 0.104 0.071 0.076 0.084

Table 2 Mean tissue viability as % of negative control in the in vitro skin irritation

Exposure 15-minutes SD
%
Negative control 100 3.8
Test substance 7.5 2.0
Positive control 15.9 3.4
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test is complies with the acceptability criteria and the test substance is considered an irritant in the in vitro skin irritation test under the experimental conditions used.
Executive summary:

An in vitro skin irritation test was conducted on a human skin model according to the OECD guideline 439. The basis of the test is that the cell viability was determined by using the MTT reduction assay. The test substance was applied undiluted on top of the skin tissue for 15 minutes. After the exposure, the tissue was incubated with MTT and the formazan that was produced from MTT was spectroscopically determined at 570 nm. The relative mean tissue viability of the test substance expressed as the absorption of the test substance against the negative control showed that the test substance has a relative mean tissue viability of ≤50% compared to the negative control. The test substance is therefore considered an irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February - 5 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance an OECD guideline by a GLP accredited laboratory.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 438 (7 September 2009)
Qualifier:
according to
Guideline:
other: EU Method B.48 (ICE test 8 December 2010)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Spring chicken
Details on test animals or tissues and environmental conditions:
The eyes collected from chickens obtained from a slaughterhouse. Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with isotonic saline.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
Amount / concentration applied:
30 µl of pure test substance or control was put onto enucleated chicken eyes during 10s.
Duration of treatment / exposure:
The incubation period was 10±1 min at 32±1°C.
Observation period (in vivo):
Corneal opacity, swelling and morphological effects were evaluated at 30, 75, 120, 180 and 240 minutes post-dosing. Fluorescein retention was determined at pretreatment and 30 minutes post-dosing.
Number of animals or in vitro replicates:
3 eyes per dose group (except negative control: 1 eye).
Details on study design:
The eyelids were excised and dissected from the skull. The eyeball was pulled from the orbit and placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The cornea of the enucleated eye was positioned vertically by a steel clamp and placed in a superfusion apparatus in such a way that the entire cornea was supplied with the isotonic saline drip.
The eyes were then examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was measured at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. Individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
The approved eyes were incubated between 60 and 76 minutes prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline. The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
Following the zero reference measurements, the eye was removed from the superfusion apparatus, placed in a horizontal position. 30 μL of the test substance was applied to the cornea for 10 seconds, such that the entire surface of the cornea is evenly covered with the test item. The test substance was applied and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature. The eye was subsequently returned to the superfusion apparatus in the original upright position for further measurements.

Irritation parameter:
cornea opacity score
Remarks:
ICE class swelling
Basis:
mean
Score:
2
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
corneal opacity
Basis:
mean
Remarks:
maximum
Score:
0.5
Max. score:
4
Irritation parameter:
other: ICE class corneal opacity
Basis:
mean
Remarks:
maximum
Score:
1
Max. score:
4
Irritation parameter:
other: Fluorescein retention
Basis:
mean
Score:
1
Max. score:
3
Irritation parameter:
cornea opacity score
Remarks:
ICE class fluorescein retention
Basis:
mean
Score:
1
Max. score:
4
Irritant / corrosive response data:
The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as non corrosive/severe irritant.
The combination of the three endpoints for the positive control, 10% acetic acid solution, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant.
The combination of the three endpoints for the test substance was 1 x I and 2 x II.

Assessment eye corrosion/severe irritation of an eye exposed to physiological saline (negative control), 10% acetic acid (positive control) and the test substance.

    time / min
End-point 0 30 75 120 180 240
Negative
control
Corneal opacity 0 0 0 0 0 0
Fluorescein retention 0.5 0.5        
Corneal thickness 0.51 0.50 0.50 0.53 0.50 0.50
Positive
control
Corneal opacity 0 2.7 3.0 3.0 3.0 3.0
Fluorescein retention 0.5 3.0        
Corneal thickness - 56 45 43 47 40
Test
substance
Corneal opacity 0 0.5 0.5 0.5 0.5 0.5
Fluorescein retention 0 1        
Corneal thickness - 8 6 9 9 6

ICE classification of the negative control, positive control and the test substance.

  End-point ICE
Negative
control
Corneal opacity I (=1)
Fluorescein retention I (=1)
Corneal thickness I (=1)
Positive
control
Corneal opacity IV (=4)
Fluorescein retention IV (=4)
Corneal thickness IV (=4)
Test
substance
Corneal opacity I (=1)
Fluorescein retention II (=2)
Corneal thickness II (=2)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance must not be classified as causing “irreversible effects on the eye”.
Executive summary:

The eye irritancy of Tropicate was assessed according to the Isolated Chicken Eye Test as described in OECD method 438. 30ml test substance was applied to 3 enucleated chicken eyes during 10s. The eyes were subsequently rinsed with 10ml of physiological saline. Three other eyes were treated with a positive control and one eye with a negative control. The damage by the test substance was assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The ocular reactions observed in the eyes treated with the test substance were slightly to moderate. Based on the results of the test, the test substance does not need to be classified as causing “irreversible effects on the eye”.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Study conducted on the substance by GLP accredited laboratory. Method according to OECD guideline.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification