Registration Dossier

Administrative data

Description of key information

Skin irritation: not irritating (OECD 439, GLP)

Eye irritation: causes serious eye irritation (OECD 405, GLP)

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:
2017-08-02 to 2017-08-04
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:
2015-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012-07-06
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MatTek Corporation Protocol for: 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:
2014-11-07
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-06-05
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, < 30 °C
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:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25834

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 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 ODtest item / positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.
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 mg/cm²) of the test item

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

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution
Duration of treatment / exposure:
60 ± 1 minute
Duration of post-treatment incubation (if applicable):
approx. 42 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
99.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.773).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.4 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.8 % - 3.9 %).
Please also refer to the field "An other information on results incl. tables" below.

Table 1: Result of the test item calcium dibenzoate

Name

Negative Control (NK)

Positive Control (PC)

Test material (TM)

Tissue

1

2

3

1

2

3

1

2

3

absolute OD570

1.755

1.853

1.950

0.112

0.104

0.087

1.729

1.858

1.799

1.819

1.847

1.671

0.120

0.106

0.092

1.754

1.902

1.833

OD570(blank-corrected)

1.712

1.811

1.907

0.069

0.061

0.044

1.686

1.816

1.756

1.776

1.804

1.628

0.078

0.063

0.049

1.711

1.859

1.790

mean OD570of the duplicates (blank-corrected)

1.744

1.808

1.767

0.073

0.062

0.047

1.699

1.837

1.773

total mean OD570of 3 replicate tissues (blank-corrected)

1.773*

0.061

1.770

SD OD570

0.032

0.013

0.069

relative tissue viability [%]

98.4

101.9

99.7

4.1

3.5

2.6

95.8

103.6

100.0

mean relative tissue viability [%]

100.0

3.4**

99.8

SD tissue viability [%]***

1.8

0.8

3.9

CV [% viabilities]

1.8

22.1

3.9

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is  20 %.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is  18%

Table 2: Historical data

 

Mean OD570±30nmNK

Mean Relative Viability [%] PC

SD Viability [%]

Mean

1.843

4.3

4.2

SD

0.286

2.2

4.7

n

22

22

84

Historical data were generatedfrom 2015 to 2016

Interpretation of results:
GHS criteria not met
Conclusions:
According to the criteria of REGULATION (EC) No 1272/2008 and its subsequent adaptions, calcium dibenzoate does not have to be classified for skin irritation.
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:
2018-01-08 to 2018-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2017-10-09
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-06-05
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry; < 30 °C
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: animal #1: approx. 31 weeks old; animal #2: approx. 40 weeks old
- Weight at study initiation: animal #1: 4.2 kg; animal #2: 4.3 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm²
- Diet (ad libitum): autoclaved hay and Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: at least 10x / hour
- 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): 0.1 g of the test item in the conjunctival sac of one eye. Untreated eye served as control.
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
animal #1: 1, 24, 48 and 72 hours as well as 4, 5, 6, 7, 8 and 9 days after test item application
animal #2: 1, 24, 48 and 72 hours as well as 4, 5, 6 and 7 days after test item application
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
2 male rabbits
Details on study design:
PREPARATION OF THE ANIMALS
Within 24 hours before the test and immediately prior to the application both eyes of each animal were examined.
Approx. 23 hours before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo®). The eyes were rinsed with physiological saline 0.9 % NaCl after the examination. None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

INITAL TEST AND CONFIRMATORY TEST
The in vivo test was performed initially using one animal. The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, one additional animal was treated in the same manner.

USE OF TOPICAL ANESTHETICS AND SYSTEMIC ANALGESICS
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Temgesic® 0.3 mg/mL) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approx. 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (Proparakaine-POS® hydrochloride ophthalmic 0.5% solution) were administered in both the treated and the control eye of each animal.
To prevent pain and distress after the application of the test item both animals were treated with doses of buprenorphine and meloxicam (Metacam® 5 mg/mL) to provide a continued therapeutic level of systemic analgesia. Treatment with the analgesic medication was conducted from 11 hours post-application (day 0) upto 4 or 5 days post-application.

REMOVAL OF TEST SUBSTANCE
- Washing: treated eyes were rinsed with physiological saline 0.9 % NaCl.
- Time after start of exposure: 1 hour after the application

SCORING SYSTEM: according to Draize scale

TOOL USED TO ASSESS SCORE: slit lamp biomicroscope / fluorescein
24 hours post-application and from then on daily until end of the observation period, the treated eyes were was examined with the aid of a fluorescein solution and a slit lamp biomicroscope. After the examination the eyes were was rinsed with physiological saline 0.9 % NaCl.

OBSERVATIONS
- body weight: prior to the administration and at least at the end of the observation period
- clinical observations: nature, severity and duration were recorded
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
2.33
Max. score:
3
Reversibility:
fully reversible within: 9 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 5 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritant / corrosive response data:
Animal #1:
- conjunctival redness: one hour post-application slight conjunctival redness (grade 1) was observed. 24 hours after application severe conjunctival redness (grade 3) was observed. From 48 hours until 72 hours post-application a moderate conjunctival redness (grade 2) was observed, which changed to a slight conjunctival redness (grade 1) from day 4 until day 8 post-application. The effect was fully reversible within day 9 after application of the test material.

- conjunctival chemosis: one hour and 24 hours after application a moderate conjunctival chemosis (grade 2) was observed. Between 72 hours and day 4 after application the animal showed a slight conjunctival chemosis (grade 1). The effect was fully reversible within day 5 after application of the test material.

- iris: 24 hours post-application the animal showed a slight iris lesion (grade 1). The effect was fully reversible within 48 hours after application of the test material.

- cornea opacity: one hour until 48 hours post-application slight corneal opacity (grade 1) was found in the animal. The effect was fully reversible within 72 hours after application of the test material.

Animal #2:
- conjunctival redness: moderate conjunctival redness (grade 2) was observed one hour after application as well as on day 4 and day 6 post-application. A severe conjunctival redness (grade 3) was observed in the animal from 24 hours until 72 hours post-application and on day 5 post-application. The effect was fully reversible within day 7 after application of the test material.

- conjunctival chemosis: a moderate conjunctival chemosis (grade 3) was detected in the animal one hour post-application before severity level decreased 24 hours after application (grade 2) and from 48 until 72 hours post-application (slight conjunctival chemosis; grade 1). The effect was fully reversible within day 4 after application of the test material.

- iris: slight iris lesion (grade 1) was observed in the animal from one hour until 48 hours after application. The effect was fully reversible within 72 hours after application of the test material.

- cornea opacity: slight corneal opacitiy (grade 1) was found in the animal from one hour until day 6 post-application. The effect was fully reversible within day 7 after application of the test material.


Local effects were observed in both animals. The observed local effects mainly comprise slight to severe hypersecretion, a dark red nictitating membrane, and a dark red spot on the nictitating membrane. All these findings were reversible within 5 or 6 days post-application.
Upon fluorescein examinations starting 24 hours post-application, the treated eyes of both animals showed corneal lesions (starting with approx. 75% of the area) which were completely reversible within 7 days.
Other effects:
- clinical observations: neither mortalities nor significant clinical signs of toxicity were observed.
- body weight: the body weight development of one rabbits was within the expected range, while the other animal showed slight weight loss.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The substance is irritating to the eyes.
According to the EC Regulation No. 1272/2008 and subsequent adaptations, the substance is classified as an eye irritant (Category 2; H319).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Eye irritation

Calcium dibenzoate was also tested in vitro in the bovine corneal opacity and permeability assay according to OECD 437. Considering the results of this study, calcium dibenzoate is not serious eye damaging (Category 1) according to the Regulation (EC) No 1272/2008 and its subsequent adaptations, but no further prediction regarding the eye irritation potential can be made according to the evaluation criteria since the IVIS was between 3 and 55.

Justification for classification or non-classification

Calcium dibenzoate did not show a skin irritation potential in an in vitro test according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method).

According to the criteria of REGULATION (EC) No 1272/2008 and its subsequent adaptions, calcium dibenzoate does not have to be classified and has no obligatory labelling requirement for skin irritation.

Calcium dibenzoate induced serious but reversible eye irritation in an in vivo study according OECD guideline 405 and is therefore classified as Eye irritant category 2 with labelling H319: Causes serious eye irritation in accordance with Regulation (EC) No 1272/2008 and its subsequent adaptions.