Slimes and Sludges, blast furnace and steelmaking

Endpoint summary

Description of key information

Test substance Slimes and Sludges was assayed for the in vitro skin corrosion in human epidermal model EpiDerm. The test was performed according to Method B. 40. Skin corrosion (in vitro). Under the above-described experimental design, the test substance Slimes and Sludges was non corrosive in In vitro Skin Corrosion Test on EpiDerm tissues.
Test substance Slimes and Sludges was assayed for the in vitro skin irritation in human epidermal model EpiDerm. The test was performed according to draft OECD and EC Test Guidelines and Standard Operating Procedure for EpiDerm published by ZEBET. According to study results, the test substance was non-irritating in EpiDerm model.
The test substance Slimes and Sludges 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. No symptoms of systemic toxicity were observed in the animals during the test period. No skin irritation was caused by 4-hour exposure to Slimes and Sludges. According to study results, the test substance was non-irritating.
Test substance Slimes and Sludges was assayed for the in vitro eye irritation in human corneal model EpiOcular. The test was performed according to MatTekOcular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/01 (1). Under the above-described experimental design, the test substance Slimes and Sludges was non-irritating for EpiOcular tissues. 
The test substance Slimes and Sludges 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. According to study results, the test substance was non-irritating. 
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

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8.2.2010-12.2.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeding farm VELAZ, s.r.o., Kolec u Kladna, Czech republic
- Age at study initiation: 4-5 months
- Weight at study initiation: 3.3-4.1 kg
- Housing: individually in cages without bedding in conventional animal room
- Diet (e.g. ad libitum): pelleted standard diet TM-MAK 1 for rabbits ang guinea-pigs ad libitum (producer: Ing. Mrkvicka Miroslav - Výroba krmných smesí, Mlýn Kocanda, Jesenice u Prahy)
- Water (e.g. ad libitum): drinking tap water ad libitum (quality corresponding to regulation No. 252/2004 Czech Coll. of Law)
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C (permanently monitored)
- Humidity (%): 30-70 % (permanently monitored)
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

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

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 hours

Number of animals:

3 rabbits (female)

Details on study design:

TEST SITE
- Area of exposure: 6×6 cm
- Type of wrap if used: the test substance in the dose of 0.5 g was applied on intact skin and then it was covered by gauze patch and cellulose cotton and held in place with non-irritating tape - Spofaplast


REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with water
- Time after start of exposure: 3 min, 1 hour and 4 hours


SCORING SYSTEM:
Erythema:
0 - no erythema
1 - very slight erythema (barely perceptible)
2 - well defined erythema
3 - moderate to severe erythema
4 - severe erythema (beet redness) to eschar formation preventing grading of erythema

Oedema:
0 - no oedema
1 - very slight oedema (barely perceptible)
2 - slight oedema (edges of area well defined by definite raising)
3 - moderate oedema (raise approximatey 1 mm)
4 - severe oedema (raised more than 1 mm and extending beyond area of exposure)

Irritation parameter:

erythema score

Basis:

animal: No. 7

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: No. 8

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: No. 9

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: No. 7

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: No. 8

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: No. 9

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

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 1: Results of initial test

exposure time	observation
3 minutes	no erythema, no oedema
1 hour	no erythema, no oedema
4 hours	no erythema, no oedema

Table 2: Skin reactions in experimental animals

time after 4-hours exposure	rabbit No.
	7	8	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:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance, Slimes and Sludges, blast furnace and steelmaking, 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 symptoms of systemic toxicity were observed in the animals during the test period.
No skin irritation was caused by 4-hour exposure to Slimes and Sludges, blast furnace and steelmaking.

Executive summary:

The test substance,Slimes and Sludges, blast furnace and steelmaking,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 Slimes and Sludges, blast furnace and steelmaking.No other signs of intoxication were observed.

Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC, Annex VI, part 3.2.6.1.), the test substanceSlimes and Sludges, blast furnace and steelmaking,does not have to be classified for skin irritationand hasnoobligatory labelling requirement for skin irritation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

5.2.2010-6.2.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

One of OD570 values (test substance after 3 min) lie out of the two other. The outlying value was omitted according to the rules given in par. 3.7e. Criteria for SDs were not fulfilled punctually in both positive controls, but the differences were so negl

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

TISSUES:
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA); lot No. 12948, kit G

Vehicle:

physiological saline

Details on test system:

TEST METHOD:
The actual test procedure was taken from ECVAM Protocol for EPIDERMTM (EPI-200): an In Vitro Assay for Assessing Dermal Corrosivity (1997). In: ICCVAM Review of in vitro dermal corrosivity methods, 62-76. The procedure is described in internal SOP M-46-1 (VUOS-CETA, 2008).

TEST SYSTEM:
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm System is manufactured according to defined quality assurance procedures. The EpiDerm tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.

TEST PROCEDURE:
a) Test substance application:
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.
b) Direct MTT reduction:
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility was performed as follows: 25 mg of the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistured) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes color from red to blue, other steps to correction have to be done.
c) Procedure:
On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, tissues are topically exposed to the test chemicals for 60 minutes. Three tissues are used per test chemical and for the positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium.
After a 24 hr incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hr MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M-46-1 (VUOS-CETA, 2009).
d) OD570 measuring:
OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.
e) Assay acceptance criteria:
The assay meets the acceptance criterion if the mean OD570 of the negative control tissues is = 1.0 and = 2.5.
The assay meets the acceptance criterion if the mean viability of positive control tissues expressed as % of the negative control tissues is = 20%.
The assay meets the acceptance criterion if the stanvdard deviation from 3 identically treated tissues is =15%.

NEGATIVE CONTROL:
The absolute OD of the negative control (NC) tissues (treated with sterile water for injection) 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:
An 8N KOH (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.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

89.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

see Table 1

Direct MTT reduction

Before the test itself direct MTT reduction was assayed.

After 60 min incubation of the test substance with MTT medium, colour was compared with control solution of MTT medium only. Due to presence of dark suspended test substance colour was not clearly appreciable. After filtration it was assured, that the test substance did not changed colour from red to blue. 

The test substance did not reduce MTT directly (see Table 1).

Table 1: MTT test results

time	treatment		OD570values in experiment:			mean	SD	interval of values allowed		% negative control
			1	2	3
3 min	negative control	water	1.911	1.963	1.826	1.900	0.056	1.615	2.185	100.0
	test substance	106/09	1.596	0.463	1.797	1.697	0.587	1.442	1.951	89.3
	positive control	8N KOH	0.231	0.295	0.233	0.253	0.030	0.215	0.291	13.3
60 min	negative control	water	1.723	1.782	1.724	1.743	0.028	1.482	2.004	100.0
	test substance	106/09	1.936	1.646	1.594	1.725	0.150	1.467	1.984	99.0
	positive control	8N KOH	0.172	0.149	0.126	0.149	0.019	0.127	0.171	8.5

data in gray field was omitted data omitted at calculation according to rules given in section “assay acceptance criteria” (Test procedure)

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, the test substance, Slimes and Sludges, blast furnace and steelmaking, was non corrosive in In vitro Skin Corrosion Test on EpiDerm tissues.

Executive summary:

The test substance, Slimes and Sludges, blast furnace and steelmaking, was assayed for the in vitro skin corrosion in human epidermal model EpiDerm. 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 Slimes and Sludges, blast furnace and steelmaking was not corrosive in EpiDerm model.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5.2.2010-7.2.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

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 (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

physiological saline

Details on test system:

TISSUES
- the reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA); lot No. 12918, kit F

CHEMICALS AND MEDIA
Chemicals:
- phosphate buffered saline (PBS) (prepared in laboratory)
- MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) (lot No.: 18733)
- isopropylakohol (lot No.: 070108)
Positive control:
- sodium dodecyl sulfate 5%: (lot No. 090909)
Media:
- EPI-100-NMM-SIT/Assay medium; lot No. 012810TTD, MatTek

TEST METHOD
The test method is available in advanced draft of OECD Guideline and as Method B.46 of EC. Details of routine are available in Liebsch M., Traue D., Kandarova H. (2007): Standard Operating Procedure EpiDerm Skin Irritation Test, Model EPI-200, Version 7, Bfr –ZEBET October 30th, 2007, 1-36

PRINCIPLE OF THE ASSAY
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential.
Evaluation is determined by measuring of optical density of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Skin irritation potential of the test material is predicted if the remaining relative cell viability is below 50%.

TEST SYSTEM
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm system is manufactured according to defined quality assurance procedures.
The EpiDerm tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped world-wide as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.

TEST PROCEDURE
- Test substance application:
The 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 spread on the tissue surface.
- Direct MTT reduction:
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility was performed as follows: 25 mg of the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistured) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes color from red to blue, other steps to correction have to be done.
- Procedure:
On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, tissues are topically exposed to the test chemicals for 60 minutes. Three tissues are used per test chemical and for the positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium.
After a 24 hr incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After a 3 hr MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M-48-1 (VUOS-CETA, 2009).
- OD570 measuring:
OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.
- 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% sodium dodecyl sulfate (in water) 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): phosphate buffered saline (moistening)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): sterile phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight): sodium dodecyl sulfate
- Concentration (if solution): 5 % w/w

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Test material: 3 replicants
Negative control: 5 replicants
Positive control: 4 replicants

Irritation / corrosion parameter:

% tissue viability

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

see Table 1

Table 1: MTT-test results

treatment		OD570values in experiment:					mean	SD	% negative control
		1	2	3	4	5
negative control	phosphate buffered saline	1.891	1.792	2.000	1.775	1.925	1.877	0.084	100,0
	% negative control	100.8	95.5	106.6	94.6	102.6	100.0	4.5	-
test substance	106/09	1.644	1.878	1.544	not tested	not tested	1.689	0.140	90.0
	% negative control	87.6	100.1	82.3	-	-	90.0	7.5	not irritant
positive control	5% sodium lauryl sulfate	0.118	0.105	0.103	0.131	not tested	0.114	0.011	6.1
	% negative control	6.3	5.6	5.5	7.0	-	6.088	0.600	R 38

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Average viability of tissues treated by the test substance Slimes and Sludges, blast furnace and steelmaking was 90.0 % of negative control average value (i.e. viability is >50%). According to study results, the test substance was non irritating in EpiDerm model.

Executive summary:

Test substance Slimes and Sludges, blast furnace and steelmaking was assayed for the in vitro skin irritation in human epidermal model EpiDerm. The test was performed according to draft OECD and EC Test Guidelines and Standard Operating Procedure for EpiDerm published by ZEBET.

Test substance (25 mg) was placed directly atop the tissue previously moistened with 25 µl of sterile PBS. Length of exposition was 60 minutes. Twelve 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 postincubated 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 Slimes and Sludges, blast furnace and steelmaking was non irritating in EpiDerm model.

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:

8.4.2010-9.4.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: MatTek Ocular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/2001

Deviations:

yes

Remarks:

A 1:1 slurry/paste of material and H2O for injection has been prepared as an application form of the test substance. The mixture was very slurry so it could not be applied with the aid of application pin (a deviation from Study Plan and MatTek instruction

GLP compliance:

yes

Details on test animals or tissues and environmental conditions:

TISSUES:
The reconstructed human epidermal corneal tissue EpiOcular(OCL-200, MatTek, Ashland, USA); lot No. 13126, kit A.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg

Duration of treatment / exposure:

3, 30 and 60 minuts

Number of animals or in vitro replicates:

3

Details on study design:

TEST SYSTEM:
MatTek's EpiOcular corneal model (OCL-200, MatTek, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell culture inserts using serum free medium, differentiate to form a multi-layered structure which closely parallels the corneal epithelium. The EpiOcular System is manufactured according to defined quality assurance procedures.

TEST METHOD:
The test consists of a topical exposure of the test chemical to a reconstructed human corneal model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential.
Evaluation is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Viability percentage is calculated fort each application time. The % viability (linear y axis) is then plotted versus the dosing time (log x axis). ET-50 value is calculated from slope of the line obtained.

TEST PROCEDURE:
a) Test substance application:
100 mg of the test substance was applied onto previously moistened (100 µL of sterile distilled water) tissue. At application it was assured that the tissues were completely covered by the test substance.
b) Direct MTT reduction:
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility was performed as follows: 25 mg of the test substance was added to 1 ml MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.
c) Procedure:
On the day of receipt, EpiDerm tissues were conditioned for one hour by incubation to release transport stress related compounds and debris. After pre-incubation, tissues were topically exposed to the test chemical for 3, 30 and 60 minutes. Three tissues were used per each time interval three for the positive control (PC) and three for negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to 5 ml of fresh medium. After 10 minutes of soaking the tissues were transferred to 24-well plates containing MTT medium (1 mg/ml). After 3 hr MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted overnight, without shaking with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm.
d) OD570 measuring:
OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.

ACCEPTANCE CRITERIA:
a) 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.
b) Positive Control:
A 0.3% Triton X-100 solution is used as positive control (PC) and tested concurrently with the test chemicals.
The assay meets the acceptance criterion if the ET-50 of PC is between 15 and 45 min.
c) Inter tissue viability difference:
Since in each test eye 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 difference among viabilities of three identically treated tissues <30% (i.e. average ±15%). Single tissue lying out of the interval can be omitted. In the case of two outliers, the substance should be re-tested.

Irritation parameter:

other: % tissue viability

Run / experiment:

60 min

Value:

63.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: % tissue viability

Run / experiment:

30 min

Value:

64.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: % tissue viability

Run / experiment:

3 min

Value:

86.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

see table 1

Table 1: Results of MTT-test

time (min)	treatment		OD570 values			mean	SD	% NC
			1	2	3
60	negative control	water	1.882	14.717	1.815	1.805	0.068	100.0
3	C1	106/09	1.541	1.538	1.621	1.567	0.038	86.8
30	C1	106/09	0.946	1.143	1.411	1.167	0.191	64.6
60	C1	106/09	1.041	1.289	1.130	1.153	0.103	63.9
15	positive control	0.3% Triton X-100	1.165	1.661	1.070	1.118	0.259	61.9
45	positive control	0.3% Triton X-100	0.432	0.463	0.487	0.461	0.023	25.5

data in grey-coloured windows were excluded from evaluation

Table 2: Corellation of ET-50 with Draize score and classification

Draize Score	Irritancy Classification	EpiOcular ET-50 (min)
0-15	Non-irritating, Minimal	>60
15.1-25	Mild	30-60
25.1-50	Moderate	3-29.99
50.1-110	Severe, Extreme	<3

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, the test substance, Slimes and Sludges, blast furnace and steelmaking, was non-irritating for EpiOcular tissues.

Executive summary:

Test substance Slimes and Sludges, blast furnace and steelmaking was assayed for the in vitro eye irritation in human corneal model EpiOcularTM. The test was performed according to MatTek Ocular 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 255.4 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 Slimes and Sludges, blast furnace and steelmaking was non irritating in EpiOcular model.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.2.2010-26.2.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

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., Kolec u Kladna, Czech Republic
- Age at study initiation: 4-5 months
- Weight at study initiation: 3.6-3.8 kg
- Housing: conventional animal room - individually in metallic cages
- Diet (e.g. ad libitum): standard pelleted diet TM-MAK 1 for rabbits and guinea pigs ad libitum (producer: ing. Mrkvicka Miroslav - Výroba krmných smesí, mlýn Kocanda, Jesenice u Prahy)
- Water (e.g. ad libitum): drinking tap water ad libitum (quality corresponding to the regulation No.: 252/2004 Czech Coll. of Law)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3°C (permanently monitored)
- Humidity (%): 30-70 % (permanently monitored)
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

1, 24, 48 and 72 hours,
next every day till 8th day

Number of animals or in vitro replicates:

3 rabbits (male)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 24 hours

SCORING SYSTEM: see legend in next window

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

>= 0 - <= 1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

>= 0 - <= 1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

see Table 1

Table 1: Results of acute eye reaction

Animal No.	Ocular lesions	Time interval of examination
		1 h	24 h	48 h	72 h
4	Cornea	0	0	0	0
	Iris	0	0	0	0
	Conjunctivae	1	1	0	0
	Chemosis	1	0	0	0
5	Cornea	0	0	0	0
	Iris	0	0	0	0
	Conjunctivae	1	1	0	0
	Chemosis	0	0	0	0
6	Cornea	0	0	0	0
	Iris	0	0	0	0
	Conjunctivae	2	0	0	0
	Chemosis	1	0	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Examination of eye irritation after single application demonstrated, that the test substance, Slimes and Sludges, blast furnace and steelmaking, is not irritating for eye of rabbit.
No clinical signs of systemic intoxication were detected.
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.

Executive summary:

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

Changes at 1 hour after application: redness of conjunctivae – diffuse, crimson colour, individual vessels not easily discernible in one animal, and redness of conjunctivae – some blood vessels hyperaemic in two animals; chemosis – some swelling above normal in two animals.

Changes at 24 hours after application: conjunctivae – some blood vessels hyperaemic (injected) in two animals.

Changes at 48 hours after application: no alterations.

No clinical signs of systemic intoxication were detected.

Examination of eye irritation after single application demonstrated, that the test substance, Slimes and Sludges, blast furnace and steelmaking, is not 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:

no adverse effect observed (not irritating)

Additional information

The results of skin irritation/corrosion were negative. Test substance Slimes and Sludges may be considered as non-irritating for skin.

The results of eye irritation were negative. The test substance Slimes and Sludges may be considered as non-irritating for eyes.

Justification for classification or non-classification

The results of skin irritation/corrosion were negative. Test substance Slimes and Sludges may be considered as non-irritating for skin.

The results of eye irritation were negative. The test substance Slimes and Sludges may be considered as non-irritating for eyes.