Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09.-11.08.2017
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Source: Ajinomoto Co., Inc. (Kawasaki, Japan)
- CAS n°: 60-18-4
- Lot n°: 555012G

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: normal human epidermal keratinocytes
Source strain:
other: not applicable
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.63 cm.
- Pre-incubation: 60 ± 5 minutes in the incubator (37 ± 1 °C, 5% CO2).
Then transferred into new wells and incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Negative control: 30 µL DPBS
- Positive control: 30 µL 5% SDS solution
- Test Item: 25 mg + 25 µL DPBS

Duration of treatment / exposure:
60 ± 1 minutes
Duration of post-treatment incubation (if applicable):
approx. 42 hours
Number of replicates:
The test was performed on a total of 3 tissues per dose group.

Test system

Details on study design:
Details of the test procedure used:
- Washing:15 times with DPBS
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 0.3 mL of MTT solution 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
- Data evaluation: the following was calculated: The mean OD of the three negative control tissues was calculated after blank correction. The mean of the photometric absorbance of the negative control is set to 100%.

Data Analysis:
For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = (mean OD test item / positive control / mean OD negative control) x 100.
For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated

Prediction model:
see table below

Test Acceptance Critria:
The test meets acceptance criteria if:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.


Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Single test with three tissues
Value:
94.9
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The controls confirmed the validity of the study.

Any other information on results incl. tables

Result of the Test Item L-Tyrosine

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

absolute OD570 

1.635

1.509

1.498

0.106

0.096

0.102

1.365

1.502

1.552

1.595

1.511

1.510

0.110

0.098

0.102

1.389

1.425

1.565

OD570(blank-corrected)

1.592

1.466

1.455

0.062

0.053

0.059

1.322

1.459

1.508

1.552

1.468

1.467

0.066

0.055

0.059

1.346

1.382

1.522

mean OD570 of the duplicates (blank-corrected)

1.572

1.467

1.461

0.064

0.054

0.059

1.334

1.420

1.515

total mean OD570 of 3 replicate tissues (blank-corrected)

1.500*

0.059

1.423

SD OD570 

0.062

0.005

0.091

relative tissue viability [%]

104.8

97.8

97.4

4.3

3.6

3.9

88.9

94.7

101.0

mean relative tissue viability [%]

100.0

3.9**

94.9

SD tissue viability [%]***

4.2

0.4

6.0

CV [% viabilities]

4.2

8.9

6.4

 

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is  20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is 18%

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 irritant effects.
The test item is therefore considered as non-irritant to skin.
Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. The test was performed according to OECD TG 439 and in compliance to GLP.

In the present study L-Tyrosine was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.9%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD570of the three negative control tissues was > 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.9%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4% - 6.0%).

In conclusion, in this study under the given conditions the test item showed no irritant effects.

The test item is therefore considered as non-irritant to skin.