Diammonium tetrachlorozincate(2-)

Endpoint summary

Description of key information

The in vitro skin irritation/corrosion test in the EpiDerm model with Diammonium tetrachlorozincate indicates that the test item is non-corrosive and non-irritant to skin .

Based on the in vitro eye irritation assay in the isolated chicken eyes test with Diammonium tetrachlorozincate, the test item is a severe eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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:

version 28 July 2015

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM) (Manufacturer: SkinEthic, France
- Tissue batch number(s):18-EKIN-017
- Expiry Date: 30 April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 22.1-24.2 °C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1wahsing step: rinsing thoroughly with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE (see any other info on mat and meth)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required

NEGATIVE CONTROL: PBS
- Concentration (if solution): 50µl

POSITIVE CONTROL: SDS 5%
- Concentration (if solution): 50µl

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

74.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented below: 

 

Table: Optical Density mean (OD) and the calculated relative viability % of the samples

Substance		Optical Density (OD)			Viability  (% RV)
			Measured	Blank corrected
Negative Control:		1	0.837	0.790	100.0
Phosphate buffered saline		2	0.835	0.788	99.9
		3	0.837	0.790	100.1
		mean	--	0.789	100.0
Positive Control:		1	0.085	0.038	4.8
5% (w/v) SDS solution		2	0.105	0.058	7.4
		3	0.094	0.047	6.0
		mean	--	0.048	6.0
Test Item:		1	0.650	0.603	76.4
diammonium tetrachlorozincate(2 -)		2	0.622	0.575	72.9
		3	0.637	0.590	74.7
		mean	--	0.589	74.7

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKINTM (SM) model test with diammonium tetrachlorozincate(2-), the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

Executive summary:

An in vitro skin corrosivity and irritation test of diammonium tetrachlorozincate(2-) was performed in a reconstructed human epidermis model.EPISKIN^TM(SM) is designed to predict and classify thecorrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity and irritation potential of the test item was evaluated according to theOECD No. 431 and No. 439 guidelines. 

Disks of EPISKIN^TM(SM) were treated with the test item and incubated for 15 minutes (irritation testing) and 4 hours (corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO_2,in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO₂protected from light,in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. Two additional disks were used to provide in each case an estimate of colour contribution (NSC_living) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Corrosivity testing:

Following exposure with diammonium tetrachlorozincate(2-), the mean cell viability was 81.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive to skin.

Irritation testing:

Following exposure with diammonium tetrachlorozincate(2-), the mean cell viability was 74.7% compared to the negative control. This is above the threshold of 50%, thereforethe test item was considered as being non-irritant to skin.

 

In conclusion, in thisin vitroEPISKIN^TM(SM)model test withdiammonium tetrachlorozincate(2-), the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

 

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

version 29 July 2016

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (Manufacturer: SkinEthic, France, Catalogue Number: EPISKIN/S/13
- Tissue batch number(s):18-EKIN-017
- Expiry Date: 30 April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23-24.6°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1wahsing step: rinsing thoroughly with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2.2 mL of 0.3 mg/mL MTT
- Incubation time: 3h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES:2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE (see any other info on mat and meth)


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement):

- The test substance is considered to be corrosive to skin, if the mean relative viability after 4 hours of exposure is below 35% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required

NEGATIVE CONTROL: NaCI (9 g/l saline)
- Concentration (if solution): 50µl

POSITIVE CONTROL: glacial acetic acid
- Concentration (if solution): 50µl

Duration of treatment / exposure:

4h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

81.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

The results of the optical density (OD) measured at 570 nm of each extract and the calculated % viability of the cells is presented below:

 

Substance			Optical Density (OD)				Viability
				Measured	Blank corrected		(% RV)
Negative Control:			1	0.837	0.790		99.1
Physiological saline			2	0.852	0.805		100.9
(0.9% (w/v) NaCl)			mean	--	0.798		100.0
Positive Control:			1	0.049	0.002		0.3
Glacial acetic acid			2	0.050	0.003		0.4
			mean	--	0.003		0.4
Test Item:			1	0.682	0.635		79.6
diammonium tetrachlorozincate(2-)			2	0.709	0.662		83.0
			mean	--	0.649		81.3

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKINTM (SM) model test with diammonium tetrachlorozincate(2-), the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

Executive summary:

An in vitro skin corrosivity and irritation test of diammonium tetrachlorozincate(2-)was performed in a reconstructed human epidermis model.EPISKIN^TM(SM) is designed to predict and classify thecorrosivity and irritationpotential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity and irritation potential of the test item was evaluated according to theOECD No. 431 and No. 439guidelines. 

Disks of EPISKIN^TM(SM)were treated withthe test itemand incubated for 15 minutes (irritation testing) and4 hours (corrosivity testing)at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). Theepidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO_2,in a > 95% humidified atmosphere(irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO₂protected from light,in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. Two additional disks were used to provide in each case an estimate of colour contribution (NSC_living) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Corrosivity testing:

Following exposure withdiammonium tetrachlorozincate(2-), the mean cell viability was 81.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive to skin.

Irritation testing:

Following exposure withdiammonium tetrachlorozincate(2-), themean cell viability was 74.7% compared to the negative control. This is above the threshold of 50%, thereforethe test item was considered as being non-irritant to skin.

 

In conclusion, in this in vitro EPISKIN^TM(SM) model test with diammonium tetrachlorozincate(2-), the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

 

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

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 09 October 2017

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 diammonium tetrachlorozincate
-positive control chicken eye: treated with 30 mg imidazole
-negative control eye: treated with 30µL physiological saline (0.9% (w/v) NaCl solution

REMOVAL OF TEST SUBSTANCE
- Washing (if done): cornea surface was rinsed thoroughly with 20ml physiological 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:

4.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 240 minutes

Value:

11.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: slight indication of irritation

Irritation parameter:

cornea opacity score

Value:

3.3

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:

3

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	4.3 %	I
Mean maximum corneal swelling at up to 240 min	11.2 %	II
Mean maximum corneal opacity	3.33	IV
Mean fluorescein retention	3.00	IV
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.
Overall ICE Class	1xII 2xIV

Based on this in vitro eye irritation in the isolated chicken eyes test with diammonium tetrachlorozincate(2-),the test item is irritant,UN GHS Classification: Category 1.

 

Positive Control

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9.6 %	II
Mean maximum corneal swelling at up to 240 min	24.1 %	II
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 controlImidazole 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	0.0 %	I
Mean maximum corneal swelling at up to 240 min	0.0 %	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 classified as 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

Conclusions:

Based on this in vitro eye irritation assay in isolated chicken eyes with diammonium tetrachlorozincate(2-), the test item is irritant, UN GHS Classification: 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 guideline (09 October 2017).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test itemwas applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL ofphysiological 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.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test item treated eyes. Severe cornea opacity change (severity 2 on one eye and severity 4 on two eyes) was observed on three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes.Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on this in vitro eye irritation assay in isolated chicken eyes with diammonium tetrachlorozincate(2-),the test item is irritant,UN GHS Classification: 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

Based upon OECD 431/439 in vitro skin irritation/corrosion data, diammonium tetrachlorozincate does not require classification as a skin irritant

Based upon OECD 438 in vitro eye irritation data, diammonium tetrachlorozincate does require classification as an severe eye irritant.