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Description of key information

Skin corrosion (OECD 431): not corrosive
Skin irritation (OECD 439): not irritating
Eye irritation (OECD 405): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 13 Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
(2004)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: No data given
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C and 5% CO2 in air
- Temperature of post-treatment incubation: 37 °C and 5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Duplicate tissues were treated with the test item and the concurrent positive and negative controls for an exposure period of 3, 60 and 240 min. At the end of the exposure period, each tissue was removed from the well and rinsed with Dulbecco´s Phosphate Buffered Saline (DPBS) containing calcium and magnesium.
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: microplate reader
- Wavelength: 540 nm

CELL VIABILITY MEASUREMENTS
- For determining alterations in cell viability, MTT reduction assays were performed immediately after the exposure period. The rinsed tissues were transferred to other wells of the same 12-well plate containing 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium in each well. The tissues were incubated for 3 h at room temperature protected from light. At the end of the 3 h incubation period each tissue was placed onto absorbent paper to dry. The epidermis was carefully separated from the collagen matrix and both parts (epidermis and collagen) placed into labeled 1.5 mL tubes containing 850 μL of acidified isopropanol. The tubes were plugged, mixed thoroughly and stored overnight at room temperature, protected from light, allowing the extraction of formazan crystals out of the MTT-loaded tissues. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a 96-well plate. The optical density was measured at 540 nm wave length in a microplate reader.
Control samples:
other: concurrent control tissues treated with sodium chloride solution served as negative controls, positive controls were exposed to glacial acetic acid
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 mg (moistened with 100 µL 0.9% (w/v) NaCl solution)

NEGATIVE CONTROL
- Amount(s) applied: 50 μL NaCl solution, 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied: 50 μL glacial acetic acid
Duration of treatment / exposure:
3, 60 and 240 min
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
The test was performed in duplicates for each treatment and control group.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 min exposure
Value:
90.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 min exposure
Value:
97.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
240 min exposure
Value:
98.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
The relative mean viability of the test item treated reconstructed human-derived keratinocyte tissues after a 3, 60 and 240 min exposure period was 90.7, 97.7 and 98.1%, respectively compared to the negative control. Therefore, the test item is considered to be non-corrosive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥ 0.115 and ≤ 0.400.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240 min exposure period.




Table 1: Mean OD540 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

Exposure period

(min)

Mean OD540 of

duplicated tissues

Relative mean

viability (%)

Negative Control

Item

240

0.214

100*

Positive Control

Item

240

0.019

8.9

Test Item

240

0.210

98.1

60

0.209

97.7

3

0.194

90.7

*: the mean viability of the negative control tissues is set at 100%

Interpretation of results:
other: non-corrosive
Conclusions:
CLP: not classified
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 - 15 Oct 2012
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:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: No data available
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C and 5% CO2 in air
- Temperature of post-treatment incubation: 37 °C and 5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Triplicate tissues were treated with the test item and the concurrent positive and negative controls for an exposure period of 15 min. At the end of the exposure period, each tissue was removed from the well and rinsed with DPBS containing calcium and magnesium. The rinsed tissues were transferred to other wells of the same 12-well plate containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C and 5% CO2 in air for 42 h.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: microplate reader
- Wavelength: 540 nm

CELL VIABILITY MEASUREMENTS
- For determining alterations in cell viability, MTT reduction assays were performed immediately after the 42 h incubation period. Therefore, tissues were transferred to new wells of the same 12-well plate containing 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium. The tissues were incubated for 3 h at 37 °C and 5% CO2 in air. At the end of the 3 h incubation period each tissue was placed onto absorbent paper to dry. The epidermis was carefully separated from the collagen matrix and both parts (epidermis and collagen) placed into labeled 1.5 mL tubes containing 500 μL of acidified isopropanol. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a 96-well plate. The optical density was measured at 540 nm wave length in a microplate reader.
Control samples:
other: concurrent control tissues treated with DPBS served as negative controls, positive controls were exposed to 5% SDS
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 10 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL SDS
- Concentration: 5% (w/v)
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
The test was performed in triplicates for each treatment and control group.
Irritation / corrosion parameter:
% tissue viability
Value:
90.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The relative mean viability of the test item treated reconstructed human-derived keratinocyte tissues after a 15 min exposure period was 90.9% compared to the negative control. Therefore, the test item is considered to be a non-irritant.

- Acceptance criteria:
The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥ 0.6, and the standard deviation value of the percentage viability is ≤ 18%. The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.
- Evaluation criteria:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18%.

Table 1: Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540 of tissues

Mean OD540

of triplicate tissues

± SD of OD540

Relative individual

tissue viability (%)

Relative mean

viability (%)

± SD of Relative

mean viability (%)

Negative Control Item

0.763

0.843

0.090

90.5

100*

10.6

0.827

98.1

0.940

111.5

Positive Control

Item

0.053

0.070

0.017

6.3

8.3

2.0

0.070

8.3

0.086

10.2

Test Item

0.776

0.766

0.019

92.1

90.9

2.2

0.744

88.3

0.777

92.2

SD = standard deviation

* = the mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified
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:
07 Jul - 01 Aug 2014
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:
(2012)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: Albino (Zika)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kaninchenbetrieb Kock, Warnkenhagen, Germany
- Age at study initiation: 15 - 16 weeks
- Weight at study initiation: 2980 and 3210 g
- Housing: the test animals were housed individually in cages.
- Diet: conventional laboratory diet (a half-and-half blend of "Holstenstolz Kaninchenverbrauchsfutter 2, Type 038" [Wilhelm Stroeh jr. GmbH & Co. KG, Germany] and "Rabbit maintenance, MuesliMash", [ssniff Spezialdiäten GmbH, Germany], ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: the untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 100 mg
Duration of treatment / exposure:
Single application
Observation period (in vivo):
8 days
Reading time points: 1, 24, 48 and 72 h and 8 days
Number of animals or in vitro replicates:
2 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The solid test substance had not been removed from the eye of the test animals by physiological mechanisms at the first observation time point after treatment, therefore the eyes were rinsed with physiological saline.
- Time after start of exposure: 1h

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: reversibility not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: reversibility not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Slight conjunctival redness and chemosis (grade 1) were observed in 2/2 animals 1 h post-application of the test material, which were fully reversible within 24 h. No effects on cornea and iris were recorded. One animal was further observed for up to 8 days, which showed no effects in the treated eye.
Other effects:
No further local or systemic effects and no effects on body weight gain were observed during the study period.
Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Two in vitro skin irritation studies with boron orthophosphate (CAS 13308-51-5) are available.

In the in vitro key skin irritation study performed according to OECD 431 (adopted 2004) and in compliance with GLP human-derived epidermal keratinocytes (EpiSkin) were exposed to 20 mg of the neat test material for 3, 60 and 240 minutes (Warren, 2014b). The corrosion potential of the test material was predicted from the relative mean cell viabilities compared to the mean viability of the negative control. Positive (glacial acetic acid) and negative (0.9% (w/v) sodium chloride solution) controls were included in the study and gave the expected results. The relative mean cell viability for the test material was calculated to be 90.7, 97.7 and 98.1% for the 3, 60 and 240 min exposure period, respectively. Based on the evaluation criteria, the test material is considered to be non-corrosive under the conditions of the test method.

In the in vitro key skin irritation study performed according to OECD 439 (adopted 2010) and in compliance with GLP human-derived epidermal keratinocytes (EpiSkin) were exposed to 10 mg of the neat test material for 15 minutes followed by a post-treatment incubation period of 42 hours (Warren, 2014a). The irritation potential of the test material was predicted from the relative mean cell viabilities compared to the mean viability of the negative control. Positive (5% SDS) and negative (DPBS) controls were included in the study and gave the expected results. The relative mean cell viability for the test material was calculated to be 90.9% when compared to the negative control after an incubation period of 42 hours. Based on the evaluation criteria, the test material is considered to be non-irritating under the conditions of the test method.

Taken together and based on the above study results and according to CLP classification criteria, the test substance is considered to be neither corrosive nor irritating to skin.

 

Eye irritation

Two eye irritation studies with boron orthophosphate (CAS 13308-51-5) are available.

The eye irritation properties of boron orthophosphate (CAS 13308-51-5) have been investigated in an in vitro study performed equivalent or similar to OECD 437 and in compliance with GLP using the bovine corneal opacity and permeability test (BCOP, Warren, 2014c). For the assessment of the eye irritation properties 750 µL of the test material was applied to the corneal surface of bovine eyes for 240 min. Positive (20% (w/v) imidazole in 0.9% (w/v) sodium chloride solution) and negative (0.9% (w/v) sodium chloride solution) controls were included in the study. It should be noted that the IVIS cut-off value (≤ 3)

for identifying substances as not requiring classification for eye irritation or serious eye damage is exceeded by the negative control (IVIS = 3.9; 0.9% (w/v) sodium chloride solution). Thus, IVIS value of the negative control is considered to be questionable.

The positive control fulfilled the evaluation and acceptance criteria of the BCOP test method. Mean values of cornea opacity (99.7), permeability (0.060) and In Vitro Irritancy Score (100.6) for the test item were calculated based on 3 treated corneas. Boron orthophosphate (CAS 13308-51-5) induced an IVIS of 100.6 and fulfils the classification criteria for causing irreversible effects on the eye (Category 1) according to Regulation (EC) No 1272/2008. 

 

In the in vivo key eye irritation study performed according to OECD 405 and in compliance with GLP 100 mg of the neat test material (CAS 13308-51-5) was instilled in the eye of two female Albino (Zika) rabbits (Prietzsch, 2014). The eyes were examined and the changes were graded according to the Draize scoring system 1, 24, 48 and 72 hours and 8 days post-application. Slight conjunctival redness and chemosis (grade 1) were observed in 2/2 animals 1 h post-application of the test material, which were fully reversible within 24 h. No effects on cornea and iris were recorded. One animal was further observed for up to 8 days, which showed no effects in the treated eye. No further local or systemic effects and no effects on body weight gain were observed during the study period.

The available in vivo eye irritation study is selected for classification since it is considered to be more relevant to human health compared to the in vitro study. Based on the above in vivo study results and according to CLP classification criteria, the test substance is not considered to be irritating to eyes and thus, not to be classified. 

 

Conclusion

Taken together, the available data on irritation/corrosion with boron orthophosphate (CAS 13308-51-5) do not indicate any hazardous potential on skin and eyes. Thus, the test substance is not to be classified.


Justification for classification or non-classification

The available data on irritation/corrosion with boron orthophosphate (CAS 13308-51-5) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.