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Physical & Chemical properties

Appearance / physical state / colour

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Administrative data

Endpoint:
appearance / physical state / colour
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-04-25 to 2018-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-25 to 2018-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same 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: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
Version / remarks:
2014-11-07
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-06-05
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, tightly closed, store away from incompatible materials (e.g. strong oxidising agents)
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25899

TEST FOR MTT INTERFERENCE
- to check the non-specific MTT-reducing capability of the test item 30 µl of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- after incubation verification of the colour by the unaided eye

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 30 µl of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- after incubation verification of the colour by the unaided eye

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

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 corresponded 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 (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µl (47 mg/cm²) of the test item
Prior to treatment, all EpiDerm™ tissues were gently blotted to remove moisture. The test item was applied undiluted. 30 µL (47 µL/cm²) of the test item were dispensed directly atop the EpiDerm™ tissue. The test item was gently spread to match size of the tissue using a bulb-headed Pasteur pipette.

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution
Duration of treatment / exposure:
60 ± 1 minute
Duration of post-treatment incubation (if applicable):
approx. 42 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
95.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
TEST FOR MTT INTERFERENCE
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

TEST FOR COLOUR INTERFERENCE
The mixture of 30 µL of the test item per 300 µl aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.849).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.2 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4 % - 6.3 %).
- The absorbance values were not below historically established boundaries.

Please also refer to the field "An other information on results incl. tables" below.

Table1:  Result of the test item zirconium, acetate lactate oxo ammonium complexes

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.824

1.879

1.802

0.090

0.106

0.099

1.767

1.896

1.651

1.898

1.893

1.800

0.094

0.105

0.109

1.794

1.871

1.663

Mean Absolute OD570

1.849****

0.101

1.774

OD570(Blank Corrected)

1.781

1.836

1.759

0.047

0.063

0.056

1.724

1.853

1.608

1.855

1.849

1.757

0.051

0.062

0.066

1.751

1.828

1.620

Mean OD570of the Duplicates (Blank Corrected)

1.818

1.843

1.758

0.049

0.063

0.061

1.738

1.841

1.614

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.806*

0.058

1.731

SD OD570

0.044

0.007

0.113

Relative Tissue Viability [%]

100.6

102.0

97.3

2.7

3.5

3.4

96.2

101.9

89.4

Mean Relative Tissue Viability [%]

100.0

3.2**

95.8

SD Tissue Viability [%]

2.4***

0.4***

6.3***

CV [% Viabilities]

2.4

12.9

6.6

 

*Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is ≤ 20%

***Standard deviation (SD) of relative tissue viability obtained from the three concurrently tested tissues for test

item, positive control and negative control is 18%.

****Mean absolute OD570of the negative control tissues is0.8 and2.8.

Table2:  Historical data

 

Mean Absolute OD570±30nmNC

MeanAbsoluteOD570±30nmPC

Mean Relative Viability [%] PC

SD Viability [%]
NC, PC, TI

Mean

1.861

0.114

3.7

4.4

SD

0.247

0.033

1.5

4.1

Range of
LCL – UCL

1.367 – 2.355

0.048 – 0.181

0.7 – 6.8

0.0 – 12.5

n

25

25

25

117

LCL:      Lower control limit (95%, mean – 2*SD)

UCL:      Upper control limit (95%, mean + 2*SD)

n:            number of control values

Historical data were generated in 2017.


 


 

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item, zirconium, acetate lactate oxo ammonium complexes, showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
visual inspection
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-06-05

Test material

Constituent 1
Reference substance name:
Zirconium, acetate lactate oxo ammonium complexes
EC Number:
272-702-7
EC Name:
Zirconium, acetate lactate oxo ammonium complexes
Cas Number:
68909-34-2
IUPAC Name:
Zirconium, acetate lactate oxo ammonium complexes
Test material form:
liquid
Details on test material:
State of aggregation: clear liquid, colourless to slightly yellow
Specific details on test material used for the study:
Storage condition of test material: ambient, tightly closed, store away from incompatible materials (e.g. strong oxidising agents)

Results and discussion

Physical state at 20°C and 1013 hPa:
liquid
Form / colour / odour
Form:
liquid
Colour:
colourless to slightly yellow
Odour:
other: no data
Substance type:
inorganic

Applicant's summary and conclusion

Conclusions:
Zirconium, acetate lactate oxo ammonium complexes is an inorganic, colourless to slightly yellow, clear liquid.