Registration Dossier

Administrative data

Description of key information

Skin irritation: not irritating (OECD 404, GLP)

Eye irritation: causes serious eye irritation (OECD 437, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-01 to 2018-01-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
August 1998
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 test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: approx 26 and 27 weeks old
- Weight at study initiation: >2 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 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test item
Because of testing a solid, the test chemical was moistened with the smallest amount of water, sufficient to ensure good skin contact.
Duration of treatment / exposure:
initial animal: 4 hours
confirmatory animal: 4 hours
Observation period:
Initial animal: 72 hours after the patch removal
Confirmatory animal: 14 days
Number of animals:
2 male rabbits
Details on study design:
TEST SITE
- Area of exposure/Type of wrap if used: approx. 24 hours before the test, the fur was removed from the dorsal area of the trunk by using an electric clipper. The test item was applied to a small area (approx. 6 cm²) of skin on one side of the dorsal area and covered with a gauze patch, which was held in place with a non-irritating tape. The untreated other side served as control. The test item was applied to the patch first and then applied to the skin. The patch was fixed with a semi-occlusive dressing. The limits of the application site were marked with an ink marker.

INITIAL AND CONFIRMATORY TESTING
The results of the initial test indicate that the test item is neither corrosive nor irritant to the skin using the procedure described. In order to confirm the negative response, one additional animal was treated in the same manner. According to OECD 404, section 17, treatment of a third animal can be omitted when animal no. 1 and 2 exhibit the same response. Also, considering the classification directives the results of animal no. 1 and 2 were sufficient for classification of the test item. Moreover, as the test item showed no signs of irritation in two animals, it is considered that adding a third animal would not change the outcome of the study.

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period, for animal no.1 the residual test item was removed with aqua ad injectionem, without altering the existing response or the integrity of the epidermis For animal no.2 the test item had not to be removed

OBSERVATION TIME POINTS
- initial animal: immediatley and 1 hour, 24, 48 and 72 hours after patch removal
- confirmatory animal: 1 hour, 24, 48 and 72 hours after patch removal and thereafter once per day (14 observation days in total)

SCORING SYSTEM: according to the Draize scale
In addition, all local effects such as hyperplasia, scaling, discolouration, fissures and scabs were also recorded.

FURTHER OBSERVATIONS
- body weights: prior to the administration and at the end of the observation period
- any systemic effects were also recorded.
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Neither erythema nor oedema was observed in two male rabbits (strain NZW) after a contact time of 4 hours during the complete observation period of 72 hours for animal no.1 and 14 days for animal no. 2.
No severe changes were observed at the skin sites. The circular redness at application site of animal no. 2 from 24h after removal of the patch until day 14 was not diagnosed as erythema or oedema, therefore it is not mentioned as dermal irritation according to Annex I of Regulation (EC) 1272/2008. It is assumed that this finding is an irrelevant and not test item related biological response.
Other effects:
- body weight: there were no significant body weight changes during the observation period
- clinical signs: no mortality was observed.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not irritating to the skin.
According to Annex I of Regulation (EC) 1272/2008, the test item Zinc dipropionate is not irritating to skin, hence no classification and labelling 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:
2017-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
2017-10-09
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Council Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-06-05
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals: cattle was between 19 and 34 months.
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.
Vehicle:
physiological saline
Remarks:
0.9 % NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the test substance mixture
- Concentration (if solution): 20 % w/v concentration

Duration of treatment / exposure:
4 hours ± 5 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. It was ensured that no bubbles were formed within the compartments.
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 μL of the test substance mixture was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment.
- 750 µL of the control substance was introduced into the anterior chamber (closed-chamber method).
- after 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
- each cornea was observed visually and pertinent observations were recorded.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated annually. This I0 value is than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance can be calculated and corneas below this value were discarded.
- change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
For the IVIS cut-off values for identifying test substances as inducing serious eye damage and test substances not including eye irritation or serious eye damage please refer to table 1 in the field "Any other information onmaterial and methods incl. tables" below.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Remarks:
(mean)
Value:
87.69
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All 3 corneas treated with zinc dipropionate showed a severe, whitish opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2017 was used (please refer to the tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 were used (please refer to the tables in the field "Any other information on results incl. tables" below)

Please also refer for results to the field "Any other information on results incl. tables" below

Table 1: Opacity

Cornea No.

Test Item

Initial Opacity

Final Opacity

Change of Opacity Value

Corrected Opacity Value

1

Negative Control

1.98

2.96

0.98

 

2

2.09

6.01

3.92

 

3

2.16

3.78

1.62

 

MV

2.08

4.25

2.27

 

4

 

Positive Control

3.50

112.05

108.55

106.38

5

3.54

83.03

79.49

77.32

6

3.74

87.36

83.62

81.45

MV

3.59

94.15

90.55

88.38

7

 

Test Item

0.77

129.01

128.24

126.07

8

0.42

76.63

76.21

74.04

9

1.01

66.61

65.60

63.43

MV

0.73

90.75

90.02

87.85

MV = mean value

Table 2: Permeability

Cornea No.

Test Item

OD490

Corrected OD490 Value

1

Negative Control

0.019

 

2

0.022

 

3

0.028

 

MV

0.023

 

4

 

Positive Control

1.473

1.450

5

2.465

2.442

6

3.065

3.042

MV

2.334

2.311

7

 

Test Item

0.018

-0.005

8

0.010

-0.013

9

0.010

-0.013

MV

0.013

-0.010

MV = mean value

Table 3: In vitro irritation score

Cornea No.

Test Item

Corrected Opacity

Corrected OD490 Value

IVIS

1

Negative Control

0.98

0.019

 

2

3.92

0.022

 

3

1.62

0.028

 

MV

2.17

0.023

2.52

4

 

Positive Control

106.38

1.450

 

5

77.32

2.442

 

6

81.45

3.042

 

MV

88.38

2.311

123.05

7

 

Test Item

126.07

-0.005

 

8

74.04

-0.013

 

9

63.43

-0.013

 

MV

87.85

-0.010

87.69

MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until August 2017

 

IVIS Positive Control – Imidazole 20%

Mean Value (MV)

125.12

Standard Deviation (SD)

17.75

MV-2xSD

89.63

MV+2xSD

160.62

Number of Replicates providing Historical Mean: 28

Positive controls are updated after every single experiment or at least every 3 months

Table 5: Historical data on opacity and permeability of the positive control (Imidazole 20 %) from August 2017 until October 2017

Incubation; 240 min

Number of Replicates providing Historical Mean

 

 

Cornea No.

Opacity

Permeability

 

 

IVIS

 

Change of Opacity Value

 

Corrected Opacity Value

 

OD490 Value

 

Corrected OD490 Value

1

4

122.785

121.861

0.662

0.624

 

 

5

117.173

116.249

1.220

1.182

133.42

 

6

102.655

101.731

2.260

2.222

 

2

4

108.553

106.381

1.473

1.450

 

 

5

79.491

77.319

2.465

2.442

123.05

 

6

83.618

81.446

3.065

3.042

 

3

4

55.644

56.308

2.200

2.209

 

 

5

71.511

72.175

1.348

1.337

92.64

 

6

65.148

65.812

2.040

2.029

 

Mean Value (MV)

89.620

88.809

1.859

1.837

116.370

Standard Deviation (SD)

23.998

23.370

0.740

0.745

21.195

MV-2xSD

41.624

42.069

0.379

0.347

73.980

MV+2xSD

137.615

135.549

3.340

3.328

158.760

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until August 2017

 

IVIS Negative Control – NaCl 0.9 %

Mean Value (MV)

1.23

Standard Deviation (SD)

0.77

MV-2xSD

-0.31

MV+2xSD

2.78

Number of Replicates providing Historical Mean: 28

Negative controls are updated after every single experiment or at least every 3 months

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until October 2017

Number of Replicates providing Historical Mean

 

 

Cornea No.

 

Opacity

 

Permeability

 

 

IVIS

 

Change of Opacity Value

 

OD490 Value

1

1

0.234

0.008

 

1.49

 

2

1.738

0.008

 

3

0.800

0.098

2

1

0.978

0.019

 

2.52

 

2

3.920

0.022

 

3

1.617

0.028

3

1

-0.149

0.009

 

-0.50

 

2

-0.415

0.015

 

3

-1.427

0.009

1

1

-0.57

0.009

 

-0.27

 

2

-0.11

0.013

 

3

-0.53

0.004

2

1

-0.25

0.004

 

0.66

 

2

0.80

0.005

 

3

1.17

0.008

Mean Value (MV)

0.520

0.017

0.780

Standard Deviation (SD)

1.296

0.023

1.254

MV-2xSD

-2.072

-0.029

-1.727

MV+2xSD

3.112

0.064

3.287

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
According to the bovine corneal opacity and permeability assay, since the IVIS was > 55 (IVIS score: 87.69), the test item zinc dipropionate can be considered as requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (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

Initially, the substance was tested in an in vitro Human Skin Model Test (Epiderm study, OECD Guideline No. 439, GLP) to assess its skin irritation activity. However, the Epiderm study could not provide a prediction for dermal classification (mean relative tissue viability 47.7 %). Therefore, further in-vivo testing of the test item for assessing acute dermal irritation/corrosion was necessary. In this study (OECD Guideline No. 404, GLP) the substance showed no skin irritating potential as it induced neither erythema nor edema at the rabbit skin.

Eye irritation

The substance was tested in vitro in a bovine corneal opacity and permeability assay (OECD 437, GLP) for its eye irritation potential. Since the mean in vitro irritation score was calculated to be 87.69, the substance can be considered to induce serious eye damage (Category 1).

Justification for classification or non-classification

Zinc dipropionate did not show a skin irritation potential in an in vivo test according to OECD Guideline 404. According to the criteria of REGULATION (EC) No 1272/2008 and its subsequent adaptions, zinc dipropionate does not have to be classified and has no obligatory labelling requirement for skin irritation.

Zinc dipropionate induced a mean in vitro irritation score (IVIS) of 87.69 in a bovine corneal opacity and permeability assay according to OECD Guideline 437 indicating on irreversible effects on the eye and is therefore classified in category 1 with labelling H318: Causes serious eye damage in accordance with Regulation (EC) No 1272/2008 and its subsequent adaptions.

Zinc dipropionate is not classified for respiratory irritation (STOT SE category 3) because of lacking data.