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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: Council Regulation 440/2008, Method B.40 BIS: “In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
EC Number:
615-231-8
Cas Number:
70983-58-3
Molecular formula:
UVCB
IUPAC Name:
Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Test material form:
liquid: viscous
Details on test material:
Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light




Specific details on test material used for the study:
Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. The EpiDerm skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.


The test meets acceptance criteria if:
- mean OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test item was applied undiluted. Firstly, 100 µL aqua dest. was applied topically on the tissue surface. Then, 50 µL of the test item was dispensed directly atop the EpiDerm tissue.

Dose Groups

Negative control: 50 µL distilled water
Positive control: 50 µL 8 N KOH
Test Item: 50 µL (undiluted)
Duration of treatment / exposure:
The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min and 60 min exposure time).
Duration of post-treatment incubation (if applicable):
3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.
Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
Number of replicates:
The test was performed on a total of 4 tissues per dose group.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min experiment - mean of 2 tissues
Value:
61.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min experiment - mean of 2 tissues
Value:
47.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
3 min experiment: After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Since the test item could not be removed completely from the tissue surface by rinsing, a Q-tip was used to remove the sticky test item and additional rinsing steps with PBS were performed. However, it was not possible to remove the test item completely. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.

60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Since the test item could not be removed completely from the tissue surface by rinsing, a Q-tip was used to remove the sticky test item and additional rinsing steps with PBS were performed. However, it was not possible to remove the test item completely. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.

Test Acceptance Criteria:

The test item could not be removed completely from the tissue surface after 3 min and 60 min treatment. Therefore, in consultation with the sponsor, 100 µL aqua dest was applied to the tissue surface before application of the test item and additional rinsing steps and manual removal by using a Q-tip were included. However, residuals of the test item were still visible. After 60 min treatment, viability of the two tissues treated identically with the test item showed high variability, exceeding the 30% threshold (32.7%). This is accepted regarding the stickiness of the test item. Under these exaggerated treatment conditions, the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was >= 50% (61.6%) after 3 min treatment and >= 15% (47.9%) after 60 min treatment. The worst case value of the two tissues of the 60 min part of the experiment was 36.8% tissue viability, which indicates no corrosive effect.
The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was >= 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (8.7%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of the control groups was >= 30% (0.2% - 4.0%).
All other test acceptance criteria were fulfilled except CV [%] in the range of 20 – 100% viability exceeded for test material (60 min experiment only).

Any other information on results incl. tables

Pre-Experiments

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 50 µL test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equalled 0%.

Results and discussion

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EpiDerm, comprising a reconstructed epidermis with a functional stratum corneum.

In the present study the test item was applied topically to the EpiDerm tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The test item shows no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

The test item could not be removed completely from the tissue surface after 3 min and 60 min treatment. Therefore, in consultation with the sponsor, 100 µL aqua dest was applied to the tissue surface before application of the test item and additional rinsing steps and manual removal by using a Q-tip were included. However, residuals of the test item were still visible. After 60 min treatment, viability of the two tissues treated identically with the test item showed high variability, exceeding the 30% threshold (32.7%). This is accepted regarding the stickiness of the test item. Under these exaggerated treatment conditions, the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was >= 50% (61.6%) after 3 min treatment and >= 15% (47.9%) after 60 min treatment. The worst case value of the two tissues of the 60 min part of the experiment was 36.8% tissue viability, which indicates no corrosive effect.

The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was >= 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (8.7%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of the control groups was <= 30% (0.2% - 4.0%).

Table 1: Results of 3 min Experiment

 Name  Negative control     Test item     Positive control   
 Tissue  1  2  1  2  1  2
 Absolute OD570

1.244

1.244

1.260

1.301

1.311

1.345 

0.730

0.797

0.793 

0.829

0.852

0.856 

0.188

0.197

0.198 

 0.261

0.261

0.274

OD570 - Blank Corrected

1.198

1.197

1.214

1.255

1.264

1.299

0.684

0.750

0.746

0.783

0.805

0.810

0.142

0.150

0.152

0.215

0.214

0.228

Mean OD570 of 3 Aliquots (blank corrected) 1.203   1.273  0.727  0.799  0.148  0.219
SD OD570  of 3 Aliquots  0.010  0.033  0.042  0.028  0.026  0.026
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)  1.238*     0.763  0.184
SD OD570 of 2 Replicate Tissues  0.049  0.051  0.050

Mean Relative Tissue Viability [%]

100.0 61.6     14.8   
Coefficient Of Variation [%]***  4.0  6.7     27.3   

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%.

Table: Results of 60 min Experiment

 Name  Negative control        Test item    Positive control
 Tissue  1  2  1  2  1  2
 Absolute OD570

1.390

1.439

1.431

1.420

1.429

1.423

0.867

0.846

0.860

0.546

0.546

0.568

0.154

0.159

0.157

0.172

0.179

0.175

 OD570 - Blank Corrected

1.344

1.393

1.385

1.374

1.383

1.377

0.820

0.799

0.814

0.499

0.499

0.521

0.108

0.113

0.110

0.125

0.133

0.128

 Mean OD570 of 3 Aliquots (blank corrected)  1.374  1.378  0.811  0.507  0.110  0.129
 SD OD570  of 3 Aliquots 0.026   0.026  0.027  0.028  0.025  0.026
 Total Mean OD570 of 2 Replicate Tissues (Blank Corrected) 1.376*  0.669     0.120   
 SD OD570  of 2 Replicate Tissues  0.003     0.215     0.013   

Mean Relative Tissue Viability [%]

 100.0     47.9     8.7**   
 Coefficient Of Variation [%]***  0.2     32.7     10.9   

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%. This criterion failed for the test item treated tissues.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.
Executive summary:

In the present Klimisch 1 OECD 431 GLP study the test item was applied topically to the EpiDerm tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues. In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.