Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
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:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (R)-3-Ammonium-1-hydroxybutyl (S)-mandelat
- Physical state: solid / white
- Analytical purity: 99.8%
- Lot/batch No.: 15-0010

Test animals

Species:
other: human epidemis model
Strain:
other: in vitro
Details on test animals and environmental conditions:
Three dimensional human epidermis model (SCT and SIT)
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, 10 mm ) and are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Test system

Type of coverage:
other: in vitro
Preparation of test site:
other: in vitro test
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro test
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Corrosion test: 50 µl (25 µl de-ionized water, an 25 µl of the solid test material )
Irritation test: 50 µl (25 µl de-ionized water, an 25 µl of the solid test material )
Duration of treatment / exposure:
Corrosion test: 3 min and 1 hour
Irritation test: 1 hour folloed by a 42-hours post-incubation period
Details on study design:
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, 10 mm ) and are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: EPI-200
Tissue Lot Number: 21699 (1st test run, SIT) and 23301 (2nd test run SIT; SCT) (Certificates of Analysis see appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Corrosion test: From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The pre-incubation medium was replaced with fresh medium immediately before application. Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. 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. Control tissues were concurrently treated with 50 µL of de-ionized water (NC) or with 50 µL of 8 N potassium hydroxide (PC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated 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 4 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.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 ca. 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently treated with 30 µL of sterile PBS (NC) or with 30 µL of 5% SDS (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 placed into 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 post-incubation 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 4 microtiter wells filled with isopropanol for each microtiter plate.


Results and discussion

Any other information on results incl. tables

Corrosion test

Exposure period 3 min: Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation

Exposure period: 3 min

Test substance

 

tissue 1

tissue 2

Mean

SD

CV[%]

NC

mean OD570

2.204

2.091

2.147

 

 

viability

[% of NC]

102.6

97.4

100.0

3.7

3.7

15/0396-1

mean OD570

1.888

2.102

1.995

 

 

viability

[% of NC]

87.9

97.9

92.9

7.0

7.6

PC

mean OD570

0.314

0.290

0.302

 

 

viability

[% of NC]

14.6

13.5

14.1

0.8

5.5

 

Exposure period 1 h: Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation

Exposure period: 1 h

Test substance

 

tissue 1

tissue 2

Mean

SD

CV[%]

NC

mean OD570

2.076

2.135

2.106

 

 

viability

[% of NC]

98.6

101.4

100.0

2.0

2.0

15/0396-1

mean OD570

1.673

1.458

1.566

 

 

viability

[% of NC]

79.5

69.3

74.4

7.2

9.7

PC

mean OD570

0.111

0.131

0.121

 

 

viability

[% of NC]

5.3

6.2

5.7

0.7

11.4

 

Irritation test

1sttest run: Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation

Test substance

 

tissue 1

tissue 2

tissue 3

mean

SD

CV[%]

NC

mean OD570

2.152

2.297

2.184

2.211

 

 

viability

[% of NC]

97.3

103.9

98.8

100.0

3.4

3.4

15/0396-1

mean OD570

1.164

0.896

1.573

1.211

 

 

viability

[% of NC]

52.7

40.5

71.1

54.8

15.4

28.2

PC

mean OD570

0.069

0.081

0.076

0.075

 

 

viability

[% of NC]

3.1

3.7

3.4

3.4

0.3

8.0

 

 

2ndtest run: Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation

Test substance

 

tissue 1

tissue 2

tissue 3

mean

SD

CV[%]

NC

mean OD570

2.330

2.267

2.599

2.399

 

 

viability

[% of NC]

97.1

94.5

108.3

100.0

7.3

7.3

15/0396-1

mean OD570

1.592

1.646

1.666

1.635

 

 

viability

[% of NC]

66.4

68.6

69.5

68.1

1.6

2.4

PC

mean OD570

0.074

0.072

0.072

0.072

 

 

viability

[% of NC]

3.1

3.0

3.0

3.0

0.0

1.6

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information
Conclusions:
Based on the observed results and applying the evaluation criteria described in chapter 3.8 it was concluded, that (R)-3-Ammonium-1-hydroxybutyl (S)-mandelat does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen. Hence, (R)-3-Ammonium-1-hydroxybutyl (S)-mandelat is not assigned to a transport category.
Executive summary:

Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 92.9%, and it was 74.4% after an exposure period of 1 hour.

Irritation test:

1sttest run: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hourwith about 42 hours post-incubationwas 54.8%.  

Due to non-concordant replicate measurements of the test-substance treated tissues (values for single tissues: 52.7%, 40.5% and 71.1%) and the borderline result another test run was performed to clarify the result.

2ndtest run: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 68.1% (values for single tissues: 66.4%, 68.6% and 69.5%). All acceptance criteria were met.