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Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Semi Dry Absorption (SDA) Product
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available

Composition of test substance:
Calcium sulphate 6.49 %
Calcium sulphite 41.40 %
Calcium carbonate 29.50 %
Calcium hydroxide 2.27 %
Calcium chloride 17.98 %
Calcium fluoride 0.57 %
Oxides (SiO2 + Al2O3 + Fe2O3 + TiO2) sum 0.97 %
Sum of toxic metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum < 0.1 %

Batch No.: SDA/0609/Sk
Appearance: White solid powder
Stability / Expiration date: 15 years (06/2024)
- Stability under test conditions: stable
- Storage condition of test material: the substance was stored in PE container at room temperature.
- Other: pH 11 approx. (by contact of application form with universal indicator pH strip moistened with water, strip producer Lach-Ner, s.r.o. Neratovice)

Test system

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

Results and discussion

In vitro

Results
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

In vivo

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.