Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trilithium orthophosphate
EC Number:
233-823-0
EC Name:
Trilithium orthophosphate
Cas Number:
10377-52-3
Molecular formula:
H3 O4 P. 3Li
IUPAC Name:
trilithium orthophosphate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Trilithiumphosphat
- Physical state: solid
- Lot/batch No.: MKBD3219V

Test animals

Species:
other: reconstructed three dimensional human epidermis
Strain:
other: in vitro assay

Test system

Vehicle:
other: test substance was moistened with PBS.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a single topical application of 25 μL bulk volume (about 13 mg) of the undiluted test substance to PBS moistened skin model.
Duration of treatment / exposure:
1 hour
Observation period:
42-hours post-incubation period
Details on study design:
TEST SYSTEM:
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, diameter: 10 mm) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: Epi-200
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Irritation test:
Three tissues were treated with the test substance, the PC and NC, respectively. 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

CONTROLS:
Negative control (NC): PBS, sterile
Positive control (PC): 5% (w/v) sodium dodecyl sulfate (SDS), sterile (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia)

Results and discussion

In vivo

Irritant / corrosive response data:
The mean viability of the test-substance treated tissues determined after an exposure period
of 1 hour with about 42 hours post-incubation was 102%.
Other effects:
The test substance is not able to reduce MTT directly.

Any other information on results incl. tables

Test substance

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

NC

mean OD570

 

2.889

2.505

2.806

2.734

 

Viability    [% of NC]

105.7

91.6

102.7

100

7.39

12/0706 -1

mean OD570

 

2.770

2.846

2.772

2.796

 

Viability    [% of NC]

101.3

104.1

101.4

102

1.59

PC

mean OD570

 

0.058

0.058

0.068

0.061

 

Viability    [% of NC]

2.9

2.1

2.5

2

0.21

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information