Boric acid (H3BO3), solid soln. with strontium oxide, europium-doped

Endpoint summary

Description of key information

Skin irritation: Not irritating (OECD 439/GLP)

Serious eye damage/eye irritation: No prediction (OECD 437/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

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Zhejiang Jingneng Phosphor Material Co., Ltd; 170825
- Expiration date of the lot/batch: 24 August 2019
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis

Vehicle:

other: Dulbecco’s phosphate buffered saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: in vitro EpiDerm™ Skin Irritation Test (EPI-200-SIT), MatTek
- Tissue batch number(s): 28646

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 ± 1 min at 37 ± 1 °C and 25 ± 1 min at room temperature
- Temperature of post-treatment incubation (if applicable): 42 ± 2 h at 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter and bandwidth: filter band pass of maximum ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. The 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, NSMTT equalled 0%.

Colour Interference
To check the colouring potential of the test item 25 mg of the test item were 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. No colouring was detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test item is considered to be irritant to skin in accordance with Regulation EC 1272/2008, if the tissue viability after exposure and post-incubation is less or equal to 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 + 25 µL DPBS

VEHICLE :
Dulbecco’s phosphate buffered saline (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067).

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


POSITIVE CONTROL
- 30 µL 5% sodium dodecyl sulfate (TC-SDS-5%; MatTek, CAS No.: 151-21-3, Lot No.: 081418ALA).

Duration of treatment / exposure:

35 ± 1 min at 37 ± 1 °C and 25 ± 1 min at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 2 h at 37 ± 1 °C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance

Value:

93.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None specified
- Direct-MTT reduction: The 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, NSMTT equalled 0%.
- Colour interference with MTT: No colouring was detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD570 of the three negative control tissues was  0.8 and ≤ 2.8 (1.789).
- Acceptance criteria met for positive control: The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.7%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.3% – 4.3%).
- Range of historical values if different from the ones specified in the test guideline: Historical data were generated from 2015 to 2018 (n=40).

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test using a human epidermal model (EPI-200), the substance is not irritating.

Executive summary:

In an in vitro skin irritation assay in a human epidermal model EPI-200 (185424), reconstructed human epidermis tissue was exposed to 25 mg of boric acid (H3BO3), solid soln. with strontium oxide, europium-doped (99%) + 25 µL DPBS for 35 ± 1 min at 37 ± 1 °C and 25 ± 1 min  at room temperature. DPBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2-hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance does not directly reduce MTT. The average viability of tissues treated by the test substance was 93.6% compared to the negative control i.e. viability was > 50 %. The average viability of tissues treated by the positive control was 4.7% of negative control average value. According to these results, the test substance is not irritating.

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

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 Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Zhejiang Jingneng Phosphor Material Co., Ltd; 170825
- Expiration date of the lot/batch: 24 August 2019
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.

Vehicle:

other: physiological saline 0.9% NaCl

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% concentration.

VEHICLE
- Concentration (if solution): 0.9% NaCl
- Lot/batch no. (if required): B. Braun Melsungen, lot no. 18095403, expiry date: 02/2021

Duration of treatment / exposure:

4 hours ± 5 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The 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 RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

CORNEAS QUALITY CHECK OF THE ISOLATED CORNEAS:
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 were used for the assay.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 3 corneas as negative controls treated with physiological saline 0.9% NaCl

POSITIVE CONTROL USED: 3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium 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 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed.

- POST-EXPOSURE INCUBATION: After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 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).

METHODS FOR MEASURED ENDPOINTS:
- Corneal 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= ( I-0/I-b)/a

with a = 0.025 and b = 0.9894

The 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 periodically.
The 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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry / microtiter plate reader (OD490). Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

- Others (e.g, pertinent visual observations, histopathology): Each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are:
IVIS ≤ 3 = No Category
IVIS > 3; ≤ 55 = No prediction can be made
IVIS> 55 = Category 1

Irritation parameter:

cornea opacity score

Run / experiment:

Test substance

Value:

37.46

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

79.05

Irritation parameter:

fluorescein retention score

Run / experiment:

Test substance

Value:

0.007

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.998

Irritation parameter:

in vitro irritation score

Run / experiment:

Test substance

Value:

37.56

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

109.01

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None of the 3 corneas treated with Boric acid (H3BO3), solid soln. with strontium oxide,europium-doped showed any opacity of the tissue by visual observation after treatment. Residual test item stuck tightly to the cornea. Further washing steps were not possible and a more invasive removal could damage the cornea.

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.
- Range of historical values if different from the ones specified in the test guideline: Historical control data from February 2015 – August 2018 was provdided (n=38).

Interpretation of results:

study cannot be used for classification

Conclusions:

In the Bovine Corneal Opacity and Permeability (BCOP) assay, the IVIS for boric acid (H3BO3), solid soln. with strontium oxide,europium-doped was 37.56. As the IVIS is > 3 but ≤ 55, no prediction can be made.

Executive summary:

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (185425), isolated bovine corneas were exposed to 20% boric acid (H3BO3), solid soln. with strontium oxide,europium-doped (99%) in physiological saline (0.9% sodium chloride) for 4 hours ± 5 minutes using the closed chamber method. 0.9% sodium chloride was used for the negative control and 20% imidazole in 0.9% sodium chloride was used for the positive control. The corneas were rinsed four times and then the opacity of each cornea was recorded. Permeability (after incubation with sodium fluorescein dye) of each cornea was also recorded.

None of the 3 corneas treated with boric acid (H3BO3), solid soln. with strontium oxide,europium-doped showed any opacity of the tissue by visual observation after treatment. Residual test item stuck tightly to the cornea. Further washing steps were not possible and a more invasive removal could damage the cornea. The cornea opacity score for the test substance was 37.46 and the permeability was 0.007. The mean in vitro irritation score was 37.56. The positive control gave the appropriate response. No prediction can be made regarding the classification of boric acid (H3BO3), solid soln. with strontium oxide, europium-doped as the IVIS is > 3 but ≤ 55.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

There is one in vitro skin irritation study available.

In an in vitro skin irritation assay in a human epidermal model EPI-200 (OECD 439/GLP), reconstructed human epidermis tissue was exposed to 25 mg of boric acid (H3BO3), solid soln. with strontium oxide, europium-doped (99%) + 25 µL DPBS for 35 ± 1 min at 37 ± 1 °C and 25 ± 1 min  at room temperature. DPBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2-hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. The colour of the test substance did not interfere with the endpoint. The test substance does not directly reduce MTT. The average viability of tissues treated by the test substance was 93.6% compared to the negative control i.e. viability was > 50 %. The average viability of tissues treated by the positive control was 4.7% of negative control average value. According to these results, the test substance is not irritating.

Serious eye irritation/eye damage

There is one in vitro eye irritation study available.

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), isolated bovine corneas were exposed to 20% boric acid (H3BO3), solid soln. with strontium oxide,europium-doped (99%) in physiological saline (0.9% sodium chloride) for 4 hours ± 5 minutes using the closed chamber method. 0.9% sodium chloride was used for the negative control and 20% imidazole in 0.9% sodium chloride was used for the positive control. The corneas were rinsed four times and then the opacity of each cornea was recorded. Permeability (after incubation with sodium fluorescein dye) of each cornea was also recorded. None of the 3 corneas treated with boric acid (H3BO3), solid soln. with strontium oxide,europium-doped showed any opacity of the tissue by visual observation after treatment. Residual test item stuck tightly to the cornea. Further washing steps were not possible and a more invasive removal could damage the cornea. The cornea opacity score for the test substance was 37.46 and the permeability was 0.007. The mean in vitro irritation score was 37.56. The positive control gave the appropriate response. No prediction can be made regarding the classification of boric acid (H3BO3), solid soln. with strontium oxide, europium-doped as the IVIS is > 3 but ≤ 55.

Justification for classification or non-classification

Based on the available information in the dossier, the substance boric acid (H3BO3), solid soln. with strontium oxide, europium-doped (CAS No. 102110-29-2) is not classified for skin irritation/corrosion and is inconclusive for serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.