Zinc nitrate

Endpoint summary

Description of key information

The in vitro skin irritation/corrosion test in the EpiDerm model with zinc nitrate indicates that the test item is a skin irritant and non-corrosive.

 

Based on the in vitro eye irritation assay in the isolated chicken eyes test with zinc nitrate the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification. To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showedfrom slight to severe vacuolation of the epithelium in 6/6 cases. Slight erosion of the corneal epithelium in 5/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, zinc nitrate was classified as Category 1.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure: approximately 25 mg of the test article + 25 µl tissue culture water (negative control) were applied on the top of each EpiDerm tissue (9mm in diameter)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffered saline (PBS)
- Time after start of exposure: test article remained in contact with the EpiDerm tissue for 3 and 60 minutes.

SCORING SYSTEM:
After washing EpiDerm tissue was transferred to a 24 well plate contaiing 300µl MTT solution. Afterwards the tissues were returned to the incubator for a three-hour MTT incubation period. Following MTT incubation period, each EpiDerm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbancy of an aliquot of the extracted MTT formazan was measured at 540nm using a microplate reader.

Analysis of data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability= 100x(OD sample/OD negative control)

Quality controls: negative controls meets the acceptance criterion if the mean OD of the 2 tissues each time point is greater than or equal to 0.8. The positive controls meets the acceptance criterion if the mean relative tissue viability at the 3 minute time point is less than or equal to 30%
Inter-tissue viability meets the acceptance criterion if the difference between two identically treated tissues is no greater than 30%

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg of the test article, plus 25 ul tissue culture water were applied to the top of each EpiDerm tissue

Duration of treatment / exposure:

3 and 60 min

Details on study design:

TEST SITE
- Area of exposure: approximately 25 mg of the test article + 25 µl tissue culture water (negative control) were applied on the top of each EpiDerm tissue (9mm in diameter)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffered saline (PBS)
- Time after start of exposure: test article remained in contact with the EpiDerm tissue for 3 and 60 minutes.

SCORING SYSTEM:
After washing EpiDerm tissue was transferred to a 24 well plate contaiing 300µl MTT solution. Afterwards the tissues were returned to the incubator for a three-hour MTT incubation period. Following MTT incubation period, each EpiDerm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbancy of an aliquot of the extracted MTT formazan was measured at 540nm using a microplate reader.

Analysis of data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability= 100x(OD sample/OD negative control)

Quality controls: negative controls meets the acceptance criterion if the mean OD of the 2 tissues each time point is greater than or equal to 0.8. The positive controls meets the acceptance criterion if the mean relative tissue viability at the 3 minute time point is less than or equal to 30%
Inter-tissue viability meets the acceptance criterion if the difference between two identically treated tissues is no greater than 30%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 3 min

Value:

79.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 60 min

Value:

49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPIDERM model test with zinc nitrate hexahydrate, the results indicate that the test item is not a skin corrosive.

Executive summary:

A study was conducted to predict and classify the skin corrosivity potential of zinc nitrate hexahydrate by using a three-dimensional human epidermis model (OECD 431). MatTek EpiDerm tissue samples were treated in duplicate with the test article, negative control and positive control for 3 minutes and 60 minutes. Following treatment, the viability of the tissues was determined using MTT uptake and conversion, and the absorbance of each sample was measured at 540nm. The viability was then expressed as a percent of control values.

In this in vitro EPIDERM model test with zinc nitrate hexahydrate, the results indicate that the test item is not a skin corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

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)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure: after moistening the EpiDerm tissue with 25µl PBS before dosing, 25 mg of the test article was added into the Millicell atop the tissue

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffered saline (PBS)
- Time after start of exposure: test article remained in contact with the EpiDerm tissue for 60 minutes.

SCORING SYSTEM:
After washing EpiDerm tissue was transferred to a 24 well plate contaiing 300µl MTT solution. Afterwards the tissues were returned to the incubator for a three-hour MTT incubation period. Following MTT incubation period, each EpiDerm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbancy of an aliquot of the extracted MTT formazan was measured at 540nm using a microplate reader.

Analysis of data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability= 100x(OD sample/OD negative control)

Quality controls: negative controls meets the acceptance criterion if the mean OD of the 2 tissues each time point is greater than or equal to 1.0 and smaller than or equal to 2.5. The positive controls meets the acceptance criterion if the mean relative tissue viability, expressed as percentage of the negative control tissues is less than or equal to 20%
the SD calculated from individual percent tissue viabilities of the three identically treated replicates is less than 18%

Amount/concentration applied:

25mg

Duration of treatment / exposure:

60 min

Irritation / corrosion parameter:

% tissue viability

Value:

38.9

Remarks on result:

positive indication of irritation

Remarks:

Basis: mean. Time point: 60min. Remarks: recovery period: 42h. (migrated information)

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Following exposure with zinc nitrate hexahydrate, the mean treated skin value was 38.9% and therefore irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Executive summary:

A study was conducted to predict dermal irritation potential of Zinc nitrate hexahydrate in the context of identification and classification of skin irritation hazard according to the EU classification (R38 or no label) and GHS classification system (category 2 and non-irritants) by using a three-dimensional human epidermis model (OECD 439). MatTek EpiDerm tissue samples were treated in triplicate with the test article, negative control and positive control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using MTT uptake and conversion, and the absorbance of each sample was measured at 540nm. The viability was then expressed as a percent of control values.

In this in vitro EPIDERM model test with Zinc nitrate hexahydrate, the results indicate that the test item is irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

version 26th July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30mg

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

not applicable

Details on study design:

treatments:
-test substance treated chicken eye: treated with 30 mg zinc nitrate hexahydrate
-positive control chicken eye: treated with 30 mg imidazole
-negative control eye: treated with 30µL isotonic saline



REMOVAL OF TEST SUBSTANCE
- Washing (if done): cornea surface was rinsed thoroughly with 20ml isotonic saline
- Time after start of exposure: after 10'' of exposure

SCORING SYSTEM:
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within ±5 minutes were considered acceptable.

The cornea thickness and cornea opacity were measured at all time points.
Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.


TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope/fluorescein

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 75 minutes

Value:

3.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 240 minutes

Value:

12.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

histopathological observations

Remarks:

slight to severe vacuolation of the epithelium in 6/6 cases. Slight erosion of the corneal epithelium in 5/6 sections. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity and fluorescein retention are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.9 %	I
Mean maximum corneal swelling at up to 240 min	12.2 %	II
Mean maximum corneal opacity	2.00	III
Mean fluorescein retention	2.00	III
Other Observations	Test item was stuck on all cornea surfacesafter the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Overall ICE Class	1xII 2xIII

 Based on this in vitro eye irritation in the isolated chicken eyes test with ZINC NITRATE HEXAHYDRATE,the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

 

Positive Control

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10.9 %	II
Mean maximum corneal swelling at up to 240 min	29.0 %	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.

 

NEGATIVE Control

 

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.6 %	I
Mean maximum corneal swelling at up to 240 min	1.6 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

The negative control Physiological saline was classifiedas non-irritating, UN GHS Classification:No Category.

Table:Assessment of the general IN VITRO eye irritancy and regulatory GHS classification.

 

UN GHS Classification	Combinations of the three ICE Classes
No Category	3×I 2×I, 1×II
No prediction can be made	Other combinations
Category 1	3×IV 2×IV, 1×III 2×IV, 1×II^* 2×IV, 1×I^* Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) Corneal opacity = 4 at any time point (in at least 2 eyes) Severe loosening of epithelium (in at least 1 eye)

Remark:^*:combinations of categories less likely to occur

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.

Conclusions:

Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg of powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline(0.9% (w/v) NaCl solution).

In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.
Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid. Slight corneal swelling was observed during the four-hour observation period on test item treated eyes. Moderate corneal opacity change (severity 1 or 2 or 3) was noted on all three eyes. Moderate fluorescein retention change (severity 2.0) was noted on all three eyes.

 

Based on this in vitro eye irritation assay in the isolated chicken eyes test with ZINC NITRATE HEXAHYDRATE the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showedfrom slight to severe vacuolation of the epithelium in 6/6 cases. Slight erosion of the corneal epithelium in 5/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, ZINC NITRATE HEXAHYDRATE was classified as Category 1.

Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation: In vitro data shows that zinc nitrate was irritating to skin and therefore requires, according to GHS criteria, skin irrit. 2: H315: Causes skin irritation

Eye irritation: taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion that the classification of UN GHS is eye irritant, Category 1,H318: Causes serious eye damage