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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:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
Name: Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine
Batch/Lot number: 0002293298
Appearance: Highly viscous, colourless to yellowish liquid
Purity**: Considered as 100%
Expiry date: 22 November 2019
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to ensure personnel health and safety.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
at least 50 μL of the test item
Duration of treatment / exposure:
The plates with the treated epidermis units will be incubated for the exposure time of 4 hours (± 10 minutes) at room temperature (20-28°C).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 6.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
General validity criteria:
After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues in the corrosivity and irritation test was in the recommended range (0.745 and 0.824).
The mean OD value of the blank samples (acidified isopropanol) in the corrosivity and irritation test was 0.047.
High viability results (>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.

Specific criteria for corrosivity testing:
The positive control treated tissue showed 0.8% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 33.3%.
The difference of viability between the two negative control tissue samples in the MTT assay was 9.9%.

Any other information on results incl. tables

Table 3: Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical Density (OD)

Viability

(% RV)

 

Measured

Blank corrected

Negative Control: Physiological saline

(0.9% (w/v) NaCl)

1

2

0.828

0.754

0.782

0.708

105.0

95.0

mean

--

0.745

100.0

Positive Control: Glacial acetic acid

1

2

0.047

0.058

0.001

0.011

0.1

1.5

mean

--

0.006

0.8

Test Item:

Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N- dimethylcyclohexanamine

1

2

0.103

0.087

0.056

0.040

7.5

5.4

 

mean

 

--

 

0.048

 

6.4

Notes:

1.        Mean blank value was0.047.

2.        Optical density means the mean value of the duplicate wells for each sample (rounded to three decimalplaces).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
In conclusion, in this in vitro EPISKIN TM (SM) model test with Ethanol, 2 -(2- butoxyethoxy) -, reaction products with phosphorus oxide (P2O5), compds. with
N,N-dimethylcyclohexanamine , the results indicate that the test item is corrosive and non -irritant to the skin, UN GHS Classification: Category 1.
Executive summary:

An in vitro skin corrosivity and irritation test of Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) assay (detailed in section 3.6.). The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines [1, 2].

Disks of EPISKINTM(SM) were treated with the test item and incubated for 15minutes (irritation testing) and 4 hours (corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5%CO2, in a > 95% humidified atmosphere (irritation testing). The viability of each diskwas assessed by incubating the tissues for 3 hours with MTT solution at 37°C in anincubator with 5% CO2protected from light, in a > 95% humidified atmosphere. Theprecipitated formazan crystals were then extracted using acidified isopropanol and quantifiedspectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant toskin.

 

Corrosivity testing:

Following exposure withEthanol,2-(2-butoxyethoxy)-,reaction productswithphosphorusoxide(P2O5), compds.withN,N-dimethylcyclohexanaminefor4hours,the mean cell viability was 6.4% compared to the negative control. This is below the threshold of 35%, therefore the test item was considered as being corrosive.

Irritation testing:

Following exposure withEthanol,2-(2-butoxyethoxy)-,reaction productswithphosphorusoxide(P2O5), compds.withN,N-dimethylcyclohexanaminefor15minutes,the mean cell viability was 114.9% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

The experiment met the validity criteria, therefore the study was considered to bevalid (the Sponsor did not want a full classification to beperformed).

In conclusion, in thisin vitro EPISKINTM(SM) model test with Ethanol, 2 -(2- butoxyethoxy) -, reaction products with phosphorus oxide (P2O5), compds. with

N,N-dimethylcyclohexanamine , the results indicate that the test item is corrosive and non -irritan t to the skin , UN GHS Classification: Category 1.