Lead dinitrate

Endpoint summary

Description of key information

Lead nitrate crystals are not irritating or corrosive to skin, but severly irritating to eyes.  

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:

4 February 2014 - 6 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or test system and environmental conditions:

HUMAN SKIN MODEL
The 0.50 cm2 reconstructed epidermis (SkinEthic RHE, Batch No. 14 RHE 0107) were received on 04 February 2014. On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in a 24 wells culture plated which has been previously filled with 1 mL of growth medium (Skinethic, batch No. 14-MPE-009) during 2 hours and 30 minutes.

ENVIRONMENTAL CONDITIONS
Temperature (°C): room temperature

Type of coverage:

other: to ensure good contact with the epidermis, the test item was covered with nylon mesh during the treatment

Preparation of test site:

other: the reconstructed human epidermis were moistened with 10 μL of distilled water prior to application of the test item

Vehicle:

unchanged (no vehicle)

Controls:

other: positive control (5% SDS, SIGMA Batch No.BCBF8969V) and negative control (PBS – PAN BIOTECH GmbH - Batch No. 1110913)

Amount / concentration applied:

16 mg of pure test item (lead nitrate crystals)

Duration of treatment / exposure:

42 minutes

Observation period:

42 hours and 15 minutes post-treatment

Number of animals:

3 human epidermis per treatment

Details on study design:

TEST SITE
The test item was applied, as supplied, at the dose of 16 mg, to the epidermal surface of 3 human skin models previously moistened with 10 μL of distilled water during 42 minutes at room temperature. To ensure a good contact with the epidermis, during all the treatment period, the test item was recovered with a nylon mesh provided by SkinEthic.

REMOVAL OF TEST SUBSTANCE
42 minutes after the test item application, the nylon mesh was removed and the human epidermis was washed with PBS (PAN BIOTECH GmbH, Batch No. 1110913) (25 x 1 mL) and was incubated for a 42 hours and 15 minutes post-treatment incubation period of the rinsed tissues in fresh medium at 37°C, 5% CO2.

SCORING SYSTEM
The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme is responsible for MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal. The skin sample was placed in 300 μL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product is then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extractions in isopropanol (1:2). The absorbance was measured in triplicate of MTT extract. The measured absorbances are proportional to the number of live cells. The measurement of optical density was performed using the ELx800 absorbance microplate reader supplied by Biotek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek. The results were expressed as a viability percentage compared with the negative control.

Irritation / corrosion parameter:

other: other: viability of epidermis (%)

Value:

72.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42h and 15 min after exposure. Remarks: 1.1% in the positive control (5% SDS). (migrated information)

Irritant / corrosive response data:

The mean viability of the epidermis skins was 72.9%, versus 1.1% in the positive control (5% Sodium Dodecyl Sulfate).

	skin	mean OD per disc	mean OD per treatment	viability (%)	mean viability (%)	SD	conclusion
	1	1.566		98.4
negative control (PBS)	2	1.599	1.592	100.4	100.0	1.5	/
	3	1.612		101.2
	4	0.024		1.5
positive control (5% SDS)	5	0.015	0.018	1.0	1.1	0.3	irritant
	6	0.015		0.9
	7	1.215		76.3
test item (lead nitrate)	8	1.484	1.160	93.2	22.2	22.2	not irritant
	9	0.783		49.2

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: OECD 439

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that lead nitrate crystals must not be classified R38 “irritating to skin”, according to the criteria for classification, packaging and labelling of dangerous substances and preparations in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol or risk phrase is required.
In accordance with the Regulation EC No. 1272/2008, the test item must not be classified in category 2 “irritant”. No signal word or hazard statement is required.

Executive summary:

The aim of this in vitro skin irritation study was to evaluate the possible irritating effects of lead nitrate crystals after topical administration on in vitro human reconstructed epidermis (RHE® model).

The test item was applied, as supplied, at the dose of 16 mg, to 3 Reconstructed Human epidermis, previously moistened with 10 μL of distilled water, during 42 minutes, followed by a 42 hours and 15 minutes post-incubation period at 37°C, 5% CO₂ and by a rinse with 25 mL of PBS. The experimental protocol was established in accordance withOECD guideline No. 439 adopted 26 July 2013 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean viability of the epidermis skins was 72.9%, versus 1.1% in the positive control (5% Sodium Dodecyl Sulfate).

The results obtained, under these experimental conditions, enable to conclude that lead nitrate crystals must not be classified R38 “irritating to skin”, according to the criteria for classification, packaging and labelling of dangerous substances and preparations in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol or risk phrase is required.

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified in category 2 “irritant”. No signal word or hazard statement is required.

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:

04.05.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17). The method followed that described in the OECD Guidelines for Testing of Chemicals, Updated Guideline No 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September, 2009).

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

OECD 437

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Details on study design:

according to guideline

Irritation parameter:

other: Opacity and Permeability at 490 nm

Basis:

mean

Time point:

other: 240 min

Score:

434.46

Irritant / corrosive response data:

positive control: 191.42
negative control: 1.31

Test Group	Opacity value = Difference (t240-t0) of Opacity^*		Permeability at 490 nm (OD490)		In vitroScore		Proposedin vitroIrritation Scale
	Individual	Mean	Individual	Mean	Individual	Mean
Negative Control	0	0.33	0.070	0.065	1.05	1.31	Non eye irritant
	1		0.060		1.90
	0		0.065		0.98
Positive Control	187.67 *		0.106 *		189.26	191.42	Very severe eye irritant
	182.67 *		0.046 *		183.36
	198.67 *		0.198 *		201.64
Lead dinitrate	434.67 *		-0.011 *		434.50	434.46	Very severe eye irritant
	432.67 *		-0.019 *		432.38
	436.67 *		-0.012 *		436.49

* corrected values

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The study performed shows that the test item is considered to be a very severe eye irritant in vitro under the described conditions.

Executive summary:

Purpose

This study should provide a rational basis for risk assessment in humans. Ocular contact is a possible route of human exposure. This in vitro study was performed to assess the corneal irritation and damage potential of lead dinitrate by means of the BCOP assay using fresh bovine corneae.

Study Design

Damage is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively. Both measurements are used to calculate an in vitro irritation score, which is used to assign an in vitro irritancy category. After a first opacity measurement of the fresh bovine corneae (t0), the test item/saline (0.9% (w/v) NaCl in deionised water) stock, the positive, or the negative controls were applied to the epithelial surface of three corneae each and incubated for 240 minutes at 32 +/- 2 °C. After the incubation phase, the cornea were each rinsed and opacity was measured again (t240). Thereafter the permeability of the corneae was determined. Therefore, 1 mL of a fluorescein solution was added to the anterior chamber of the corneal holder and after 90 min incubation in a horizontal position at 32 +/- 2 °C, the permeability of the corneae was measured quantitatively using the increase in fluorescence in the posterior chamber medium at 490 nm.

Results

The negative control (0.9% NaCl solution) yields no increase of opacity nor permeability of the corneae, while the the positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae, thus demonstrating the validity of the assay. The test item caused caused heavy opacity of the corneae compared with the results of the negative control. The calculated mean in vitro score was 434.46 and therefore, the test item was classified as very severe eye irritant.

Conclusion

The study performed shows that the test item is considered to be a very severe eye irritant in vitro under the described conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro skin irritation study was performed with lead nitrate crystals, using a three-dimensional reconstructed human epidermis model. This model comprises non-transformed human-derived epidermal keratinocytes that have been cultured to form a multilayered and highly differentiated model of the human epidermis. From this test, which measures the viability of the keratinocytes after application of a chemical, it was concluded that lead nitrate crystals are not irritating to skin.

From an in vitro skin corrosion test, conducted with the same test system, it was concluded that lead nitrate crystals are not corrosive to skin.

Eye irritation

In a Bovine Corneal Opacity and Permeability Assay, it was shown that lead nitrate crystals cause opacity of the corneae. The calculated score indicates that lead nitrate crystals are severly irritating to eyes, with irreversible effects.

Respiratory irritation

No studies assessing the irritating potential of lead nitrate crystals after inhalation are available. Inhalation is not considered a relevant route of exposure for lead nitrate crystals (see also waiver for acute inhalation toxicity study).

Justification for selection of skin irritation / corrosion endpoint:
OECD Guideline 439 study, compliant with GLP

Justification for selection of eye irritation endpoint:
OECD Guideline 437 study, compliant with GLP

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Lead nitrate crystals should be classified as follows:

• in accordance with EEC Directives 67/548, 2001/59 and 99/45: Xi "Risk of serious damage to eyes" (R41)
• in accordance with Regulation (EC) No 1272/2008: Category 1 "Eye irritation" with signal word "Danger" and hazard statement H318 "Causes seriouse eye damage".

Based on the available data, a classification for skin irritation/corrosion and respiratory irritation is not required.