Registration Dossier

Administrative data

Description of key information

Skin irritation: not irritating (OECD 439; GLP)

Eye irritation: no prediction can be made based on a BCOP study (in vitro OECD 437; GLP). Hence, lead di(acetate) trihydrate has been tested in an in vivo eye irritation study (OECD 405; GLP). Based on this result lead di(acetate) trihydrate does require classification as serious damaging to eyes (CLP (Cat.1)).

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:
2018-02-07 to 2018-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
yes
Remarks:
Pre-test for colour interference was only performed with deionised water instead of isopropanol and water.
Qualifier:
according to
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 / remarks:
2017-07-11
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature, dry, tightly closed in original container
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 25882
- Delivery date: 2018-02-20

TEST FOR COLOUR INTERFERENCE
Before the test started, functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.

TEST FOR MTT INTERFERENCE
The test item was evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (24 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with assay medium. Tissues were incubated for 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium was changed (pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were rinsed three times with DPBS. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well ensuring that the tissues were completely covered and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 2 hours while shaking at room temperature.
After the extraction period was completed, 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 was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minute exposure. The optical density was determined with a microplate reader. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (mean OD test item or positive control/ mean OD of negative control) x 100
For the test item and the positive control, the mean relative viability ± standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium dodecyl sulfate (SDS) solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
85.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
TEST FOR COLOUR INTERFERENCE
since the test item did not dye water or did not change colour in the presence of water, an additional test with viable tissues (but without MTT addition) was not necessary to be performed.

TEST FOR MTT INTERFERENCE
since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus showing the quality of the tissues (acceptance criteria: tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8 in accordance with OECD TG 439..
- Acceptance criteria met for positive control: treatment with the positive control induced a sufficient decrease in the relative absorbance compared to the negative control to 2.8 %, thus assuring the validity of the test system (acceptance criteria: an assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % viability values of the test item, the positive and negative controls in the main test were below 18 (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: ≤ 18), thus ensuring the validity of the study.
- optical density values should not be below historically established boundaries.

Concurrent negative controls and positive controls will be used in each run to demonstrate that viability (with the negative control), barrier function and resulting tissue sensitivity (with the positive control) of the tissues are within a defined historical acceptance range.
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness of the test system, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDermTM lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.8 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria)

Please also refer to the field "Any other information on results incl. tables" and "Attached background material" below.

Table 1: Results after treatment with Lead di(acetate) trihydrate and the controls

Treatment Group

Tissue No.

OD 570 nm
Well 1

OD 570 nm
Well 2

OD 570 nm
Well 3

Mean OD of 3 Wells

Mean OD

of 3 Wells blank

corrected

Mean

OD

of 3 tissues

blank corrected

Rel. Viability [%] Tissue
1, 2 + 3*

Standard Deviation

Mean Rel. Viability

[%]**

Blank

 

0.038

0.038

0.037

0.037

 

 

 

 

 

Negative Control

1

1.707

1.603

1.613

1.641

1.603

1.706

94.0

5.4

100.0

2

1.796

1.776

1.746

1.773

1.735

101.7

3

1.862

1.809

1.782

1.817

1.780

104.3

Positive Control

1

0.080

0.079

0.078

0.079

0.042

0.047

2.5

0.4

2.8

2

0.093

0.093

0.094

0.093

0.056

3.3

3

0.082

0.082

0.079

0.081

0.043

2.5

Test Item

1

1.658

1.551

1.572

1.594

1.556

1.462

91.2

7.8

85.7

2

1.376

1.318

1.349

1.348

1.310

76.8

3

1.584

1.549

1.534

1.556

1.518

89.0

* Relative viability [rounded values]: (100 x (absorbance test item / positive control / negative control)) / mean absorbance negative control

 ** Mean relative viability [rounded values]: (100 x (mean absorbance test item / positive control / negative control)) / mean absorbance negative control

Table 2: Historical control data

Positive Control; OD at 570 nm after
exposition to 5 % SDS solution in deionised
water (MatTek)

Negative Control OD at 570 nm

DPBS (MatTek)

Mean Viability

4.28%

Mean Absorption

1.66

Standard Deviation

1.00 p.p.

Standard Deviation

0.20

Rel. Standard Deviation

23.44%

Rel. Standard Deviation

11.96%

Range of Viabilities

2.24%—6.19%

Range of Absorbance*

1.28—2.00

Mean Absorption

0.07

* should be 0.8—2.8 (OECD 439)

or 1.0—2.5 (MatTek)

Standard Deviation

0.02

Rel. Standard Deviation

25.96%

Range of Absorbance

0.03—0.11

Data of 36 sets of controls shared between 147 studies performed from January 2017 until January 2018. (p.p.—percentage points)

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this study and under the experimental conditions reported, the test item is non-irritant to skin and does not require classification and labelling for skin irritation according to UN GHS and EU CLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-05-02 to 2018-05-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2017-10-09
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2017-05-08
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25 °C, in the closed original container in a dry, cool and well-ventilated place.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Age at study initiation: approx. 5.5 months
- Weight at study initiation: 3.7 kg
- Housing: kept singly in cages with dimensions of 823 mm x 660 mm x 500 mm (Scanbur, Denmark)
- Diet (ad libitum): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C ± 3 °C (maximum range)
- Relative humidity: 30 % - 70 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item into the conjunctival sac of the right eye (left eye served as control)
Duration of treatment / exposure:
not applicable
Observation period (in vivo):
prior to administration and 1, 24, 48, and 72 hours after administration
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
one male rabbit
Details on study design:
INITIAL AND CONFIRMATORY TEST
The test was performed initially using one animal.
Due to absence of a light reflex (iridial response grade 2) the study was terminated 72 hours after the test item administration. No further animal was employed.

USE OF TOPICAL ANAESTHETICS AND SYSTEMIC ANALGESICS:
- topical anaesthetics and systemic analgesics were used according to the procedure recommended by the OECD TG 405.
- approx. sixty minutes prior to test item administration, 0.01 mg Buprenovet®/kg bw were administered by subcutaneous injection to all animals to provide a therapeutic level of systemic analgesia to avoid or minimize pain and distress.
- approx. five minutes prior to the test item administration, two drops of Novesine, a topical anaesthetic, were applied to both eyes of all animals.
- approx. 8 and 24 hours after test administration 0.01 Buprenovet® and Acticam® 0.5 mg/kg, were administered subcutaneously to provide a continued therapeutic level of systemic analgesia.
- thereafter, Buprenovet was administered once every 12 hours, Acticam was administered once every 24 hours until 72 hours after administration of the test item.

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were not rinsed

SCORING SYSTEM: Draize scale
Any further lesions are listed.

TOOL USED TO ASSESS SCORE: slit lamp / fluorescein (starting 24 hours after administration)

OBSERVATIONS
- body weight of the animal was measured at the beginning and at the end of the study.
- behaviour and food consumption were monitored.
- any adverse systemic effects were recorded.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Remarks on result:
other: Due to absence of a light reflex (iridial response grade 2) the study was terminated 72 hours after the test item administration. An absence of a light reflex of the iris are generally not reversible.
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
2.67
Max. score:
3
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
2.67
Max. score:
4
Irritant / corrosive response data:
- mild or moderate corneal opacity (grade 1 or 2) was observed 24 to 72 hours after administration.
- the fluorescein test revealed corneal staining (whole surface; grade 4) 24 to 72 hours after instillation.
- iridial response was not evaluable (grade 2; absence of a light reflex) 24 to 72 hours after administration.
- mild to severe conjunctival redness (grade 1 to 3) was observed 60 minutes until 72 hours after administration.
- moderate chemosis (grade 2 or 3) was observed 24 hours until 72 hours after administration.
- lesions such as an absence of a light reflex of the iris are generally not reversible; hence, the study was stopped 72 hours after administration and the animal was humanely killed.
Other effects:
not specified
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The substance causes serious eye damage.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does require classification as serious eye damaging (Category 1; H318).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

No prediction can be made based on a BCOP study (in vitro OECD 437; GLP).

Hence, lead di(acetate) trihydrate has been tested in an in vivo eye irritation study (OECD 405; GLP). Based on this result lead di(acetate) trihydrate does require classification as serious damaging to eyes (CLP (Cat.1)).

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritation potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

A study on eye irritation in accordance with OECD 437 was performed resulting in no prediction (IVIS score >3 <= 55%, i.e., 33%) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Hence, lead di(acetate) trihydratehas been tested in an in vivo eye irritation study (OECD 405; GLP). Based on this result lead di(acetate) trihydrate does require classification as serious damaging to eyes (CLP (Cat.1)).