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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 July 2016 - 16 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 BIS.: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test; Official Journal of the European Union, No. L 142
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: (IATA) for skin corrosion and irritation, Series on Testing and Assessment No. 203
Version / remarks:
11 July 2014
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Constituent 1
Reference substance name:
reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate
Molecular formula:
C5H11N2O2Na and C8H14N2O4Na2
IUPAC Name:
reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Do_0164

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under nitrogen
- Stability under test conditions: guaranteed

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit ( EPI-200 tissues with surface of 0.6 cm²), MatTek In Vitro LifeScience Laboratories, Bratislava, Slovakia
- Tissue batch number(s): 23348

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 hour in the incubator at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: washed with PBS to remove residual test material

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570) 1.850 ± 0.053
- Barrier function:ET50 = 6.18 hours
(Lower acceptance limit: ET50 = 4.0 hours and Upper acceptance limit: ET50 = 8.7 hours)
- Morphology: functional stratum corneum, a viable basal cell layer, intermediate spinous and granular layers
- Contamination: no, sterile

NUMBER OF REPLICATE TISSUES: 2 tissues per exposure time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: EPI-200 tissue that is killed by freezing at -20°C
- N. of replicates : 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
Mean tissue viability
(% of negative control)
3 min: < 45 corrosive (Optional Sub-category 1A) (1)
3 min: 45-55 borderline (2)
3 min: >55 and 1 hour: 10-20 borderline (2)
3 min: >55 and 1 hour: < 10 Corrosive (Optional Sub-category 1B and 1C) (1)
3 min: >55 and 1 hour: >20 Non-corrosive
(1)According to the current OECD Guideline 431 a sub-categorization is possible based on the results. However, the sub-categorization into 1A is highly over-predictive as stated in the guideline and differentiation into sub-category 1B or 1C is not possible. If the test substance is identified to be corrosive by SCT and a transport classification is needed, the Corrositex® test should be performed, if applicable, to confirm classification as 1A or to differentiate between 1B and 1C.
(2) The „borderline“-evaluation (50 ± 5% and 15 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 431.




Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 μL de-ionized water was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3 min or 1 hour
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st Experiment (3 min)
Value:
74.7
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
14.2%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st Experiment (1 hour)
Value:
38.6
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
5.5%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria met for positive control: yes
A tissue viability of ≤ 30% is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.
- Acceptance criteria met for variability between replicate measurements: no
In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of %-viability is ≤ 30%. The CV of %-viability of the test substance is out of the acceptance range (46% , 1 hour exposure only). Since all other quality criteria of the test were met and the viability values are well above the cut off for skin corrosion, this deviation is not considered to adversely affect the result of this study.

Historic control data:
Historic Range of NC (Period Jan 2014 - Jun 2016)
3 min exposure: Mean OD570 1.945 ± 0.169
60 min exposure: Mean OD570 1.936 ± 0.172
Historic Range of PC (Period Jan 2014 - Jun 2016)
3 min exposure: Mean OD570 0.312 ± 0.074
60 min exposure: Mean OD570 0.130 ± 0.036
Viability % (Period Jan 2014 - Jun 2016)
3 min exposure: Mean % 16.1 ± 3.8
60 min exposure: Mean % 6.6 ± 1.7

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met

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