Calcium dihydroxide

Endpoint summary

Description of key information

Calcium dihydroxide is irritating to skin and to the respiratory system, and entails a risk of serious damage to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-01-13 to 2004-02-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study reliable with restrictions Minor deviations with no effect on the results: - Stability was not given - Animal weights at conclusion of test were missing

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

, adopted 2002-04-24

Deviations:

yes

Remarks:

, see "rationale for reliability"

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2003-06-04

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: DIMED Schönwalde GmbH, 16352 Schönwalde
- Age at study initiation: approx. 11 to 17 months
- Weight at study initiation: 2.6-2.9 kg b.w.
- Housing: The rabbits were caged individually in PPO cages (floor area: 2576 cm²) with perforated floor.
- Diet (ad libitum): A pelleted complete rabbit diet "Alromin 2123" from Altromin, D-32791 Lage, Lippe.
- Water (ad libitum): Domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C +/- 3 °C
- Relative humidity: 55 % +/- 15 %
- Air changes: 10 times/hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test item
No further information on the test item was stated.

Duration of treatment / exposure:

Initial test (one rabbit): 3 minutes, 1hour and 4 hours
Confirmatory test (2 rabbits): 4 hours

Observation period:

Immediatly and 1 h, 24 h, 48 h, 72 h as well as on day 7 and 14 after termination of exposure

Number of animals:

3 rabbits

Details on study design:

Initial test:
On the experimental day one rabbit was physically restrained on a test table and its back was divided into six test fields. The left side of the body served as the test area for the test item. The test item was applied to a 16-layer gauze patch (2.5 X 2.5 cm), the patch was placed on the appropriate anterior test field and secured with adhesive Gothaplast tape (2.5 cm). After an exposure time of 3 min, the first patch was removed. Since no corrosive effects were observed a second patch with the test item was applied on the median test field of the rabbit back. In order to improve the patch securing the trunk of the animal was additionally wound with 5 cm wide adhesive Gothaplast tape. After 1 h the second patch was removed. Also, after 1 h no corrosive effects were observed so that the exposure time could be extended to 4 hours. The test item was applied on a third patch and put on the posterior test field of the animal. Adhesive Gothaplast tape (2.5 cm X 5 cm wide) served again as securing. After a 4-hour exposure time the third patch was removed.
Since the animal did not show any corrosive effect within a seven day observation period it was decided to carry out a confirmatory test with two additional animals.
Confirmatory test:
Likewise for the confirmatory test the animals were physically restrained on a test table and the back of each rabbit was divided in four test fields. Always the anterior left field was selected as the test area for the test item.
The test item was applied on a 16-layer gauze patch (2.5 cm X 2.5 cm) and the patch was placed on the appropriate test field. The gauze patch was secured with adhesive Gothsplast tape (2.5 cm wide) and fixed with Gothaplast tape (5 cm wide) loosely wound round the trunk of the animals.
TEST SITE
- Area of exposure: The day before the treatment the rabbits were weighed and the skin area on the back was clipped as closely as possible with an electric clipper.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin was cleaned with mild soap and lukewarm water
- Time after start of exposure: Immediately

SCORING SYSTEM: Draize scoring system
No further information on the study design was stated.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

2

Remarks on result:

other: Average scores recorded after 24, 48 and 72 hours after a 4-hour application.

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48, and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Average scores recorded after 24, 48 and 72 hours after a 4-hour application.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Average scores recorded after 24, 48 and 72 hours after a 4-hour application.

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Remarks on result:

other: Average scores recorded after 24, 48 and 72 hours after a 4-hour application.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

2

Remarks on result:

other: Average scores recorded after 24, 48 and 72 hours after a 4-hour application.

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

1

Remarks on result:

other: Average scores recorded after 24, 48 and 72 hours after a 4-hour application.

Irritant / corrosive response data:

After a maximum 4-hour exposure of calcium hydroxide (hydrated lime) in putty form (60 % H2O, 40 % Ca(OH)2) to the skin of the three test animals (Nos. 2692, 2632 and 2616) no skin corrosion but the following symptoms of irritation were observed during the subsequent 14-day observation period.
The animal No. 2692 used in the initial test showed a very slight erythema on the middle (1 h exposure period) and posterior (4 h exposure period) left test field immediately after the application.
1 h after termination of exposure a well defined erythema was observed on the posterior (4 h exposure period) left test field of the animal No. 2692 used in the initial test. Animal No. 2616 showed very slight erythema on the anterior left test field, whereas no skin reaction was observed at the remaining animal No. 2632.
24 and 48 h after termination of exposure the animal No. 2692 used in the initial test showed a well defined erythema on the middle (1 h exposure period) and posterior (4 h exposure period) left test field. A well defined erythema and a very slight oedema were oberved on the anterior left test field of another animal No. 2632. The remaining animal No. 2616 showed no signs of skin irritation.
72 h after termination of exposure a very slight erythema was observed on the middle (1 h. exposure period) left test field of the animal No. 2692 used in the initial test and a well defined erythema on the posterior left test field. Another animal No. 2632 showed a well defined erythema and a very slight oedema on the anterior left test field, whereas no skin reactions were observed at the remaining animal No. 2616.
7 days after termination of exposure animal No. 2632 showed a well defined erythema on the anterior left test field. The animal No. 2692 used in the initial test was free of any skin reactions, whereas on day 6 a very slight erythema was still observed on the middle (1 h exposure period) and posterior (4 h exposure period) left test field. The remaining animal No. 2616 did not show any signs of skin irritation, too.
14 days after the termination of exposure animal No. 2632 was also free of any skin reactions.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results of the study described in this report and accroding to the Directive 67/548/EEC and subsequent regulations, calcium hydroxide (hydrated lime) in putty form (60 % H2O, 40 % Ca(OH)2), shall be classified as irritating to the skin but shall not be classified as corrosive.
Based on the results of the study described in this report and according to the EC Regulation No. 1272/2008 and subsequent regulations, calcium hydroxide (hydrated lime) in putty form (60 % H2O, 40 % Ca(OH)2), shall be classified as non-irritating.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-03-29 to 1994-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study Minor deviations to the guideline with no effect on the results: - The test substance was not moistened before application in order to ensure good contact with the skin of the animals, as recommended by OECD 404. - Sex, age and weight of the animals are not indicated. - It is not indicated if the test substance was removed after treatment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

, adopted 1992-07-17

Deviations:

yes

Remarks:

, see "rationale for reliability"

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 1991-10-21

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kaninchenhof Süstedt, Alter Pohl 8, 27305 Süstedt
- Housing: Individual housing (50 X 45X 40 cm, L X B X H) in a battery of cages, each equipped with a paper roll disposal system.
- Diet (ad libitum): Pellets; Ssniff K-Haltung (Zuchtdiät für Kaninchen) (Ssniff Spezialdiäten GmbH, 59494 Soest/Westfalen)
- Water (ad libitum): drinking water as for human consumption

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 3 °C
- Relative humidity: 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animal was stated.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of calcium hydroxide.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

4 hours

Observation period:

At 60 min, 24, 48, and 72 hours after patch removal

Number of animals:

3 animals

Details on study design:

TEST SITE
- Area of exposure: 24 h before treatment, fur was removed with electric clippers from an area of roughly 8 X 15 cm on the back of each animal. The skin was examined for abrasions and only animals with healthy, intact skin were used in the test. The test article was applied to the test site (ca. 6 cm² in size) an adjacent area of untreated skin served as control.
- Type of wrap if used: Each skin area was covered with a semi-occlusive dressing consisting of Ypsiplast® (Holthaus Medical, Remscheid-Lüttringhausen), which was held in place by non-irritating tape (Elastoplast®, Beiersdorf AG, Hamburg), and Stülpa® (P. Hartmann AG, Heidenheim-Brenz), which enveloped the whole of the animal's trunk.

SCORING SYSTEM: Draize scoring system
No further information on the study design was stated.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritant / corrosive response data:

No test article-dependent findings were observed.

Other effects:

No toxic effects were observed.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test article was classified on the basis of the mean skin reactions at 24, 48 and 72 h after patch removal.
According to the criteria specified by Directive 67/548/EEC and subsequent regualtions, the test item is not a skin irritant.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not a skin irritant.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th - 16th June, 2016

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:

July 28, 2015

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 (Bis)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epidermis

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

water

Details on test system:

Epi-200 Kit Components Needed for the Assay:
- EpiDerm™ Kit Lot No.: 23341
1 Sealed 24-well plate; Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates; For MTT viability assay
4 6-well plates; For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium; DMEM-based medium
1 bottle DPBS Rinse Solution; For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM); For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol); For extraction of formazan crystals

Cell Culture
- Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in
patterns analogous to those found in vivo.
- The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).
- EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 14 June 2016 and stored in the refrigerator until use.
- Next day the pre-incubation phase of the EpiDerm™ tissues started.

Test for Direct MTT Reduction and Colour Interference
Step 1:
- 25 mg of the test item were added to 0.3 ml of deionised water (transparent glass test-tube).
- The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min.
- At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.
- If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue.
- An additional test with viable tissues (without MTT addition) should be performed (step 2).
- Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3:
- All test items (including those already evaluated in Step 1 and Step 2) should be further evaluated for their potential to interfere with MTT assay. To test if an item directly reduces MTT, 25 mg of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution
(1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test with freeze-killed tissues (step 4) must be performed.
- Since the test item proved to be a MTT reducer, an additional test with freeze-killed tissues had to be performed (step 4).
Step 4:
- The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item similar to viable tissues.
- Each MTT reducing chemical was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

Data correction procedure
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue; kt = killed tissues; tkt = treated killed tissue; ukt = untreated killed tissue (NC treated tissue)
Although the interference by the test item was greater than 30% of the negative control value, the test item was not considered incompatible with this test system according to an expert judgment.

Experimental Performance
- Pre-warming of EpiDerm™ Tissues: At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates
containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 μL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

- Treatment: Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control (see below for treatment levesl and times).
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

- MTT Assay: Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 μL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the MTT solution was blotted from the wells and the wells were rinsed three times with DPBS. The inserts were transferred into new 24-well plates. The inserts were immersed in extractant solution by gently pipetting 2 mL of extractant solution (isopropanol) into each insert ensuring that the tissue was completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for about 20.5 hours without shaking in the refrigerator.
After the extraction period 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. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96- well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

- Evaluation: The mean OD of the duplicate 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/positive control / mean OD negative control]x100

- Interpretation of results: For the test item and the positive control the mean relative viability ± rel. standard deviation of the two individual tissues for both exposure periods was calculated and used for classification according to the following prediction model:
Viability measured after exposure time points/ Prediction to be considered
< 50% after 3 minutes exposure/ Corrosive: Optional Sub-category 1A*
≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure/ Corrosive: Optional Sub-category 1B and 1C
≥ 50% after 3 minutes exposure AND ≥ 15% after 60 minutes exposure/ Non-corrosive

- Acceptability of the Assay: An assay met the acceptance criterion if
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39.7 mg/cm2)

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL were applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL were applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods.
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes or 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

91.6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

77.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed blue colour, therefore, an additional test with freeze-killed tissues had to be performed to gain a correction factor to calculate the true viability.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.784 and 1.948)
- Acceptance criteria met for positive control: yes. the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% (7.4%) compared to the negative control
- Acceptance criteria met for variability between replicate measurements: yes. the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 1.49% and 27.85% for the negative and positive control and the test item in the main experiment)
- Range of historical values if different from the ones specified in the test guideline: Historical values are attached for reference.

Absorbance results table is attached.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, Calcium dihydroxide was non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

An in vitro study was performed to assess the corrosive potential of Calcium dihydroxide by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the colour interference pre-test, but an additional test with freeze-killed tissues had to be performed due to the MTT-reducing property of the test item to gain a correction factor for calculating the true viability in the main test.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour,

respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then

removed from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 20.5 hours at room

temperature.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (28.8%) and for the 1 hour

exposure period (7.4%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item Calcium dihydroxide the corrected relative absorbance value decreased slightly to 91.6% after 3 minutes exposure. After 1 hour exposure the corrected

viability was reduced to 77.4%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

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:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Minor deviations: - Exposure time was 15 minutes and then observation after 1 h instead of 20 seconds exposure time and a 5-minute observation period.

Qualifier:

according to guideline

Guideline:

other: HET-CAM

Deviations:

yes

Remarks:

, see "rational for reliability"

GLP compliance:

not specified

Details on test animals or tissues and environmental conditions:

In vitro eye irritation testing using chicken eggs

Fifty two (52) fresh, intact hen's eggs (White Leghorn, incubation day 11; 50-60 g) were were incubated in the Clinic for Poultry at the Hanover College of Veterinary Medicine up to the test day at 37.5 +/- 0.5 °C and 62.5 +/-7.5 % humidity.
No further information on the eggs was stated.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

250 mg of calcium hydroxide (calcium lime mixture) were applied on the Chorion-Allantois membrane. Thirteen (13) embryonated he's eggs were used.

Duration of treatment / exposure:

15 minutes

Observation period (in vivo):

One hour

Number of animals or in vitro replicates:

Not applicable, see "amount/concentration applied"

Details on study design:

Test performance:
The air chamber on the shell of the eggs was identified by means of candling (UV lamp) and the shell was then opened in the area of the air chamber. The Chorion-Allantois membrane was prepared by removing the inner membrane. 250 µl of 0.9 % NaCl (negative control), 0.1 N NaOH (positive control), or 250 mg of calcium hydroxide (calcium lime mixture) were applied on the Chorion-Allantois membrane and, after 15 minutes, were washed off with 10 ml of 0.9% NaCl. Thirteen (13) embryonated hen's eggs were used each time. As symptoms of irritation of the Chorion-Allantois membrane the following parameters were visually assessed by two persons independently from each other, before and after one hour (incubation at 37 °C) after the application of the test substance:
- Development of oedema
- Colouration of the Chorion-Allantois membrane
- Changes in the blood vessels
No further information on the study design was stated.

Irritant / corrosive response data:

In the embryonated hen's eggs, redness occurred on the treated Chorion-Allantois membrane within 15 minutes after appication of the calcium hydroxide (calcium lime mixture; 60 % solid content). The vessels appeared watery. There was no conspicuous swelling of the Chorion-Allantois membrane. These findings were just as concise as after the application of 0.1 N NaOH.

See "Attached background material" for a graph of the results.

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the result of this study, the test sample, calcium hydroxide (calcium lime mixture; 60 % solid content), must be attributed a mucous membrane irritating effect. In view of this result, it is proposed that the test substance be characterised with the R phrase R36 (Irritating to eyes). According to other studies (in vivo), calcium hydroxide should be classified as R41 (“Risk of serious damage to eyes”).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

An in vitro skin corrosivity test performed in a reconstructed human epidermis model (EpidDerm) according to OECD 431 indicates that calcium dihydroxide is not corrosive to skin.

The results of available in vivo skin irritation studies (rabbit) indicate that calcium dihydroxide is irritating to skin. In vivo data show that calcium carbonate is not a skin irritant. Applying the generic concentration limits specified in table 3.2.3 of the CLP guidance indicates that all grades of calcium dihydroxide, including those containing up to 35% calcium carbonate would be classified as Skin Irritant 2.

In the available eye irritation studies (in vivo, rabbit), calcium dihydroxide caused irreversible lesions in the eye. In vivo data show that calcium carbonate is not an eye irritant. Applying the generic concentration limits specified in table 3.3.3 of the CLP guidance indicates that all grades of calcium dihydroxide, including those containing up to 35% calcium carbonate would be classified as Eye Damage 1.

From human data it is concluded that calcium dihydroxide is irritating to the respiratory tract. No data are available for calcium carbonate, however based on its non-irritancy to skin and eyes, it would not be expected to be a respiratory irritant. Applying the generic concentration limits mentioned in table 3.8.3 of the CLP guidance indicates that all grades of calcium dihydroxide, including those containing up to 35% calcium carbonate would be classified as STOT SE 3.

Justification for selection of skin irritation / corrosion endpoint:
Studies summarised in section 7.10.3 provide examples of human cases of eye irritation due to accidental exposure to calcium hydroxide (Schmidt, 2008), and skin corrosion due inadvertent exposure to wet cement over several hours (Rados, 2005). In the latter case, it is important to note that aqueous cement slurry has been found to result in subtantially higher pH values (>13.5) than CaO or Ca(OH)2 alone. Therefore, the corrosive phenomena reported this case study should not be extrapolated to lime. This is supported by the findings of the in vitro study which indicates that calcium dihydroxide is not corrosive to skin. Overall, the two human case studies summarised in this section demonstrate that exposure to hydrated lime or mixtures based upon lime may result in eye or skin irritation, particularly upon prolonged skin contact

Justification for classification or non-classification

Based on experimental results and human data, calcium dihydroxide requires classification as Skin Irrit 2 (H315 – Causes skin irritation).

Based on experimental results and human data, calcium dihydroxide requires classification as Eye Damage 1 (H318 - Causes serious eye damage).

Based on human data as summarised and evaluated in the SCOEL recommendation (see section 7.2.2), it is proposed to classify calcium dihydroxide as STOT SE 3 (H335 - May cause respiratory irritation).