Zinc glycinate sulfate

Endpoint summary

Description of key information

Skin irritation: according to an OECD 439 guideline study, RL1, GLP, zinc monoglycinate sulfate is non-irritant to skin according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Eye irritation: according to an OECD 437 guideline study, RL1, GLP, zinc monoglycinate sulfate does not require classification according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) with respect to 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:

2022-11-02 to 2022-11-18

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:

14 June 2021

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Qualifier:

according to guideline

Guideline:

other: UN GHS (published 2003, last (8th) revision 2019)

Version / remarks:

2019

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 12/04/2020.

Version / remarks:

12/04/2020

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT kits and MTT-100 assays are purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- Tissue batch number(s): lot nr: 36184, keratinocyte strain: 00267
- Shipping date: 2022-11-16
- Delivery date: 2022-11-16
- Date of initiation of testing: 2022-11-16
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Each incubation of the tissues was performed under 37 ± 1.5°C and 5 ± 0.5% CO2 in DMEM Medium.
- Temperature of post-treatment incubation: The post-treatment incubation was under standard incubation conditions (37 ± 1.5°C and 5 ± 0.5% CO2).
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material.
- Observable damage in the tissue due to washing: Optical evaluation of the test item treated tissues revealed no visible damage.
- Modifications to validated SOP: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: On the day of the experiment a MTT solution of 1 mg/mL in DMEM was prepared, 300µL MTT was used.
- Incubation time: The MTT incubation time was 3 hour ± 5 min.
- Spectrophotometer: The microplate reader Versamax® Molecular Devices was used.
- Wavelength: The optical density at OD570nm was determined.
- Filter: The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC, 4 hours, n=3, Viability should be between 1.0 -3.0, viability = 1.499 +/- 0.046, Pass
- Barrier function: ET-50 assay, 100 µL 1% Triton X-100, 4 time points, n=3, MTT assay, ET-50 schould be between 4.77-8.72 hours, ET-50 = 8.05 hours, Pass.
- Contamination: no contamination detected
- Reproducibility: yes
NUMBER OF REPLICATE TISSUES:
Each group (negative control, positive control, test item) was tested in triplicates.
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not prove to be a MTT reducer in the MTT interference pre-experiment.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1 main experiment was used for prediction.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 1 hour exposure is less than or equal to 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after 1 hour exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

DPBS (MatTek)

Duration of treatment / exposure:

Pre-experiment (colour interference test): The treatment in the colour interference test was 3 hours with deionised water under standard conditions, and in parallel 3 hours with isopropanol at room temperature.
Pre-experiment (MTT-interference test): in the MTT interference test treatment was 3 hours under standard conditions.
Main-experiment: The test item and the controls, respectively, were tested in triplicate tissues with an exposure time of 60 minutes. Within this period the 6-well plates were placed in the incubator for 35 minutes at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.

Duration of post-treatment incubation (if applicable):

At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material. Afterwards, the tissues were incubated in 0.9 mL of fresh assay medium for 24 ± 2 hours. After this incubation period the medium was changed, and the tissues were incubated for another 18 ± 2 hours at standard incubation conditions. The complete incubation time was 42 ± 4 hours.
After the 42 hour incubation period was completed, the tissues were transferred to the MTT-plates. After a 3 hour ± 5 minutes incubation period at standard conditions the tissues were rinsed with PBS and carefully dried with blotting paper.
The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted within 2-4 hours while shaking at room temperature.
At the end of the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert were discarded.
Per each tissue, 3 x 200 µL aliquots of the formazan solution were transferred into a 96-well, flat bottom, microtiter plate. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.

Number of replicates:

negative control: 3
positive control: 3
test material: 3

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was tested neat.
In the pre-experiments (colour inteference test and MTT inteference test) 50 ± 2 mg of the test item was applied onto the surface of the tissue.
In the main-experiment 25 ± 2 mg (39.7 mg/cm2 according to guideline) of the test item was applied onto the surface of the tissue.

Duration of treatment / exposure:

Pre-experiment (colour interference test): 3 hours with deionised water under standard conditions, and in parallel 3 hours with isopropanol at room temperature.
Pre-experiment (MTT-interference test): 3 hours under standard conditions.
Main-experiment: Exposure time of 60 minutes in total. The plates were placed for 35 minutes in the incubator at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material.
- Time after start of exposure: 1 hour
OBSERVATION TIME POINTS
60 minutes
SCORING SYSTEM:
- Method of calculation:
1)The mean OD value of the three wells for each tissue and the blank control (ODBlk) was calculated (Mean [OD570] (well 1, well 2 and well 3).
2) The mean ODBlk was subtracted from each mean OD value of the three wells.
Mean [OD570] blank corr. (well 1, well 2 and well 3)). These values were used for all further calculations below.
3) The mean OD of the three relating tissues for each test group (negative control (NC), positive control (PC)) and the test item (TI) were calculated with the blank corrected mean OD (Mean [OD570] of T1, T2 and T3).
4) The percent viability of each test group relative to the negative control (= 100%) was calculated:
Viability (%)=100 ×〖mean OD〗_(TI⁄PC/NC)/〖mean OD〗_NC
5) The viability of each test group was calculated for each tissue replicate.
6) The standard deviation between the OD values within one test group was calculated (not reported). In addition, the standard deviation between the viability values within one test group was calculated.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

57.88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Pre-experiment:

 

Assesment of Colour Interference:

Treatment Group	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Evaluation Mean OD570 (blank corrected) > 0.08
Blank Aqua Deion.	0.037	0.036	0.037
Test Item + Aqua Deion.	0.042	0.041	0.042	0.005	no
Blank Isopropanol	0.037	0.038	0.037
Test Item+ Isopropanol	0.043	0.044	0.044	0.007	no

The mean OD of the test item in deionised water or isopropanol was < 0.08 and therefore, an additional test with viable tissues without MTT addition was not necessary in the main experiment.

 

 

Assesment of MTT Interference:

In the pre-experiment for MTT interference the test item did not reduce MTT to Formazan salt after optical evaluation. Therefore, an additional test with freeze-killed tissues was not necessary for the quantitative correction of the test item viability.

 

 

Main-experiment:

Results after treatment with Zinc Monoglycinate Sulfate Hydrate and the controls

Treatment Group	Tissue No.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
		Well 1	Well 2	Well 3
Blank		0.040	0.040	0.040	0.040
Negative Control	1	1.737	1.700	1.669	1.702	1.662	1.631	101.942	1.7	100.0
	2	1.647	1.649	1.655	1.650	1.611		98.784
	3	1.648	1.670	1.658	1.658	1.619		99.274
Positive Control	1	0.134	0.123	0.117	0.125	0.085	0.073	5.217	0.7	4.47
	2	0.106	0.104	0.102	0.104	0.064		3.941
	3	0.109	0.110	0.108	0.109	0.069		4.238
Test Item	1	0.967	0.954	0.949	0.957	0.917	0.944	56.227	1.7	57.88
	2	0.989	0.978	0.983	0.983	0.944		57.865
	3	0.999	1.016	1.018	1.011	0.971		59.553

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported the test item is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the skin irritation potential of Zinc Monoglycinate Sulfate Hydrate by means of the Human Skin Model Test.

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. The OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Three tissues each of the human skin model EpiDerm^™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD => 0.8 and <= 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item Zinc Monoglycinate Sulfate Hydrate the mean relative viability value was 57.88% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%.

Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, Zinc Monoglycinate Sulfate Hydrate is non-irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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:

From 2022-10-28 to 2022-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (Reach).

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Version / remarks:

April 1997

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:

26th June 2020

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

09 Dec 2010

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals (e.g. age, sex, weight): The slaughterhouse did not confirm the age of the donor cattle despite repeated requests.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes.
- Indication of any existing defects or lesions in ocular tissue samples:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
- Indication of any antibiotics used: 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium)
- Selection and preparation of corneas: Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
- Quality check of the isolated corneas: Only corneas with a value of the basal opacity < 7 were used.

Vehicle:

physiological saline

Remarks:

(0.9% NaCl in deionised water)

Controls:

yes

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: The test item was tested as a 20% solution (w/v) in saline.
VEHICLE
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

The incubation period was 240 minutes.

Duration of post- treatment incubation (in vitro):

The anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.

Number of animals or in vitro replicates:

Number of Corneas:
15 corneas where used for the experiment (three per group):
Negative Control I: 3
Negative Control II: 3
Positive Control I: 3
Positive Control II: 3
Test Item: 3

Details on study design:

NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: deionised water
SOLVENT CONTROL USED: Saline (0.9% NaCl in deionised water)
POSITIVE CONTROL USED
Positive Control 1: 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline using sonication.
Positive Control 2: 20 % (w/v) Imidazole in saline
APPLICATION DOSE AND EXPOSURE TIME
The test item was tested as a 20% solution (w/v) in saline.
The incubation period was 240 minutes.
TREATMENT METHOD: closed chamber
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of the test item the corneas were given a final rinse with cMEM without phenol red. Fresh cMEM was added into both compartments and opacity was measured (t240).
- POST-EXPOSURE INCUBATION:
After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer determines changes in the light transmission passing through the corneas and displays a numerical opacity value. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490).
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
DECISION CRITERIA: according to guideline

Irritation parameter:

in vitro irritation score

Run / experiment:

1 Experiment with five groups (two negative controls, two positive controls, one test item)

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

 

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation IVIS	Proposed Category
		Mean		Mean
Negative Control (Saline)	0	0.33	0.071	0.067	1.07	1.34	0.58	No Category
	1		0.067		2.01
	0		0.064		0.96
Negative Control (deionised water)	1		0.065		1.98	1.29	0.60	No Category
	0		0.060		0.90
	0		0.066		0.99
Positive Control (10 % Benzalkonium chloride)	76.67*		0.768*		88.18	91.70	3.05	Category 1
	82.67*		0.710*		93.31
	85.67*		0.529*		93.60
Positive Control (20 % Imidazole)	88.67*		0.769*		100.20	101.15	4.74	Category 1
	80.67*		1.086*		96.95
	87.67*		1.242*		106.29
Test Item	1.67*		0.00**		1.67	1.00	0.58	No Category
	0.67*		0.00**		0.67
	0.67*		0.00**		0.67

* corrected values

** Value was set to zero since the calculated value was negative

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not categorized (EU CLP/GHS No Category).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Zinc Monoglycinate Sulfate Hydrate by means of the BCOP assay using fresh bovine corneas (OECD 437, 2013-07-26).
After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) solution in saline (0.9% (w/v) NaCl in deionised water) of the test item Zinc Monoglycinate Sulfate Hydrate as well as the positive and the negative controls were each applied to different corneas fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240).
After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
With the negative controls saline and deionised water, neither an increase of opacity nor permeability of the corneas could be observed (saline mean IVIS = 1.34; deionised water mean IVIS = 1.29).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 91.70) corresponding to a classification as serious eye damaging (EU CLP/GHS (Category 1) as well as the positive control imidazole (mean IVIS = 101.15).

Based on these results, zinc monoglycinate sulfate dihydrate does not require classification according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) with respect to eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available