Reaction mass of sodium 1-methoxy-1-oxohexadecane-2-sulphonate and sodium methyl 2-sulphooctadecanoate

Endpoint summary

Description of key information

Studies performed according to OECD/EU test guidelines and GLP principles:

In vitro skin corrosion test on reaction mass: negative

In vitro skin irritation test on reaction mass: negative

In vitro eye irritation test on reaction mass: negative

In vivo eye irritation test on reaction mass: irritating Category 2

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March - 02 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 431: In vitro Skin Corrosion: Human Skin Model Test (13 April 2004)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure: human skin model
- % coverage: 0.6 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 3 minutes and 1 hour

SCORING SYSTEM: percentage viability
Cell viability measurement:
The DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the Multiskan Spectrum.

Control samples:

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, crushed and ground in a mortar with pestle to improve the consistency

Duration of treatment / exposure:

3 minutes and 1 hour exposure times

Duration of post-treatment incubation (if applicable):

After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Number of replicates:

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to MIZULAN FL-80 and two for a 1-hour exposure.

Species:

other: human-derived epidermal keratinocytes

Details on test animals or test system and environmental conditions:

EpiDerm Skin Model (EPI-200, Lot no.: 12961 kit I). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those foundin vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 91 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.1 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

EpiDerm Skin Model (EPI-200, Lot no.: 12961 kit I). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those foundin vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 91 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.1 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MIZULAN FL-80 compared to the negative control tissues was 100% and 88% respectively. Because the mean relative tissue viability for MIZULAN FL-80 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MIZULAN FL-80 is considered to be not corrosive.

MIZULAN FL-80 was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that MIZULAN FL-80 did not interact with MTT.

The absolute mean OD₅₄₀(optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 10%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 26% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%. It was therefore concluded that the test system functioned properly.

Interpretation of results:

other: not corrosive

Conclusions:

MIZULAN FL-80 is not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April - 03 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EC Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test "

Deviations:

no

Principles of method if other than guideline:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Draft Proposal for a New Guideline: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method.
- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No. 761/2009 OJ No. L220, 24 August 2009.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 87 - 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg solid, crushed and ground in a mortar with pestle to improve the consistency

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 minutes treatment with MIZULAN FL-80 compared to the negative control tissues was 75%. Since the mean relative tissue viability for MIZULAN FL-80 was above 50% after 15 minutes treatment MIZULAN FL-80 is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 4%. The absolute mean OD₅₇₀of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Interpretation of results:

other: not irritating

Conclusions:

It is concluded that this test is valid and that MIZULAN FL-80 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. Where a negative result is obtained, an in vivo test is subsequently required.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

The study procedures described in the report are also based on the following documents:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 124. Bovine Opacity and Permeability Assay - SOP of Microbiological Associates Ltd., 1999.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were used as soon as possible after slaughter on the same day.

Source: obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands) as soon as possible after slaughter, and transported in physiological saline

The isolated corneas were stored at 32 +/- 1ºC in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum).

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

750 µl of a 10% (w/v) suspension

Duration of treatment / exposure:

10 ± 1 min

Observation period (in vivo):

120 ± 10 min

Number of animals or in vitro replicates:

1 cornea per treatment group

Details on study design:

Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1ºC. After the incubation the control or test substance was removed and the epithelium was washed at least three times with cMEM. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1ºC. After the completion of the incubation period opacity determination was performed (opacitometer)

Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. For thta, the medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution and were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1ºC.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at
490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of replicates

Value:

26

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The pH of the 10% (w/v) solution of MIZULAN FL-80 was 5.5.
The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.2. The individual positive control in vitro irritancy scores ranged from 156 to 189 for Benzalkonium Chloride. The corneas treated with the positive control substances were turbid after the 10 minutes of treatment.

The corneas treated with MIZULAN FL-80 showed opacity values ranging from 0 to 2 and permeability values ranging from 1.122 to 2.304. The corneas were clear after the 10 minutes of treatment with MIZULAN FL-80. Hence, the in vitro irritancy scores ranged from 17 to 37 after 10 minutes of treatment with MIZULAN FL-80.

 

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	0	0.000	0.0
Positive control (Benzalkonium Chloride)	86	5.974	176
MIZULAN FL-80	1	1.676	26

 

1          Calculated using the negative control mean opacity and mean permeability values.

2           In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

 

Interpretation of results:

other: not irritating

Conclusions:

It is concluded that this test is valid and that MIZULAN FL-80 is a non irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 March -03 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Harlan, Belton, Leics, England
- Age at study initiation: 7-12 weeks old
- Weight at study initiation:1552, 1942 and 2306 grams
- Housing: Animals were individually housed in labeled cages with perforated floors and shelters.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 - 19.4
- Humidity (%): 43 - 73
- Air changes (per hr): approx 15x
- Photoperiod (hrs dark / hrs light): 12 -12

IN-LIFE DATES: From 31 March 2010 to 03 May 2010

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

on average, 26.0 mg (range 25.7 - 26.6 mg) per animal

Duration of treatment / exposure:

The lids were gently held together for about one second to prevent loss of the test substance.

Observation period (in vivo):

7 days for two animals
14 days for the other animal

Number of animals or in vitro replicates:

three

Details on study design:

No washing needed.
SCORING SYSTEM: At each observation, the highest scores given were recorded:

CORNEAL IRRITATION
Opacity: degree of density (area most dense taken for reading)
No ulceration or opacity (may include slight dulling of normal luster) 0
Scattered or diffuse areas of opacity, details of iris clearly visible 1
Easily discernible translucent area, details of iris slightly obscured 2
Nacreous area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4

Area of cornea involved:
No ulceration or opacity 0
One quarter or less but not zero 1
Greater than one quarter, but less than half 2
Greater than half, but less than three quarters 3
Greater than three quarters, up to whole area 4

IRIS
Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination thereof, iris still reacting to light
(sluggish reaction is positive) 1
No reaction to light, hemorrhage, gross destruction (any or all of these) 2

CONJUNCTIVAL IRRITATION
Redness (refers to palpebrae and sclera, excluding cornea and iris):
Blood vessels normal 0
Some blood vessels definitely hyperaemic (injected) 1
Diffuse, crimson color, individual vessels not easily discernible 2
Diffuse beefy red 3

Chemosis (refers to lids and/or nictitating membranes):
No swelling 0
Any swelling above normal (includes nictitating membranes) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids more than half closed 4

Discharge:
No discharge (may include small amounts observed in inner canthus of normal animals) 0
Any amount different from normal and/or lacrimation 1
Discharge with moistening of the lids and hairs just adjacent to lids 2
Discharge with moistening of the lids and hairs (considerable area around the eye) 3

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility not applicable

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility not applicable

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility not applicable

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Instillation of approximately 26 mg of MIZULAN FL-80 (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae.
The corneal injury consisted of opacity (maximum grade 1) and epithelial damage (maximum 50, 25 or 25% of the corneal area). The corneal injury resolved within 72 hours in all animals.
Iridial irritation grade 1 was observed and resolved within 24 hours in all animals.
The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in two animals and within 14 days in the other animal.

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Remarks:

Category 2 according to Regulation (EC) No. 1272/2008

Conclusions:

Based on these results of three rabbits in the eye irritation study:
according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), MIZULAN FL-80 should be classified as : mildly irritating to eyes (Category 2B).
according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, MIZULAN FL-80 should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

In an in vitro human skin corrosion test, performed according to OECD 431 and EU B.40 test guidelines, the relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the reaction mass compared to the negative control tissues was 100% and 88% respectively. Because the mean relative tissue viability for the reaction mass was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the reaction mass is considered to be not corrosive.

Subsequently an in vitro skin irritation study was performed according to EU B.46 test guidelines. The relative mean tissue viability obtained after 15 minutes treatment with the reaction mass compared to the negative control tissues was 75%. Since the mean relative tissue viability for the reaction mass was above 50% after 15 minutes treatment, the reaction mass is considered to be non-irritant. As the in vitro skin irritation test has been performed according to validated methods, it is accepted as a stand-alone test for the endpoint skin irritation and the classification without the in vivo test.

Eye:

In an in vitro eye irritation study performed according to OECD 437 test guidelines, bovine corneas were exposed to the reaction mass. The corneas treated with the reaction mass showed opacity values ranging from 0 to 2 and permeability values ranging from 1.122 to 2.304. The corneas were clear after the 10 minutes of treatment with the reaction mass. Hence, the in vitro irritancy scores ranged from 17 to 37 after 10 minutes of treatment with the reaction mass and thus was considered to be not irritating.

In an in vivo study, three rabbits were exposed to 0.1 ml of reaction mass according to OECD, EC and EPA test guidelines. Observations were made 1, 24, 48 and 72 hours, 7 and 14 days after instillation which showed effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity (maximum grade 1) and epithelial damage (maximum 50, 25 or 25% of the corneal area). The corneal injury resolved within 72 hours in all animals. Iridial irritation grade 1 was observed and resolved within 24 hours in all animals. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in two animals and within 14 days in the other animal. The reaction mass is therefore considered to be irritating.

Effects on eye irritation: irritating

Justification for classification or non-classification

The reaction mass has to be classified as Eye irritant Category 2 and labeled as H319: Causes serious eye irritation, but does not have to be classified for skin irritation according to CLP Regulation 1272/2008.