Iron titanium pseudobrookite

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 439; GLP compliant)

Eye irritation: not irritating (OECD 405; GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-23 to 2016-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200).

Version / remarks:

Rev. 07/11/2014

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from moisture

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

other: Dulbecco's phosphate buffered saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 23353
- Shipping date: 2016-09-06

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (22.5 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with fresh assay medium. Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new plates containing fresh medium. Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 42 hours. Following incubation, the tissue viablity was measured.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT/ EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the wells were rinsed three times with DPBS. Inserts were transferred onto new plates containing extractant solution (isopropanol) ensuring that the tissues are completely covered. The plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 2.5 hours while shaking (~ 120 rpm) at room temperature.
After the extraction period was completed, the inserts were pierced to allow the extract to run into the well from which the insert was taken. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Prior to the start of the test, the test item’s colour interference potential was evaluated. 25 ± 2 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 % RH.
Since the solution change colour significantly, the test item was presumed to have the potential to stain the tissue. A functional check on viable tisses has to be performed.
To check the tissue-binding of a coloured test item, one viable tissue was exposed to 25 ± 2 mg of the test item. For negative control, two tissues were exposed to DPBS. Experimental procedure was carried out according to main test procedure described above, except the tissues were incubated for 3 hours in culture media without MTT (37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 % RH), instead of incubation in media containing MTT. After incubation period, the tissues were rinsed and extracted using isopropanol. Optical densitiy was measured at 570 nm.
- Data correction procedure: since the tissues treated by coloured test item had a mean optical density (OD) between 5 % and 30 % of the negative control tissue (treated with PBS), the real MTT OD (unaffected by interference with the coloured test items) was calculated using following formula: OD = OD(coloured tissue(MTT assay)) - OD(coloured tissue (no MTT assay))

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. For this purpose,25 ± 2 mg of the test item were added to 1 mL of MTT solution (resulting: 1 mg/mL). This mixture was incubated at 37 ± 1.5 °C ,5 ± 0.5 % CO2, 95 % RHfor 60 minutes. An untreated MTT medium was used as control. If the colour of the MTT did not turn blue/purple, the test item was not considered to reduce MTT and additional test with freeze-killed tissues did not have to be performed.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (OD test item/ OD mean of negative control) x 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritauion potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg of the test item (~ 39 mg/m²) wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium Lauryl Sulfate (SLS) solution

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(corrected mean)

Value:

82.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT: the test item changed colour when mixed with deionised water (colour interference pre-experiment). Also its intrinsic colour was intensive. Consequently, additional tests with viable tissue were performed, to enabling data correction procedure.
- Direct-MTT reduction: the colour did not turn blue/purple and the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues for data correction did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (2.079, 1.941, and 1.762 (mean: 1.927)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.4 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 12 % (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%).
Please refer to the field "Any other information on results incl. tables" below

Historical Data

Positive Control		Negative Control [OD570]
Mean Viability	4.64%	Mean Absorption	1.74
Rel. Standard Deviation	11.2%	Rel. Standard Deviation	8.68%
Range of Viabilities	4.00% - 5.90%	Range of Absorbance	1.48 – 1.98
Mean Absorption	0.0803
Rel. Standard Deviation	12.6%
Range of Absorbance	0.066 - 0.097

Data of 31 studies performed from July 2015 until March 2016

Table 1: Results after treatment with Pseudobrookite; Pigment Brown 48 and the controls

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Absor-bance of 3 Wells	Mean-Absorbance of three wells blank corrected	Mean Absor-bance of 3 tissues after blank correction	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]**	Corrected mean Absorbance [% of Negative Control]
Blank		0.038	0.038	0.037	0.038	0.000
Negative Control	1	2.064	2.078	2.207	2.116	2.079	1.927	107.9	8.2	100.0
	2	1.955	2.003	1.978	1.979	1.941		100.7
	3	1.768	1.784	1.849	1.800	1.762		91.4
Positive Control	1	0.119	0.122	0.112	0.117	0.080	0.084	4.1	5.2	4.4
	2	0.126	0.125	0.128	0.126	0.088		4.6
	3	0.129	0.118	0.120	0.122	0.084		4.4
Test Item	1	1.890	1.816	1.775	1.827	1.789	1.600	92.8	11.1	83.0	82.6
	2	1.597	1.643	1.599	1.613	1.575		81.7
	3	1.473	1.474	1.477	1.475	1.437		74.5
Blank		0.038	0.038	0.037	0.038	0.000
NC Viable Tissue	1	0.048	0.049	0.048	0.048	0.011				100.0
TI Viable Tissue	2	0.047	0.047	0.044	0.046	0.008				77.3

*         relative absorbance per tissue [rounded values]: [100 x absorbance(tissue)] / [mean absorbance(negative contol)]

**       relative absorbance per treatment group [rounded values]: [100 x mean absorbance(test item/positive control)] / [mean absorbance(negative control)]

***     corrected relative absorbance [rounded values]:mean absorbance colored tissue (MTT assay-mean absorbance colored tissue (no MTT assay)

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Remarks:

in vivo study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-14 to 2016-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012-10-02

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, stored in closed original container in a dry place

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Age at dosing: approx. 7 months
- Weight at dosing: 4.4 kg to 6.1 kg
- Housing: kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany)
- Diet (ad libitum): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 30% - 70% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

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

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

1, 24, 48 and 72 hours after the administration

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

INITIAL AND CONFIRMATORY TEST
The test was performed initially using one animal. As no corrosive or severe irritant effects were observed in the animal treated in the preliminary test, 2 further animals were employed 24 hours after start of the initial test.

USAGE OF ANAESTHETICS AND SYSTEMIC ANALGESICS
Sixty minutes prior to test item administration, 0.01 mg Buprenovet®/kg bw were administered by subcutaneous (s.c.) injection to all animals to provide a therapeutic level of systemic analgesia to avoid or minimize pain and distress.
Five minutes prior to the test item administration, two drops of Conjuncain®EDO, a topical anaesthetic, were applied to each eye of all animals, to the right eye, in which the test item was to be applied, and to the left eye, which served as control.
Seven hours after administration, Buprenovet® 0.01 mg/kg, s.c. and Acticam 0.5 mg/kg, s.c. were administered to provide a continued therapeutic level of systemic analgesia.

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were rinsed with 20 mL 0.9 % aqueous NaCl solution
- Time after start of exposure: 1 hour

SCORING SYSTEM: according to the Draize scale and a scale scoring the degree of staining (Fluorescein-test)

TOOL USED TO ASSESS SCORE: slit lamp and fluorescein
The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours after the administration. The eye reactions were observed and registered.
24, 48 and 72 hours after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OBSERVATIONS
- body weight: at the beginning and at the end of the study.
- behaviour and food consumption were monitored
- any adverse systemic effects were recorded

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

- slight conjunctival redness (grade 1) and slight chemosis (grade 1) were observed in all animals 1 hour after administration.
- corneae and the irises were not affected by administration of the test item.

Other effects:

- Lesions and clinical observations: no systemic intolerance reactions were observed.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was not observed to be irritating to the eye in a reliable in vivo eye irritation study according to OECD 405.

Justification for classification or non-classification

Skin irritation

The substance does not possess an skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does not possess an eye irritating potential based on an in vivo OECD 405 (2012) test and does not require classification as eye irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

.