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Description of key information

Zinc monoglycinate sulfate is not expected to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July, 2010
Principles of method if other than guideline:
- Principle of test:
Test conducted similar to OECD guideline 429
- Short description of test conditions:
Groups of 4 CBAKa mice were treated with 25 µL of test material, or with an equal volume of the vehicle alone, on the dorsum of both ears. Treatment was performed once daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected by the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were killed 5 hours later and the draining lymph nodes excised and pooled for each experimental group. A single-cell suspension of lymph node cells was prepared by mechanical disaggregation. The lymph node cell suspension was washed twice in an excess of PBS and then precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18 hours. Pellets were resuspended in TCA and the incorporation of tritiated thymidine measured by β-scintillation counting.
- Parameters analysed / observed: cell proliferation measured by incorporation of tritiated thymidine
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Source: Sigma, Poole, UK.
Purity: 99 %
Species:
mouse
Strain:
CBA/Ca
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac, Bicester, United Kingdom)
- Age at study initiation: 7- 12 weeks
- Housing: in groups of four animals

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, 25%
No. of animals per dose:
4
Details on study design:
MAIN STUDY

Groups of 4 CBA/Ca mice were treated with 25 µL of test material, or with an equal volume of the vehicle alone, on the dorsum of both ears. Treatment was performed once daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected by the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine (2 Ci mmol/L; Amersham International, Amersham, UK). Mice were killed 5 hours later and the draining lymph nodes excised and pooled for each experimental group. A single-cell suspension of lymph node cells was prepared by mechanical disaggregation. The lymph node cell suspension was washed twice in an excess of PBS and then precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18 hours. Pellets were resuspended in TCA and the incorporation of tritiated thymidine measured by B-scintillation counting.

- Criteria used to consider a positive response:
A substance was regarded as a skin sensitizer if, at any test concentration, the proliferation in treated lymph nodes was threefold or greater than that in the concurrent vehicle treated controls.


Positive control substance(s):
other: see 'Remarks'
Statistics:
Not reported
Positive control results:
From the positive control substances, mercury and cobalt resulted in a SI value of over 3 at least in two of three concentrations and were thus considered as dermal sensitisers.
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
at 5 % zinc sulfate
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
at 10% zinc sulfate
Key result
Parameter:
SI
Value:
2.3
Test group / Remarks:
at 25 % zinc sulfate
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS: none reported
Interpretation of results:
GHS criteria not met
Conclusions:
In this study similar to OECD guideline 429 (LLNA), zinc sulfate is not considered to be a dermal sensitiser.
Executive summary:

In a dermal sensitization study similar to OECD guideline 429 (22 July, 2010) with zinc sulfate in DMSO, young adult CBA/CA mice (4/group) were tested in an LLNA. Additionally, besides zinc sulfate, other metal salts were investigated in the LLNA which are known dermal sensitizers and these metal salts are considered to be the positive controls.


No adverse clinical signs or mortality was observed during the study. Stimulation Indices (S.I.) of 1.3, 2.0 and 2.3 were determined with the test item at concentrations of 5, 10 and 25 % in DMSO, respectively. These results indicate that the test substance could not elicit an SI ≥ 3 and is therefore not regarded as skin sensitizer under the conditions of this study.


Proliferation of the treated lymph nodes was in no concentration increased to over 3-fold of that of the vehicle control lymph nodes. Thus, in this study, zinc sulfate is not a dermal sensitizer and does not need to be classified as dermal sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Source: Wako Pure Chemical Industries, Ltd., Osaka, Japan.
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6-8 weeks
Vehicle:
other: ethanol (20 %)
Concentration:
10%
No. of animals per dose:
3
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The mice (n = 3) received 25 µL of test chemical solution, or vehicle alone, on the dorsum of each ear. The application was repeated for three consecutive days. In some experiments, the dorsal surface of each ear was gently abraded by lightly dragging a 19-g needle across the dorsal surface of each ear five times (without causing bleeding) just prior to the application of test chemicals. Four days following the initial application, mice were killed and the draining lymph nodes (auricular and axillary) were excised and pooled per animal. A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200-mesh steel gauge. Lymphocyte suspensions were washed twice in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 culture medium supplemented with 10% fetal calf serum (FCS), 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), 100 µg/ml penicillin and 100 U/ml streptomycin. The cell concentration was adjusted to give 5 E+06 cells/mL. Lymphocyte suspensions were seeded into 96-well microtiter plates at a concentration of 1 E+06 cells/well (5 wells per animal), and cultured with 0.5 µCi [3H]methyl thymidine ([3H]TdR) for 18 h at 37°C in a humidified atmosphere of 5% CO2 in air.
Culture was terminated by automatic cell harvesting. The incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) ± standard deviation per node of three animals for each test group. Increases in [3H]TdR incorporation relative to vehicle-treated controls were calculated for each test group, and expressed as stimulation indices (SI).
Positive control substance(s):
other:
Positive control results:
The SI values observed with CoCl2 and K2Cr2O7 were 4.3 and 3.1, respectively.
Key result
Parameter:
SI
Value:
1.41
Test group / Remarks:
zinc sulfate, 10 %, in 20 % ethanol solution
Parameter:
SI
Value:
4.33
Test group / Remarks:
positive control CoCl2
Parameter:
SI
Value:
0
Test group / Remarks:
negative control, 20 % ethanol solution
Parameter:
SI
Value:
3.16
Test group / Remarks:
positive control K2Cr2O7
Cellular proliferation data / Observations:
In the present study only the results from one treatment group were reported.

THE LOCAL LYMPH NODE ASSAY PERFORMED WITH FIVE METAL SALTS

Chemical

[3H]TdR incorporation (mean cpm±SD (x 10-3)

SI

20% EtOH

1.52± 0.67

-

10% FeSO4

2.03±0.63

1.32

10% MnCl2

1.26±0.08

0.82

10% ZnSO4

2.14±0.77

1.41

10% CuSO4

4.08±1.20

2.67

10% NiSO4

3.56±0.25

2.32

Interpretation of results:
GHS criteria not met
Conclusions:
In the present study conducted by Ikarashi et al. (1992), zinc sulfate is not considered to be a sensitizer in the LLNA.
Executive summary:

In the present study conducted by Ikarashi et al. (1992), zinc sulfate was dissolved in 20% ethanol and applied to the dorsal surface of the ear on three consecutive days. After four days after initial application the mice were killed and the draining lymph nodes were dissected and incubated for 18 hours at 37°C in humidified air with [3H]TdR. After the incubation period the [3H]TdR incorporation was detected and the SI was determined.


Incubation with 10% ZnSO4 in 20% ethanol resulted in a SI value of 1.41. Thus, under the conditions of the present test, the substance was not considered a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
other: Information from the QSAR Toolbox database
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
not specified
Vehicle:
not specified
Key result
Parameter:
EC3
Remarks on result:
other: Value not reported in QSAR Toolbox
Key result
Parameter:
SI
Value:
< 3
Test group / Remarks:
mean of all tested groups
Remarks on result:
other:
Remarks:
Result as reported by the OECD QSAR Toolbox
Interpretation of results:
GHS criteria not met
Conclusions:
The OECD QSAR Toolbox was used in order to gather all available information about the skin sensitizing potential of glycine. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted accoding to OECD guideline 429 (LLNA). Glycine does not meet the classification criteria of Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS) and is therefore no classified as dermal sensitizer.
Executive summary:

No data are available regarding the skin sensitizing potential of glycine. Beside the QSAR prediction of skin sensitization using the VEGA-Module data was also gathered from the OECD QSAR Toolbox. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted accoding to OECD guideline 429 (LLNA). In both cases glycine was niot classified as skin sensitizer because there was no SI value above 3 in the mentioned study reports, thus, glycine is not considered to be a skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS).

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
(Q)SAR
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
VEGA-QSAR:AI inside a platform for predictive toxicology
2. MODEL (incl. version number)
1.1.5
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
O=C(O)CN
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For more detailed information please refer to the 'attached justification' section

5. APPLICABILITY DOMAIN
For more detailed information please refer to the 'attached justification' section

6. ADEQUACY OF THE RESULT
For more detailed information please refer to the 'attached justification' section
Qualifier:
no guideline followed
Principles of method if other than guideline:
Result of a QSAR prediction using VEGA-QSAR platform Skin Sensitization model (CAESAR) 2.1.6.
GLP compliance:
no
Justification for non-LLNA method:
No data are available for the source substance glycine, thus, based on a weight of evidence approach i.a. the results of a QSAR prediction were used to determine the skin sensitizing potential of glycine.
Species:
other: not applicable for an in silico system
Strain:
other: not applicable for an in silico system
Details on test animals and environmental conditions:
not applicable for an in silico system
Vehicle:
other: not applicable for an in silico system
Concentration:
not applicable for an in silico system
No. of animals per dose:
not applicable for an in silico system
Details on study design:
not applicable for an in silico system
Key result
Parameter:
other: not applicable for an in silico system
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Interpretation of results:
study cannot be used for classification
Conclusions:
In the present QSAR calculation using VEGA-QSAR platform and its Skin Sensitization model (CAESAR) 2.1.6. the skin sensitizing potential of Glycine was estimated. There are no indications for skin sensitisation by appliying this QSAR prediction method. The results are considered to be reliable because the substance falls in the applicability domain of the used model. Glycine is not a sensitizier according to this prediction.
Executive summary:

No data are available for the soure substance glycine with regard to its skin sensitizing potential. Thus, a QSAR prediction with the VEGA module was performed. It predicts the skin sensitizing potential based on atom-centered fragments of a set of structural similar molecules. Based on the evaluation summary contained in the prediction results the QSAR estimation can be regarded as reliable. The accuracy of the prediction for similar structures is considered to be good, the concordance for similar structures is good, the Atom Centered Fragments similarity is good. Thus, there are no inconclusive predictions to be discussed and Glycine is predicted as NOT SENSITIZING TO THE SKIN.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance zinc monoglycinate sulfate and the source substances zinc sulfate and zinc bisglycinate consist of the Zn2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance zinc monoglycinate sulfate and the source substance zinc bisglycinate.
It is generally accepted that the Zn2+ cation (as measure for dissolved zinc species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance zinc monoglycinate sulfate is a chelate-complex which consists of the divalent zinc ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Zinc monoglycinate sulfate and the source substance zinc sulfate are ionic and consist of the Zn2+ cation and the respective anions. It is generally accepted that the zinc cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, zinc, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Parameter:
SI
Value:
1.41
Test group / Remarks:
zinc sulfate, 10 %, in 20 % ethanol solution
Remarks on result:
other: Ikarashi et al., 1992
Parameter:
SI
Value:
2.3
Test group / Remarks:
at 25 % zinc sulfate
Remarks on result:
other: Basketter et al., 1999
Parameter:
SI
Value:
2
Test group / Remarks:
at 10 % zinc sulfate
Remarks on result:
other: Basketter et al., 1999
Parameter:
SI
Value:
1.3
Test group / Remarks:
at 5 % zinc sulfate
Remarks on result:
other: Basketter et al., 1999
Parameter:
SI
Value:
< 3
Test group / Remarks:
Glycine: mean of all tested groups
Remarks on result:
other: Result as reported by the OECD QSAR Toolbox
Interpretation of results:
GHS criteria not met
Conclusions:
Zinc monoglycinate sulfate is not considered to be a skin sensitiser.
Executive summary:

The potential of zinc monoglycinate sulfate to elicit skin sensitisation was examined in a read-across approach since the in chemico test (DPRA) is not designed to accommodate the spectrum of reaction mechanisms considered to be associated with sensitising metals. Metal compounds can be readily excluded from testing (EURL ECVAM RECOMMENDATION on the Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing, November 2013). Furthermore, Zn2+ has been shown to be cytotoxic to in vitro cultured cell lines (Borovanský and Riley, 1989). Thus, it is unlikely to yield 2 valid results out of 3 tests according to IATA with zinc monoglycinate. The endpoint is therefore covered with data on zinc sulfate, while glycine is not considered relevant for skin sensitisation.


In the dermal sensitization study by Basketter et al. (1999) similar to OECD guideline 429 (22 July, 2010) with zinc sulfate in DMSO, young adult CBA/CA mice (4/group) were tested in an LLNA. No adverse clinical signs or mortality was observed during the study. Stimulation Indices (S.I.) of 1.3, 2.0 and 2.3 were determined with the test item at concentrations of 5, 10 and 25 % in DMSO, respectively. These results indicate that the test substance could not elicit an SI ≥ 3 and is therefore not regarded as skin sensitizer under the conditions of this study. Proliferation of the treated lymph nodes was in no concentration increased to over 3-fold of that of the vehicle control lymph nodes. Thus, in this study, zinc sulfate is not a dermal sensitizer and does not need to be classified as dermal sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).


In the study conducted by Ikarashi et al. (1992), zinc sulfate was dissolved in 20% ethanol and applied to the dorsal surface of the ear on three consecutive days. After four days after initial application the mice were killed and the draining lymph nodes were dissected and incubated for 18 hours at 37°C in humidified air with [3H]TdR. After the incubation period the [3H]TdR incorporation was detected and the SI was determined. Incubation with 10% ZnSO4 in 20% ethanol resulted in a SI value of 1.41. Thus, under the conditions of the present test, the substance was not considered a sensitizer.


No data are available regarding the skin sensitizing potential of glycine. Beside the QSAR prediction of skin sensitization using the VEGA-Module data was also gathered from the OECD QSAR Toolbox. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted according to OECD guideline 429 (LLNA). In both cases glycine was not classified as skin sensitizer because there was no SI value above 3 in the mentioned study reports and thus, glycine is not considered to be a skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS).


In conclusion, zinc monoglycinate sulfate is not considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The potential of zinc monoglycinate sulfate to elicit skin sensitisation was examined in a read-across approach since the in chemico test (DPRA) is not designed to accommodate the spectrum of reaction mechanisms considered to be associated with sensitising metals. Metal compounds can be readily excluded from testing (EURL ECVAM RECOMMENDATION on the Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing, November 2013). Furthermore, Zn2+ has been shown to be cytotoxic to in vitro cultured cell lines (Borovanský and Riley, 1989). Thus, it is unlikely to yield 2 valid results out of 3 tests according to IATA with zinc monoglycinate. The endpoint is therefore covered with data on zinc sulfate, while glycine is not considered relevant for skin sensitisation.


In the dermal sensitization study by Basketter et al. (1999) similar to OECD guideline 429 (22 July, 2010) with zinc sulfate in DMSO, young adult CBA/CA mice (4/group) were tested in an LLNA. No adverse clinical signs or mortality was observed during the study. Stimulation Indices (S.I.) of 1.3, 2.0 and 2.3 were determined with the test item at concentrations of 5, 10 and 25 % in DMSO, respectively. These results indicate that the test substance could not elicit an SI ≥ 3 and is therefore not regarded as skin sensitizer under the conditions of this study. Proliferation of the treated lymph nodes was in no concentration increased to over 3-fold of that of the vehicle control lymph nodes. Thus, in this study, zinc sulfate is not a dermal sensitizer and does not need to be classified as dermal sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).


In the study conducted by Ikarashi et al. (1992), zinc sulfate was dissolved in 20% ethanol and applied to the dorsal surface of the ear on three consecutive days. After four days after initial application the mice were killed and the draining lymph nodes were dissected and incubated for 18 hours at 37°C in humidified air with [3H]TdR. After the incubation period the [3H]TdR incorporation was detected and the SI was determined. Incubation with 10% ZnSO4 in 20% ethanol resulted in a SI value of 1.41. Thus, under the conditions of the present test, the substance was not considered a sensitizer.


No data are available regarding the skin sensitizing potential of glycine. Beside the QSAR prediction of skin sensitization using the VEGA-Module data was also gathered from the OECD QSAR Toolbox. The QSAR Toolbox revealed two identical entries exhibiting the same result from a test conducted according to OECD guideline 429 (LLNA). In both cases glycine was not classified as skin sensitizer because there was no SI value above 3 in the mentioned study reports and thus, glycine is not considered to be a skin sensitizer according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS).


In conclusion, zinc monoglycinate sulfate is not expected to be a skin sensitiser.