Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 404): not irritating

Eye irritation (OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 - 19 Jan 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Each animal is additionally treated with 3 different dilutions of test substance. No analytical purity of test substance given.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
(1981)
Deviations:
yes
Remarks:
each animal is additionally treated with 3 different dilutions of test substance
Qualifier:
equivalent or similar to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
(Commission Directive 84/449/EEC)
Deviations:
yes
Remarks:
each animal is additionally treated with 3 different dilutions of test substance
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 12 - 16 weeks
- Weight at study initiation: 2.15 - 2.17 kg
- Housing: individually in metal cages
- Diet: Rabbit Diet (Preston Farmers Limited, Lincolnshire, U.K.), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 20
- Humidity (%): 59 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: diethyl phthalate/ethanol (1:1)
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g
- Concentrations: 2, 10, 50% (w/w) and 100%
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Reading time point: 1, 24, 48, 72 hours and 7 days
Number of animals:
2 males and 1 female
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of wrap if used: The treated skin was covered with a gauze patch and held in place with surgical adhesive tape.

REMOVAL OF TEST SUBSTANCE
- Washing: Skin sites were gently swabbed with cotton wool soaked in ethanol.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: undiluted test substance
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: undiluted test substance
Irritation parameter:
edema score
Basis:
animal: #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversiblitiy: not applicable
Remarks on result:
other: undiluted test substance
Irritant / corrosive response data:
Undiluted test substance: No adverse skin reactions were noted at any treated skin site 1 hour after patch removal. Very slight erythema was noted at the time points 24 and 48 hours. Desquamation was noted at two treatment sites at the 72-hour observation and at one treatment site at the 7-day observation. Very slight oedema was noted at one treatment site at the 24-hour observation.
50% dilution: Very slight erythema was noted at the reading time points 24 and 48 hours and at two treatment sites at the 72-hours observation. Desquamation was noted at two treatment sites at the 7-day observation. Very slight oedema was noted at all treatment sites at the 24-hour observation and at one treatment site at the 48-hour observation.
10% dilution: Very slight erythema was noted at one treatment site one hour after patch removal and at all treatment sites at the 24-and 48-hour observations. Very slight erythema or desquamation was noted at two treatment sites at the 72-hour observation. Desquamation only was noted at these sites at the 7-day observation. Slight oedema was noted at one treatment site at the 24-hour observation with very slight oedema at the 48-hour observation.
2% dilution: Very slight erythema was noted at one treatment site one hour after patch removal. Very slight or well-defined erythema was noted at all treatment sites at the 24-hour observation. Very slight erythema was noted at two treatment sites at the 48 and 72-hour observations with desquamation only at these sites at the 7-day observation. Slight oedema was noted at one treatment site at the 24-hour observation with very slight oedema at the 48-hour observation.

Table 1: Individual skin examination scores with 100, 50, 10 and 2% test substance

Animal

Concentration %

Erythema/Eschar formation

Oedema formation

1 h

24 h

48 h

72 h

7 d

Mean 24/48/72 h

1 h

24 h

48 h

72 h

7 d

Mean 24/48/72 h

1 (male)

100% (undiluted)

0

1

1

0D

0D

0.67

0

1

0

0

0

0.33

50%

0

1

1

0

0

0.67

0

1

0

0

0

0.33

10%

0

1

1

0D

0D

0.67

0

0

0

0

0

0

2%

0

1

0

0

0

0.33

0

0

0

0

0

0

2 (male)

100% (undiluted)

0

1

1

0

0

0.67

0

0

0

0

0

0

50%

1

1

1

1

0D

1

0

1

1

0

0

0.67

10%

0

1

1

0

0

0.67

0

0

0

0

0

0

2%

1

1

1

1

0D

1

0

0

0

0

0

0

3 (female)

100% (undiluted)

0

1

1

0D

0

0.67

0

0

0

0

0

0

50%

0

1

1

1

0D

1

0

1

0

0

0

0.33

10%

1

1

1

1

0D

1

0

2

1

0

0

1

2%

0

2

1

1

0D

1.33

0

2

1

0

0

1

D: desquamation

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of this skin irritation study in rabbits the undiluted test substance (100%) was not irritating to the skin. Also the test substance concentrations of 2, 10 and 50% in ethanol/diethylphthalat (1:1) did not show an irritation potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
7 -29 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
yes
Remarks:
no historical data of positive and negative controls
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
human
Strain:
other: EpiOcular™
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approx. 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
in duplicates for each treatment and contol group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ (MatTek Corporation, Bratislava, Slovakia), Lot No.: 23707
- Tissue viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.918 ± 0.095 (acceptance criteria: 1.1 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.12 min (acceptance criteria: 12.2 - 37.5 min).
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours exposure (37 °C), 25 min post-exposure immersion (room temperature), 18 hours post-exposure (37 °C)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a plate reader (Versamax Molecular Devices, Ismaning, Germany). No reference wavelength measurement was used.
- Decision criteria/Prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
- Acceptance criteria: The results are acceptable if 1) the negative control OD is > 0.8 and < 2.5, 2) the mean relative viability of the positive control is below 50% of the negative control viability and 3) the difference of viability between the two relating tissues of a single test substance is < 20% in the same run (for positive and negative control tissues and tissues of test substances).
Irritation parameter:
other: % mean relative absorbance of 2 tissues
Run / experiment:
6 hours exposure
Value:
85.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.352 and 1.573) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (13.0%).

The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% to 15.1%) in the same run (for positive and negative control tissues and tissues of single test items).

Table 2. Results after 6 hours incubation time

Test group

Mean absorbance*

 

 

Mean absorbance of 2 tissues*

Rel. absorbance (%)**

Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2

Rel. absorbance (% of negative control)

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative control

1.352

1.573

1.463

92.4

107.6

15.1

100.0

Positive control

0.189

0.190

0.190

13.0

13.0

0.0

13.0

Test substance

1.332

1.159

1.245

91.1

79.2

 11.9

85.1

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance is non irritating towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vivo skin irritation test (1990) in albino rabbits according to OECD Guideline 404 and in compliance with GLP. 0.5 g of the test substance moistened with ethanol/diethylphthalate 1:1 (v/v) and 0.5 mL of the test substance in dilutions of 2, 10 and 50% (w/v) in ethanol/diethyl phthalate 1:1 were applied to the clipped skin of three rabbits under semi-occlusive conditions for 4 hours. After the exposure period the patch was removed and the skin sites were wiped with ethanol. Scores were taken 1, 24, 48 and 72 hours and 7 days after patch removal using the Draize scoring system. Very slight erythema was noted at all treatment sites of the undiluted test substance after 24 and 48 hours. Desquamation was noted at two animals after 72 hours and in one animal after 7 days.Very slight edema was noted in one animal after 24 hours. For the undiluted test substance the mean score after 24, 48 and 72 hours for erythema was 0.67, 0.67 and 0.67 and for edema 0.33, 0 and 0 for the 3 animals.Test substance concentrations of 2, 10 and 50% (w/w) showed partially very slight to well defined erythema and very slight to slight edema reversible within 72 hours or 7 days. Based on the results, the undiluted test substance as well as the 2, 10 and 50% (w/w) concentrations of the test substance were not irritating to the skin under the conditions of the test.

Eye

The eye irritation potential of the test substance was determined by an in vitro eye irritation test using a human cornea model according to OECD Guideline 492 and in compliance with GLP (2016). 50 mg of the test substance, 50 µL of negative (deionized water) and positive control (methyl acetate), respectively, were applied to each of duplicate EpiOculartissue for 6 hours. After rinsing, tissues were incubated for approx. 18 hours in assay medium (post-treatment incubation). Cell viability was assessed with an MTT assay. Compared to the negative control the relative mean absorbance after incubation with the test substance was 85.1% (threshold for irritancy: ≤ 60%) and therefore the test substance is not irritating towards human-derived epidermal keratinocytes. All acceptance criteria for negative and positive control were met. The test substance had no colour change potential and no MTT-reducing capacity. Based on results of this human cornea model test, the test substance is considered to be not irritating to the eye.

Justification for classification or non-classification

The available data on skin and eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.