Tyrosine

Endpoint summary

Description of key information

The skin irritancy potential of L-Tyrosine was investigated in a human epidermis model (Epiderm) test.

The test item was found to not cause skin irritation.

The eye irritancy potential of L-Tyrosine was investigated in the bovine corneal opacity and permeability assay.

The test item was found to not cause eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

GLP compliance:

yes (incl. QA statement)

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.

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%.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

94.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study.

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 OD_{570 nm}of 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%

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.

Endpoint conclusion

Endpoint conclusion:

no study available

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.07.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 99.9%

Species:

cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: isolated corneas obtained on the test day as a by-product from animals freshly slaughtered (abattoir A. Moksel AG, Buchloe, Germany)
- Transport: in HBSS containing Pen/Strep on ice. Immediately after arrival of the eyes, cornea preparation was initiated
- Inspection: carefully examined for defects and any defective eyes were discarded
- Preparation of the tissue: the tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS
- Preparation of the corneas: corneas were mounted in corneal holders (endothelial side against the O-ring of the posterior chamber). The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI) for 1 hour at 32 ± 1 °C (equilibration period)

Vehicle:

physiological saline

Remarks:

20% concentration

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Volume: 750 μL

Duration of treatment / exposure:

4 hours ± 5 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3 corneas for the test item, the positive control and the negative control, respectively

Details on study design:

Treatment of the corneas
- An initial measurement was performed on each of the corneas (opacitometer). Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay
- The medium was removed from the anterior chamber and replaced with the test item or control
- 750 μL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method) for 4 hours ± 5 minutes incubation at 32 ± 1 °C. Then test substance or control substance was removed and the epithelium washed at least three times with MEM (containing phenol red) till the medium was free of test substance, finally the cornea was rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Corneas were observed visually and pertinent observations were recorded.

- After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Acceptance criteria
- The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- The negative control responses resulted should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Evaluation of results
- The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer: Opacity = ( I0/I-b)/a with a = 0.025 and b = 0.9894.
The value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.

- The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

- The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

- The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

-The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

- The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: IVIS ≤ 3: no category; > 3 ≤ 55: no prediction can be made; > 55: category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

3 corneas

Value:

>= 1.45

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The acceptability criteria were fullfilled and the validity of the test system was confirmed

 In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.64	0.005
2		1.18	0.019
3		0.9	0.014
MV		0.9	0.013	1.09
4	Positive Control	112.97	1.757
5		110.15	1.837
6		98.06	2.977
MV		107.06	2.191	139.92
7	Test Item	1.06	-0.002
8		0.88	-0.006
9		2.7	-0.013
MV		1.55	-0.007	1.45

MV: Mean Value

Interpretation of results:

GHS criteria not met

Conclusions:

According to the evaluation criteria the test item L-Tyrosine is classified into 'UN GHS No Category'.

Executive summary:

In the current study the eye irritation potential of the test item L-Tyrosine was investigated in the bovine corneal opacity and permeability assay. The test was performed according to OECD 437 and in compliance to GLP.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Thus the acceptability criteria were fullfilled and the validity of the test system was confirmed.

The test item was suspended with physiological saline 0.9% NaCl to obtain a 20% concentration.

All 3 corneas treated with L-Tyrosine showed no opacity of the tissue but the test item bequeathed few white spots.

The following mean in vitro irritation score was calculated:

1.45

Therefore the test item was classified into UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation:

In the in vitro Epiderm test, the mean relative tissue viability was found to be 94.9. As this is above the >= 50% threshold, classification of the test item for skin irritating effects is not necessary.

Eye irritation:

In the bovine corneal opacity and permeability assay the IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: IVIS ≤ 3: no category; > 3 ≤ 55: no prediction can be made; > 55: category 1.

All 3 corneas treated with L-Tyrosine showed no opacity of the tissue and the mean in vitro irritation score was calculated as 1.45.

Therefore the test item does not need to be classified for eye irritation effects.