Registration Dossier

Administrative data

Description of key information

The substance was not irritating to skin in a 24-hour oclusive test.

The substance was not irritating in an in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model.

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:
1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Principles of method if other than guideline:
Test substance at three dose levels was applied to the clipped intact and abraded skin on the back of 12 albino rabbits (4/dose) under occlusive conditions for 24 hours, and the skin reactions were scored.
GLP compliance:
no
Remarks:
pre-GLP
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Label on test material: RIFM 71-10-56 1-13-72
Species:
rabbit
Strain:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2-3 kg
- Housing: Individually housed in metabolism cages
- Diet (e.g. ad libitum): Rabbit pellets, ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 2 weeks
Type of coverage:
occlusive
Preparation of test site:
abraded
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0, 3.9 and 6.0 mL/kg bw
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
24 hours
Observation period:
14 days
Number of animals:
12 rabbits (4/dose)
Details on study design:
TEST SITE
- Area of exposure: Prior to placing the animals on test their backs were clipped free of all hair with small animal clippers. The backs were further prepared by making epidermal abrasions every two to three centimeters, longitudinally, over the clipped area of exposure. The abrasions were sufficiently deep so that they penetrated the stratum corneum bot not the dermis, so that no bleeding occurred.
- Type of wrap if used: Application areas were covered with a rubber sleeve or dam which fit snuggly around each animal.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Application sites were thoroughly wiped down.
- Time after start of exposure: 24 hours

OBSERVATION TIME POINTS: 24 hours after application and throughout the 14 days observation period
Irritation parameter:
other: observation of signs of irritation
Remarks on result:
no indication of irritation
Remarks:
at the end of 24-hour contact period and throughout the 14 days observation period
Irritant / corrosive response data:
Application sites on the back of rabbits did not show any erythema or oedema at the end of 24-hour contact period and remained normal throughout the study.
Other effects:
All animals consumed their daily ration, gained weight and behaved as normal laboratory acclimatized animals. A comparison of the initial and final haematogram values of each animal did not reveal any significant changes.

None

Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with Regulation EC No 1272/2008, the test substance does not require classification for skin irritation according to CLP and GHS regulations. No hazard statement and no signal word were required.
Executive summary:

Test substance at the dose levels of 2.0, 3.9 or 6.0 mL/kg bw was applied to the clipped intact and abraded skin on the back of 12 albino rabbits (4/dose) under occlusive conditions for 24 hours. Following the 24-hour exposure period, patches were removed and skin reactions were recorded. Animals were also observed for food consumption, body weights and general behaviour for 14 days.

Application sites on the back of rabbits did not show any erythema or oedema at the end of 24-hour contact period and remained normal throughout the study. All animals consumed their daily ration, gained weight and behaved as normal laboratory acclimatized animals. A comparison of the initial and final haematogram values of each animal did not reveal any significant changes.

In accordance with CLP (Regulation EC No 1272/2008) and GHS regulations, the test substance was identified as not requiring classification for skin irritation. No hazard statement and no signal word are required.

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:
09-12 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
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)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
23 October 2015
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 171893
- Date received: 22 April 2016
- Manufacturing date: 10 December 2015
- Expiry date: 09 December 2017
- Purity test date: 10 December 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Fridge (6 ± 3°C), darkness

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied
Species:
human
Details on test animals or tissues and environmental conditions:
- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23709] were received on 10 May 2016.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 2 hours at standard culture conditions
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
PRELIMINARY TESTS
- Test for direct interaction with MTT:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of MTT solution at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The colouration potential of the test item in water or isopropanol was checked by adding 50 µL of the test item to 1 mL of distilled water or 2 mL of isopropanol, respectively.


MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 17 hours and 51 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied, as supplied, at the dose of 50 µL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and for comparable colour with the negative control. This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2-hour post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 05 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD at 570 nm was measured in triplicate samples of formazan extracts using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
Main test
Value:
82.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution with deposit of the test item at the bottom of the well was observed after 3 hours of incubation between 37.3°C and 38.0°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained in water after 1 hour of incubation between 37.3°C and 38.0°C, 5% CO2. A colourless solution was obtained in isopropanol after 3 hours of incubation at room temperature. No significant colouration appeared, therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 82.80%, versus 31.99% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

1.035

1.006

1.079

93.28

100.00

13.44

1.023

0.961

2

1.158

1.151

106.72

1.128

1.167

Positive control

1

0.367

0.358

0.345

33.19

31.99

2.41

0.351

0.357

2

0.335

0.332

30.78

0.333

0.328

Test item

1

1.027

0.932

0.893

86.42

82.80

7.23

0.898

0.873

2

0.87

0.854

79.18

0.857

0.833

 #: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance does not require classification for eye irritation or serious eye damage according to Regulation EC No. 1272/2008 or according to UN GHS (No hazard Category in UN GHS). No hazard statement and no signal word were required.
Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to Guideline OECD 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test substance was 82.80%, versus 31.99% in the positive control (Methyl acetate).

Therefore, the test substance does not require classification for eye irritation or serious eye damage according to Regulation EC No. 1272/2008 or according to UN GHS (No hazard Category in UN GHS). No hazard statement and no signal word were required.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Test substance at the dose levels of 2.0, 3.9 or 6.0 mL/kg bw was applied to the clipped intact and abraded skin on the back of 12 albino rabbits (4/dose) under occlusive conditions for 24 hours. Following the 24-hour exposure period, patches were removed and skin reactions were recorded. Animals were also observed for food consumption, body weights and general behaviour for 14 days.

Application sites on the back of rabbits did not show any erythema or oedema at the end of 24-hour contact period and remained normal throughout the study. All animals consumed their daily ration, gained weight and behaved as normal laboratory acclimatized animals. A comparison of the initial and final haematogram values of each animal did not reveal any significant changes.

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to Guideline OECD 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test substance was 82.80%, versus 31.99% in the positive control (Methyl acetate).

Justification for classification or non-classification

No significant changes were observed after a 24-hour application under oclusive conditions. Therefore, in accordance with CLP (Regulation EC No 1272/2008) and GHS regulations, the test substance was identified as not requiring classification for skin irritation.

In an in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model, the mean percent tissue viability of the RhCE replicates treated with the test substance was 82.80%, versus 31.99% in the positive control (Methyl acetate).

Therefore, the test substance does not require classification for eye irritation or serious eye damage according to Regulation EC No 1272/2008 or according to UN GHS.