Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004.
Deviations:
yes
Remarks:
-reference to historical data of the model not reported.
Qualifier:
according to
Guideline:
other: OECD Guideline 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”, Paris 22 July 2010.
Deviations:
yes
Remarks:
-missing data on acceptability of the QC data with reference to historical batch data, reference to historical control data
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
yes
Remarks:
-reference to historical data of the model not reported.
Qualifier:
according to
Guideline:
other: Regulation (EC) 761/2009: B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test"
Deviations:
yes
Remarks:
- missing data on acceptability of the QC data with reference to historical batch data, reference to historical control data
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): GLUCOSE SYRUPS WHEAT HYDROLYZED, Dried wheat glucose syrup, Dried Glucose Syrup
- Physical state: Solid (white, slightly hygroscopic powder)
- Analytical purity: 38.2 (DE )
- Lot/batch No.: 5093187
- Expiration date of the lot/batch: June 2011
- Storage condition of test material: Room temperature, no protection from light
- Other: pH: neutral; Solubility: good soluble in water

Test animals

Species:
human
Strain:
other: not applicable (in vitro human skin model)
Details on test animals and environmental conditions:
MatTek´s EpiDerm System consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, 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 EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm Ø) and shipped as kits, containing 24 tissues on shipping agarose.

Test system

Type of coverage:
other: not applicable (in vitro human skin model)
Preparation of test site:
other: not applicable (in vitro human skin model)
Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg; The "spoonful" was levelled by gently scratching the excess material away, avoiding compression. 25 μL H2O were added for wetting of the test substance.
Duration of treatment / exposure:
EpiDerm Skin Corrosivity Test: 3 minutes; 1 hour
EpiDerm Skin Irritation Test: 1 hour
Number of animals:
EpiDerm Skin Corrosivity Test: 2 tissue replicates
EpiDerm Skin Irritation Test: 3 tissue replicates
Details on study design:
1) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue (MTT)-test
After incubation with the test substance, post-incubation (EpiDerm Skin Irritation Test) and washing with PBS, the tissues were incubated with MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated from all wells and the tissues were gently rinsed with phosphate buffered saline (PBS) (2 times). For extraction, the tissues were incubated with extractant solution (isopropanol) for 2 hours with shaking.
After the extraction period, the tissues were pierced with an injection needle and the extract (now a blue formazan solution) was allowed to run into the well from which the tissue was taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were homogeneous in colour.

2) Cell viability measurement
- EpiDerm Skin Corrosivity Test:
Per each tissue 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate and the optical density (OD) was measured using the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.
- EpiDerm Skin Irritation Test:
Per each tissue 2 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate and the OD was measured using the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.

3) Assay acceptance criteria
- EpiDerm Skin Corrosivity Test:
In the protocol INVITTOX n°119 by ECVAM the following assay acceptance criteria are defined:
• The mean OD 570 nm of the two negative control tissues is ≥ 0.8.
• The mean tissue viability of the 3 minutes positive control is ≤ 30 %.
• The inter tissue viability difference is < 30 % between two identically treated tissues.
- EpiDerm Skin Irritation Test:
• The mean OD 570 nm of the negative control tissues is ≥ 1.0 and ≤ 2.5.
• The mean tissue viability of the positive control is ≤ 20 %.
• The standard deviation calculated from individual percentual tissue viabilities of the 3 identically treated replicates is < 18 %.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Skin Corrosion tissue viability (%)
Basis:
mean
Time point:
other: 3 min
Score:
97.8
Max. score:
100
Reversibility:
no data
Remarks on result:
other: Non-corrosive
Irritation parameter:
other: Skin Corrosion tissue viability (%)
Basis:
mean
Time point:
other: 1 h
Score:
98.2
Max. score:
100
Reversibility:
no data
Remarks on result:
other: Non-corrosive
Irritation parameter:
other: Skin irritation tissue viability (%)
Basis:
mean
Time point:
other: 1 h
Score:
96.9
Max. score:
100
Reversibility:
no data
Remarks on result:
other: Non-irritant
Irritant / corrosive response data:
See Tables 1 to 4
Other effects:
No unforeseen events occurred which could impede the outcome of this study.

Any other information on results incl. tables

Table 1: EpiDerm Skin Corrosivity Test: Assay acceptance criteria, validity of the test

Sample

Mean tissue viability

(%)

Assay acceptance

criteria

Acceptable

Yes/No

positive control

16.8

≤ 30 %

Yes

 

mean OD

 

negative control

 

3 minutes 1.949

1 hour 1.907

≥ 0.8

Yes

 

 

Maximum inter

tissue viability

difference (%)

 

GLUCOSE SYRUPS WHEAT HYDROLYZED

3 minutes 1.6

1 hour 6.7

< 30 %

Yes

OD = optical density

 

Table 2: EpiDerm Skin Irritation Test: Assay acceptance criteria, validity of the test

Sample

Mean tissue viability

(%)

Assay acceptance

criteria

Acceptable

Yes/No

positive control

6.1

≤ 20 %

Yes

 

mean OD

 

negative control

 

2.030

≥ 1.0 and ≤ 2.5

Yes

 

 

SD viability (%)

 

GLUCOSE SYRUPS WHEAT HYDROLYZED

4.6

<18 %

Yes

OD = optical density; SD = standard deviation

 

Table 3: EpiDerm Skin Corrosivity Test: Cell viability measurement

3 minutes test

Substance

Tissue

N

OD – blank corrected

Mean of aliquots

OD mean

Viability (%)

Viability mean (%)

Negative control

1

1.986

1.949

1.949

100.0

100.0

1.918

1.942

2

1.938

1.950

100.0

1.953

1.958

Positive control

1

0.295

0.294

0.327

15.1

16.8

0.291

0.296

2

0.359

0.359

18.4

0.363

0.356

GLUCOSE SYRUPS WHEAT HYDROLYZED

1

1.897

1.891

1.906

97.0

97.8

1.883

1.893

2

1.938

1.921

98.5

1.940

1.884

1 hour test

Negative control

1

1.857

1.847

1.907

96.9

100.0

1.844

1.839

2

1.991

1.966

103.1

1.942

1.966

Positive control

1

0.182

0.185

0.212

9.7

11.1

0.187

0.187

2

0.240

0.238

12.5

0.229

0.246

GLUCOSE SYRUPS WHEAT HYDROLYZED

1

1.928

1.935

1.872

101.5

98.2

1.914

1.962

2

1.814

1.809

94.9

1.801

1.812

OD = optical density

 

Table 4: EpiDerm Skin Irritation Test: Cell viability measurement

Substance

Tissue

N

OD – blank corrected

Mean of aliquots

OD mean

Viability (%)

Viability mean (%)

SD of viability

Negative control

1

2.142

2.145

2.030

105.62

100.0

5.1

2.147

2

1.944

1.946

95.85

1.948

3

2.000

2.001

98.53

2.001

Positive control

1

0.124

0.125

0.124

6.13

6.1

0.4

0.125

2

0.114

0.115

5.64

0.115

3

0.135

0.132

6.50

0.129

GLUCOSE SYRUPS WHEAT HYDROLYZED

1

1.952

1.951

1.967

96.07

96.9

4.6

1.949

2

2.057

2.069

101.90

2.081

3

1.884

1.883

92.72

1.881

OD = optical density; SD = standard deviation

 

Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Remarks:
Criteria used for interpretation of results: other: CLP (EC 1272/2008)