Registration Dossier

Administrative data

Description of key information

Test substance Semi Dry Absorption (SDA) Product was assayed for:

The in vitro skin corrosion in human epidermal model EpiDermTM

The test was performed according to Method B. 40. Skin corrosion (in vitro).

Under the experimental design, the test substance was non corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

The in vitro skin irritation in human epidermal model EpiDermTM.

The test was performed according to draft OECD and EC Test Guidelines and Standard Operating Procedure for EpiDerm published by ZEBET.

Under the experimental design, average viability of tissues treated by the test substance was 95.1 % of negative control average value (i.e. viability is >50%).

According to study results, the test substance was non-irritating in EpiDermTM model.

The study for acute dermal irritation/corrosion.

Test was performed according to Method B.4 - Acute Toxicity: Dermal Irritation/Corrosion. Rabbits (New Zealand Albino breed) were used for the test.

No symptoms of systemic toxicity were observed in the animals during the test period. No skin irritation was caused by 4-hour exposure to test substance.

According to study results, the test substance was non-irritating.

The in vitro eye irritation in human corneal model EpiOcularTM.

The test was performed according to MatTekOcular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/01 (1).

Under the experimental design, the test substance was non-irritating for EpiOcularTM tissues.

The eye irritation/corrosion

The test was performed according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion using albino rabbit (New Zealand Albino breed).

Examination of eye irritation after single application demonstrated, that the test substance is slightly irritating for eye of rabbit.

All the tests mentioned above were performed in accordance with GLP.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.08. - 14.08.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Method B. 40. Skin corrosion (in vitro)
Version / remarks:
Published in O.J. L 142, 2008
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: human skin model a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA)
- Tissue batch number(s): No. 12233, kit E

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
tissues were thoroughly rinsed and blotted to remove the test substance

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
tissues were transferred to 24-well plates containing MTT medium (1 mg·mL-1)
- Incubation time: 3 hrs
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Direct MTT reduction: the test substance did not reduce MTT directly.
2. MTT test

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
Test substance (25 mg) was placed directly atop the tissue previously moistened with 25 μl of sterile water for injection to improve contact of the tissue surface with the test chemical. The material was not grinded and it was spread on the tissue surface.

VEHICLE
Water for injection - Ardeapharma a.s.
Lot No. 0101030309

NEGATIVE CONTROL
Water for injection - Ardeapharma a.s.

POSITIVE CONTROL
KOH 8N, solution prepared in laboratory
Duration of treatment / exposure:
3 min. and 60 min.
Duration of post-treatment incubation (if applicable):
24 + 18 hrs
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
96.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All assay acceptance criteria have been met.
Negative control: The assay meets the acceptance criterion - the mean OD570 of the NC tissues was 1.482 (3 min) and 1.441 (60 min) what is ≥ 1.0 and ≤ 2.5.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 9.1 % what is ≤ 20%.
Conclusions:
In the experiment arrangement given above, the test substance Semi Dry Absorption (SDA) Product was not corrosive in EpiDermTMmodel.
Executive summary:

Test substance Semi Dry Absorption (SDA) Product was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to Method B. 40. Skin corrosion (in vitro), Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008.

The test substance (25 mg) was placed atop the tissue previously moistened with 25 μl of sterile water. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (TS), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and extracted overnight subsequently at room temperature without shaking. OD570 of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. 

In the experiment arrangement given above, the test substance Semi Dry Absorption (SDA) Product was not corrosive in EpiDermTMmodel.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.11 - 14.11.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.
Qualifier:
according to
Guideline:
other: draft OECD and EC Test Guidelines and Standard Operating Procedure for EpiDermTM published by ZEBET
Deviations:
yes
Remarks:
24 hours preincubation period was omitted due to time shortage – a deviation from the MatTek instruction. The deviation had had no impact on the outcome of the study.
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Vehicle:
physiological saline
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
25 mg of the test substance was placed directly atop the tissue previously moistened with25 μl of sterile PBS

VEHICLE
sterile PBS

NEGATIVE CONTROL
sterile PBS
POSITIVE CONTROL
5% SDS (in H2O) solution
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
24 + 18 hrs
Number of replicates:
3
Amount / concentration applied:
TEST MATERIAL
Test substance (25 mg) was placed directly atop the tissue previously moistened with 25 μl of sterile PBS to improve contact of the tissue surface with the test chemical. The material was not grinded and it was spread on the tissue surface.

VEHICLE
PBS (phosphate buffered saline) – prepared in laboratory
Duration of treatment / exposure:
60 min.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
> 95.1
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Assay acceptance criteria
Negative Control:
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.        
 
Positive Control:
A 5% SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as %
of the negative control tissues is ≤ 20%.
 
Standard Deviation (SD):
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18.

All assay acceptance criteria have been met.

Direct MTT reduction

Before the test itself direct MTT reduction was assayed.

After 60 min incubation of the test substance with MTT medium no changes in colouring were observed compared with concurrent control without TS.

The test substance did not reduce MTT directly.

MTT test

OD570measuring was performed after 2-hour extraction. Results are given in the following table.

   

Table No. 1: OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

12/11 –14/11 2009

tissue

mean

SD

% NC

1

2

3

4

5

NC

PBS

1.577

1.559

1.848

1.631

1.490

1.621

0.122

100.0

 

% NC

97.29

96.18

114.00

100.62

91.92

100.00

7.534

 

SDA Product

1.683

1.474

1.470

1.542

0.099

95.1

% NC

103.825

90.932

90.685

95.147

6.137

NI

PC

5% SDS

0.103

0.097

0.108

 -

0.103

0.004

6.3

 

 % NC

6.35

5.98

6.66

 -

6.33

0.277

R38

Notes:

NC

negative control

PC

positive control

mean

arithmetic mean

% NC

viability of single tissues compared with negative control

SD

standard deviation calculated from individual % tissue viabilities

NI

non-irritant

Conclusions:
Under the above-described experimental design. average viability of tissues treated by the test substance Semi Dry Absorption (SDA) Product was 95.1 % of negative control average value (i.e. viability is >50%).
Executive summary:

Test substance Semi Dry Absorption (SDA) Product was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to draft OECD and EC Test Guidelines and Standard Operating Procedure for EpiDermTMpublished by ZEBET.

The test substance (25 mg) was placed directly atop the tissue previously moistened with25 μl of sterile PBS. Length of exposition was 60 minutes. Nine tissues were used for the experiment, three per test substance (TS), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were post-incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. OD570 of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

In the experiment arrangement given above, the test substance Semi Dry Absorption (SDA) Product was non-irritating in EpiDermTMmodel.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14.09. - 22.09.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 4-5 month
- Weight at study initiation: 2.8 – 3.4 kg
- Housing: individually in cages without bedding in conventional animal room
- Diet: pelleted standard diet TM-MAK 1 for rabbits and guinea-pigs ad libitum (producer: Ing.Mrkvička Miroslav - Výroba krmných směsí, Mlýn Kocanda, 252 42 Jesenice u Prahy).
- Water: drinking tap water ad libitum (quality corresponding to Regulation No. 252/2004 Czech Coll. of Law)
- Acclimation period: 5 days
- Identification of animals: code number on inner side of ear of animal, number of study on each cage
- Health condition: certificate of good health condition – from breeding farm; no signs of diseases were observed at clinical check-in, in acclimatization period and before the start of experiment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature 17 - 23+/-C, permanently monitored
- Humidity (%): 30 – 70 %, permanently monitored
- Air changes (per hr): 12 hour light/dark cycle
- Photoperiod (hrs dark / hrs light): approximately 15 air changes per hour


STUDY TIME SCHEDULE
- Animal supply: 09. 09. 2009
- Experimental part of study: 14.09. – 22. 09. 2009
- Evaluation of results and final report elaboration: 22.09. – 15. 10. 2009
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): The test substance was applied in delivered form.


Duration of treatment / exposure:
3 min, 1 hr, 4 hrs
Observation period:
1 hr, 24 hrs, 48 hrs, 72 hrs
Number of animals:
3 animals (1 female and 2 males)
Details on study design:
TEST SITE
- Area of exposure: 6 x 6 cm
- Type of wrap if used: The test substance was covered by gauze patch and cellulose cotton and held in place with non-irritating tape - Spofaplast.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of each exposure period the patch was removed and remaining sample was washed with water.
- Time after start exposure:
1st patch - 3 minutes
2 nd patch - 1 hours
3 rd patch - 4 hours

EVALUATION OF SKIN REACTION
- Rabbits were examined for signs of erythema and oedema at 1, 24, 48 and 72 hours after patch removal.

SCORING SYSTEM:
- According to Method B.4 - Acute Toxicity: Dermal Irritation/Corrosion, Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

Irritation parameter:
erythema score
Basis:
animal: 7
Time point:
other: 24,48,72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal: 7
Time point:
other: 24,48,72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal: 8
Time point:
other: 24,48,72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal: 8
Time point:
other: 24,48,72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal: 9
Time point:
other: 24,48,72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal: 9
Time point:
other: 24,48,72 h
Score:
0
Max. score:
4
Other effects:
There was no evidence of a corrosive effect on the skin.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.
No histopathology was performed.

Table No. 1: Skin reactions in experimental animals – grades

Time after

 4-hours exposure

Observation

Rabbit No. 7

Rabbit No. 8

Rabbit No.9

1 hour

Erythema       0

Oedema         0

Erythema       0

 Oedema        0

Erythema      0

 Oedema        0

24 hours

Erythema       0

Oedema         0

Erythema       0

 Oedema        0

Erythema      0

 Oedema        0

48 hours

Erythema       0

Oedema         0

Erythema       0

 Oedema        0

Erythema       0

 Oedema        0

72 hours

Erythema       0

Oedema         0

Erythema       0

 Oedema        0

Erythema       0

 Oedema        0

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance, Semi Dry Absorption (SDA) Product, was tested for acute dermal irritation/corrosion.
Three rabbits were exposed to 0.5 g of test substance, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were evaluated after patch removal and observations were made at 1, 24, 48 and 72 hours after exposure. No symptoms of systemic toxicity were observed in the animals during the test period. No skin irritation was caused by 4-hour exposure to Semi Dry Absorption (SDA) Product.
Executive summary:

The test substance, Semi Dry Absorption (SDA) Product, was tested in the study for acute dermal irritation /corrosion. Rabbits (New Zealand Albino breed) were used for the test.

Test was performed according to Method B.4 - Acute Toxicity: Dermal Irritation/Corrosion, Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

Three rabbits were exposed to 0.5 g of test substance, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were evaluated after patch removal and observations were made at 1, 24, 48 and 72 hours after exposure.

At first the test substance was applied on the skin of one rabbit (test animal No. 7). Rabbit No. 7 was investigated 3 minutes, 1 hour and 4 hours after application of the test substance. No symptoms of irritation were observed on the skin. In confirmatory test, two others rabbits (rabbit No. 8 and No. 9) were used with 4-hour exposition period. No evidence of a corrosive effect or symptoms of irritation were observed on the skin.

No skin irritation was caused by 4-hour exposure to Semi Dry Absorption (SDA) Product. No other signs of intoxication were observed.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15.10 - 16.10.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: MatTek Ocular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/01 (1)
Deviations:
yes
Remarks:
This deviation had no impact on the outcome of the study.
GLP compliance:
yes
Amount / concentration applied:
TEST MATERIAL
As the test substance is partially soluble it could not be applied as a paste. First, tissues were moistened with 100 µL of H2O for injection before layering the test substance on its surface. At application it was assured, so the tissues were completely covered by the test substance.


Duration of treatment / exposure:
3, 30, 60 mins
Irritation parameter:
other: ET-50
Remarks:
1370.0
Value:
1 370
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
As it is possible to see from the results given above, average viabilities of affected tissues are 82.8% after 3 minutes of treatment, 70.9% after 30 min and 66.5% after 60 min of administration. So, viability decreases slightly with time and value of ET-50 calculated is 1369.98 min. According to the Table 1, it responses to the Draize score 0-15 and to the to the Kay and Calandra classification category non irritating.

Direct MTT reduction

Before the test itself, direct MTT reduction was assayed.

After 60 min incubation of the test substance with MTT medium no changes in colouring were observed compared with concurrent control without test substance.

The test substance did not reduce MTT directly.

 

MTT test

OD570measuring was performed after overnight extraction. Results are given in the following table and figure.

Table No. 2: Results of the MTT test

time

 

treatment

 OD570values 

mean

SD

 Inter tissue

variability limits

% NC

 

 

 

1

2

3

 

 

 

 

NC

 water

1.757

1.503

1.690

1.650

0.107

1.403

1.898

100.0

3 min

C1

 67/09

1.481

1.330

1.290

1.367

0.082

1.162

1.572

82.8

30 min

C1

 67/09

1.248

1.175

1.087

1.170

0.066

0.995

1.346

70.9

60 min

C1

 67/09

1.061

1.135

0.358

1.098

0.350

0.933

1.263

66.5

15 min

PC

 0.3% Triton X-100

1.028

1.126

1.223

1.126

0.080

0.957

1.295

68.2

45 min

PC

 0.3% Triton X-100

0.401

0.265

0.278

0.315

0.061

0.267

0.362

19.1

NC

negative control

PC

positive control

C1

test substance

mean

arithmetic mean

% NC

viability of single tissues compared with negative control

SD

standard deviation of OD570values

0.358

value excluded according to the rules

Accuracy criteria fulfilment

Negative Control: criterion was fulfilled

Positive Control: criterion was fulfilled

Inter tissue viability difference:

One tissue treated with the test substance for 60 min had lower OD570 than the other two tissues. After administration, this tissue was also detached from base and rolled up (see primary data). This OD570value did not fulfil the criterion of being in the interval of mean value±15%, so it was excluded from evaluation.

Interval among positive control tissues after 45 min treatment was higher than ±15%. It was caused by damage of tissues after positive control treatment. At comparison of ET-50 values with and without an outlying value it was found out, that ET-50 would changed from 22.54 to 22.08 min, what are very close values and both belong into acceptability interval.

 

Evaluation of result

As it is possible to see from the results given above, average viabilities of affected tissues are 82.8% after 3 minutes of treatment,70.9% after 30 min and 66.5% after 60 min of administration. So, viability decreases slightly with time and value of ET-50 calculated is 1369.98 min. According to the Table 1, it responses to the Draize score 0-15 and to theto the Kay and Calandra classification category non-irritating.
Conclusions:
Under the above-described experimental design, the test substance Semi Dry Absorption (SDA) Product was non irritating for EpiOcularTM tissues.
Executive summary:

Test substance Semi Dry Absorption (SDA) Product was assayed for the in vitro eye irritation in human corneal model EpiOcularTM. The test was performed according to MatTekOcular Irritation Protocol: Neat Method (MTT ET-50),Rev. 1/1/01 (1).

The test substance was applied in delivered form onto a tissue moistened with water. Length of exposition was 3, 30 and 60 minutes. Three tissues were used for each time interval, six for positive control (PC) and three for negative control (NC).

After rinsing and soaking in medium, tissues were incubated with MTT for three hours and then extracted overnight without shaking. OD570 of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each three tissues as % of the mean viability of the negative control. A chart of semi-log scale - the % viability (linear y axis) versus the dosing time (log x axis) was constructed and ET-50 value was calculated from slope of the line obtained. The ET-50 calculated was 1369.98 min. It responses to the Draize score 0 - 15 and to the Kay and Calandra classification category non-irritant.

In the experiment arrangement given above, the test substance Semi Dry Absorption (SDA) Product was non irritating in EpiOcularTMmodel.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.09 - 25.09.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 4 – 5 months
- Weight at study initiation: 3.0 – 3.2 kg
- Housing: Conventional animal room – individually in metallic cages
- Diet: Standard pelleted diet TM-MAK 1 for rabbits and guinea pigs ad libitum (producer: Ing.Mrkvička Miroslav - Výroba krmných směsí, Mlýn Kocanda, 252 42 Jesenice u Prahy)
- Water: Drinking tap water ad libitum (quality corresponding to the Regulation No.: 252/2004 Czech Coll. of Law)
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature: 20+/-3°C, permanently monitored
- Humidity (%): Relative humidity: 30 – 70%, permanently monitored
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Light period: 12 hour light/12 hour dark


STUDY TIME SCHEDULE
Animal supply: 09. 09. 2009
Experimental part of study: 21. 09. – 25. 09. 2009
Evaluation of results and final report elaboration: 25. 09. - 30. 10. 2009
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g
- The test substance was used in delivered form and it was measured out immediately before application.


APPLICATION OF THE TEST SUBSTANCE
The test substance was placed in the conjunctival sac of one eye of animal after gently pulling the lower lid away from the ball.
After application the lids were gently held together for about one second to prevent loss of the test substance.
Untreated eye (on the right side) served as a control.
Duration of treatment / exposure:
24 hrs
Observation period (in vivo):
1 hr, 24 hrs, 48 hrs, 72 hrs after application and then daily.
Number of animals or in vitro replicates:
3 males (No. 10, No. 11, No. 12)
Details on study design:
INITIAL TEST
The test substance was applied initially to one animal (No.10). Because the results of this test indicated the substance not to be corrosive or a severe irritant to the eye, further testing was performed.

CONFIRMATORY TEST
The response was confirmed using two additional animals (No.11 and No.12).

CLINICAL OBSERVATION
The eyes were examined at 1, 24, 48 and 72 hours after application and then daily. After recording the observations at 24 hours, the eyes of rabbit were further examined with the aid of fluorescein and the ophthalmoscopy. The degree and nature of irritation or corrosion observed were recorded.

SCORING SYSTEM:
according to the grading system given in Method B.5 Acute Toxicity: Eye Irritation/Corrosion. Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

TOOL USED TO ASSESS SCORE: fluorescein, ophthalmoscopy
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24,48,72 h
Score:
>= 0 - < 1
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
>= 0 - <= 2
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
>= 0 - <= 3
Max. score:
4
Reversibility:
fully reversible
Irritant / corrosive response data:
Clinical examination of eye
Rabbit No. 10:
strong lacrimation was observed 1 hour after application
1 hour: Conjunctivae – diffuse, crimson colour, individual vessels not easily discernible
Chemosis – swelling with lids more than half closed
Cornea – opacity, details of iris clearly visible
Strong lacrimation
24 hours: Conjunctivae – diffuse, crimson colour, individual vessels not easily discernible
Chemosis – swelling with lids more than half closed
Cornea – opacity, details of iris clearly visible
48 hours: Conjunctivae – some blood vessels hyperaemic (injected)
Chemosis – some swelling above normal
72 hours: Conjunctivae – some blood vessels hyperaemic (injected)
Chemosis – some swelling above normal
96 hours: No alterations

Rabbit No. 11:
1 hour: Conjunctivae – diffuse, crimson colour, individual vessels not easily discernible
Chemosis – some swelling above normal
24 hours: Conjunctivae – some blood vessels hyperaemic (injected)
Chemosis – some swelling above normal
48 hours: Conjunctivae – some blood vessels hyperaemic (injected)
72 hours: No alterations

Rabbit No. 12:
1 hour: Conjunctivae – diffuse, crimson colour, individual vessels not easily discernible
Chemosis – swelling with partial eversion of lids
24 hours: Conjunctivae – some blood vessels hyperaemic (injected)
Chemosis – some swelling above normal
48 hours: No alterations
Other effects:
No symptoms of systemic toxicity were observed in the animals during clinical observation in the test period and no mortality occurred.

Table No. 1 -result of acute eye reaction (grades)

Animal No.

Ocular lesion

1 h

24 h

48 h

72 h

10

Cornea

Iris

Conjunctivae

Chemosis

1

0

2

3

1

0

2

3

0

0

1

1

0

0

1

1

11

Cornea

Iris

Conjunctivae

Chemosis

0

0

2

1

0

0

1

1

0

0

1

0

0

0

0

0

12

Cornea

Iris

Conjunctivae

Chemosis

0

0

2

2

0

0

1

1

0

0

0

0

0

0

0

0

Interpretation of results:
GHS criteria not met
Conclusions:
The changes described further were observed on eye of animals. The effects were detected at all three animals at 1 h and 24 h after application, in one animal the lesions persisted to 72 h after application.
Changes at 1 hour after application: redness of conjunctivae – crimson colour, individual vessels not easily discernible; chemosis – swelling, with lids more than half closed and strong lacrimation.
Changes at 24 hours after application: cornea – diffuse areas of opacity, detail of iris clearly visible; conjunctivae – diffuse, crimson colour, individual vessels not easily discernible; chemosis –swelling with lids about half closed.
Changes at 48 hours after application: conjunctivae – some blood vessels hyperaemic (injected); chemosis – some swelling above normal.
Changes at 72 hours after application: redness of conjunctivae – some blood vessels hyperaemic (injected) and chemosis – some swelling above normal.
Changes at 96 hours - no signs of eye irritation were observed.
No clinical signs of systemic intoxication were detected.
Examination of eye irritation after single application demonstrated, that the test substance, Semi Dry Absorption (SDA) Product, is slightly irritating for eye of rabbit.
Executive summary:

The test substance, Semi Dry Absorption (SDA) Product, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion. Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

The test was performed initially using one animal. Because a corrosive effect was not observed in initial test, the response was confirmed using two additional animals.

The changes described further were observed on eye of animals. The effects were detected at all three animals at 1 h and 24 h after application, in one animal the lesions persisted to 72 h after application.

Changes at 1 hour after application: redness of conjunctivae – crimson colour, individual vessels not easily discernible; chemosis – swelling, with lids more than half closed and strong lacrimation.

Changes at 24 hours after application: cornea – diffuse areas of opacity, detail of iris clearly visible; conjunctivae – diffuse, crimson colour, individual vessels not easily discernible; chemosis – swelling with lids about half closed.

Changes at 48 hours after application: conjunctivae – some blood vessels hyperaemic (injected); chemosis – some swelling above normal.

Changes at 72 hours after application: redness of conjunctivae – some blood vessels hyperaemic (injected) and chemosis – some swelling above normal.

Changes at 96 hours - no signs of eye irritation were observed.

No clinical signs of systemic intoxication were detected.

Examination of eye irritation after single application demonstrated, that the test substance, Semi Dry Absorption (SDA) Product, is slightly irritating for eye of rabbit.

The classification of the test substance eye irritation/corrosion effect was performed according to Directive 67/548/EEC, Annex VI (part 3.2.6.2.)

Based on the test results and according to the EC criteria for classification and labelling of dangerous substances and preparations the test substance does not have to be classified for eye irritation/corrosion and has no obligatory labelling requirement for eye irritation/corrosion.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification

The results of skin irritation/corrosion were negative. Test substance Semi Dry Absorption (SDA) Product may be considered as non-irritating for skin.The results of eye irritation were evaluated as non-irritating (in vitro) or slightly irritating (in vivo). Since the in vitro model (EpiOcular) have not been fully validated yet, there is preferable to consider in vivo results to be more reliable. Therefore, the test substance Semi Dry Absorption (SDA) Product may be considered as slightly irritating (reversible).

According to the classification criteria the test substance, Product of Semi Dry Absorption (SDA), is not classified.