Registration Dossier

Administrative data

Description of key information

Dilithium tetraborate was not corrosive in the in vitro skin corrosion test according to OECD TG 431.Dilithium tetraborate was not irritating under the conditions of the in vitro skin irritation study (OECD TG 439).

In an OECD TG 405 study in rabbits, one of the test animals had a lack of recovered effects on the cornea and conjuctivae after 21 days. Dilithium tetraborate was therefore considered to be a Category 1 : H318: Causes serious eye damage.

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
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item storage: At room temperature
Test system:
other: EpiDerm Skin Model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized 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 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM (Dulbecco’s Modified Eagle’s Medium). The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The skin was moistened with 25 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 34.3 to 42.0 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

For the negative and positive controls, 2 tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues were treated with 50 µl 8N KOH (positive control) for both the 3-minute and 1-hour time point.

After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item followed by immediate determination of the cytotoxic (corrosive) effect. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.

For the cell viability measurements, the DMEM was replaced by 300 µl MTT-medium and the tissues were incubated for 3 hours at 37°C in air containing 5% CO2. Following incubation, the tissues were washed with PBS and formazan and then extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
In the test, 34.3 to 42.0 mg of the solid test item was added directly to the top of the skin tissues. No vehicle was used. The skin was dampened with Milli-Q water prior to the test item application.

For the negative and positive controls, 2 tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues were treated with 50 µl 8N KOH (positive control)
Duration of treatment / exposure:
Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
Duration of post-treatment incubation (if applicable):
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item followed by immediate determination of the cytotoxic (corrosive) effect. The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Number of replicates:
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
not corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
not corrosive
Other effects / acceptance of results:
The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Results showed that the solutions did not turn blue / purple nor a blue / purple precipitate was observed. It was therefore concluded that the test item did not interfere with the MTT endpoint.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 99% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 12%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <17%, indicating that the test system functioned properly.


Table 1          Mean Absorption in the in vitro Skin Corrosion Test with the test item

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean

(OD570)

SD

A (OD570)

B (OD570)

Mean

(OD570)

SD

Negative control

1.773

1.920

1.847

0.104

1.958

1.616

1.787

0.242

The test item

1.822

1.629

1.725

0.136

1.608

1.937

1.772

0.233

Positive control

0.220

0.245

0.233

0.017

0.203

0.213

0.208

0.007

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0434). Isopropanol was used to measure the background absorption.

Table 2          Mean Tissue Viability in the in vitro Skin Corrosion Test with the test item

 

3-minute application

viability (percentage of control)

1-hour application

viability (percentage of control)

Negative control

100

100

The test item

93

99

Positive control

13

12

Table 3          Coefficient of Variation between Tissue Replicates

 

3 minute

1 hour

Negative control

7.7

17

The test item

11

17

Positive control

10

4.9

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:
GHS criteria not met
Conclusions:
Lithium tetraborate is not corrosive in the in vitro skin corrosion test according to OECD 431.
Executive summary:

An in vitro skin corrosion test was conducted according to OECD 431. The test item was applied directly to moistened human skin and performed on a total of 4 tissues per test item together with a negative control and positive control.  Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.  The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 99% respectively.  Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive. In addition, the acceptance criteria was met for both the positive and negative control.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2017 - 20 November 2017
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)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item storage: At room temperature


Test system:
other: EpiDerm Skin Model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Not applicable
Justification for test system used:
In the interests of animal welfare, the validated in vitro skin irritation test, EPISKIN test has been conducted initially in order to minimize the need for in vivo testing.
Vehicle:
unchanged (no vehicle)
Details on test system:
The EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-039) is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22.5 hours at 37°C.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The solid test item was applied directly to the skin tissue. The skin was initially moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item. The amount of test item applied ranged from 14.2 mg to 22.2 mg.
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
Duration of post-treatment incubation (if applicable):
The skin tissues were placed in 2 mL warmed maintenance medium until all tissues were dosed and rinsed. The skin tissues were then incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/ml in PBS) and then the tissues were incubated for a further 3 h at 37°C.
Number of replicates:
3 tissues per test item together with negative and positive controls.
Irritation / corrosion parameter:
% tissue viability
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control was determined to be 1.028 with a standard deviation of 0.052 which met the required acceptance criteria (SD<18%).

The mean relative tissue viability of the positive control was determined to be 11% with a standard deviation of 3.7 which was <50% relative to the negative control and within the Standard Deviation (SD) acceptance criteria of <18%.

The mean tissue viability for the test item was determined to be 89%. The standard deviation of the viability of the test item treated tissues was 19% which is above acceptance criteria. However, since all individual viabilities were >50%, the test outcome was considered valid.

Table 1           Mean Absorption in the In Vitro Skin Irritation Test with Dilithium tetraborate

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

 

SD

Negative control

0.978

1.082

1.025

1.028

±

0.052

Test item

1.076

0.698

0.962

0.912

±

0.194

Positive control

0.090

0.161

0.102

0.118

±

0.038

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

Table 2       Mean Tissue Viability in the In VitroSkin Irritation Test with Dilithium tetraborate

 

Mean tissue viability (percentage of control)

Standard deviation (percentage)

Negative control

100

5.1

Test item

89

19

Positive control

11

3.7


 

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, an in vitro skin irritation test was conducted according to OECD 439. Dilithium tetraborate was found to be non-irritant and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

An in vitro skin irritation test was conducted according to OECD 439. The test item was applied directly to moistened human skin for 15 minutes. After exposure, the skin was thoroughly rinsed and transferred to fresh medium. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item.  A negative and positive control were also conducted in parallel.  

 

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Dilithium tetraborate compared to the negative control tissues was 89%. Since the mean relative tissue viability was above 50% after 15 ± 0.5 minutes treatment, Dilithium tetraborate was considered as non-irritant.

 

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure.  The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 6% for the negative and positive control, indicating that the test system functioned properly.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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)
Version / remarks:
Adopted July 26th 2013
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse were used in this study. The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.
Duration of treatment / exposure:
The eye damage of dilithium tetraborate was tested through topical application for approximately 240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Two identical experiments were undertaken
Details on study design:
The test consists of topical application of dilithium tetraborate on the epithelium of the bovine cornea for 4 hours. The non-surfactant solid test item is applied neat by direct application to the surface of the cornea. After exposure the corneas were thoroughly rinsed. The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.
Irritation parameter:
cornea opacity score
Run / experiment:
Dilithium tetraborate - first experiment
Value:
2.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Dilithium tetraborate - second experiment
Value:
-1.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Dilithium tetraborate - first experiment
Value:
37
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Dilithium tetraborate - second experiment
Value:
14
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Dilithium tetraborate - first experiment
Value:
2.297
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Dilithium tetraborate - second experiment
Value:
0.99
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corneas treated with dilithium tetraborate showed opacity values ranging from 2.0 to 3.9 and permeability values ranging from 0.317 to 3.903. The corneas were translucent after the 240 minutes of treatment with dilithium tetraborate. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 7.4 to 60 after 240 minutes of treatment with Dilithium tetraborate.Since the results for dilithium tetraborate were spread over 2 categories (7.4, 60 and 44, respectively), the test was inconclusive and a repeat experiment was performed. The corneas treated with dilithium tetraborate showed opacity values ranging from -1.8 to -1.0 and permeability values ranging from 0.065 to 2.642. The corneas were partly translucent after the 240 minutes of treatment with dilithium tetraborate. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from -0.8 to 39 after 240 minutes of treatment with dilithium tetraborate. Since both tests provided inconclusive results, no conclusion can be drawn for eye irritation or serious eye damage.

First experiment

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

4.0

0.022

4.3

Positive control

116

1.744

143

Dilithium tetraborate

2.8

2.297

37

1  Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2  In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

 

Second experiment

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

2.5

0.021

2.8

Positive control

123

1.758

150

Dilithium tetraborate

-1.3

0.990

14

1    Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


 

Interpretation of results:
study cannot be used for classification
Conclusions:
Both tests (experiment 1 & 2) provided inconclusive results, and therefore no conclusion can be drawn for eye irritation or serious eye damage.
Executive summary:

In a well conducted Bovine Corneal Opacity and Permeability Assay (BCOP), dilithium tetraborate was topically applied to the epithelium of the bovine cornea for 4 hours. The non-surfactant solid test item was applied neat by direct application to the surface of the cornea. After exposure the corneas were thoroughly rinsed. The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein. The mean in vitro irritancy score was 37 and 14 after 240 minutes of treatment with the test item in the first and second experiment, respectively. The results for dilithium tetraborate were spread over 2 categories in both experiments. Because both tests provided inconclusive results, no conclusion can be drawn for eye irritation or serious eye damage. A further study on rabbits was therefore conducted (van Sas, 2017). 

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27th November 2017 - 24th December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
An additional body weight was obtained for animal 336 on Day 8. This study plan deviation did not affect the integrity of the study because the additional data does not influence the outcome of the study.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
3 males, approximately 13-15 weeks old were used. Weight at initation of dosing - 3029 to 3501 g. The animals were allowed to acclimate to the test facility toxicology accommodation for at least 5 days before the commencement of dosing. Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 20°C with an actual daily mean relative humidity of 65 to 68%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Vehicle:
water
Controls:
yes
other: The untreated eye served as the reference control
Amount / concentration applied:
61.6 mg (range 61.4 – 61.7 mg) of the test item (a volume of approximately 0.1 mL)
Duration of treatment / exposure:
One hour exposure
Observation period (in vivo):
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and 14 days, and for two animals, 21 days after instillation of the test item.
Number of animals or in vitro replicates:
3
Details on study design:
Animals were treated by instillation of, on average, 61.6 mg (range 61.4 – 61.7 mg) of the test item (a volume of approximately 0.1 mL), in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control. Immediately after the 1 hour observation, the treated eye was rinsed with approximately 100 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove any visible residual test item. For reference control, the other eye was also rinsed. Additional injections of buprenorphine 0.01 mg/kg were administered immediately after the 1-hour observation and at the end of the first day for one animal to reduce pain and distress.The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and 14 days, and for two animals, 21 days after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
Irritation parameter:
cornea opacity score
Basis:
animal: 324
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
not specified
Irritation parameter:
cornea opacity score
Basis:
animal: 335
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not fully reversible within:
Remarks:
21 days
Irritation parameter:
cornea opacity score
Basis:
animal: 336
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal: 324
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal: 335
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
not specified
Irritation parameter:
iris score
Basis:
animal: 336
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal: 324
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal: 335
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not specified
Irritation parameter:
conjunctivae score
Basis:
animal: 336
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal: 324
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal: 336
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
not specified
Irritation parameter:
chemosis score
Basis:
animal: 335
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
not specified
Irritant / corrosive response data:
The corneal injury consisted of opacity and epithelial damage which resolved within 7 days in two animals and persisted until termination in one animal.
Iridial irritation was observed and resolved within 48 hours for one animal, 7 days for one animal and 14 days for the remaining animal.

The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in one animal, 21 days in one animal and persisted until termination in the remaining animal.

Reduced elasticity of the eyelids was noted for one animal after 72 hours and 7 days and for one animal after 7, 14 and 21 days.
Other effects:
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

TABLE 1: Individual Eye Irritation Scores

Animal 

Time after dosing

Cornea

Iris

Conjunctivae

Comments

Opacity

(0-4)

Area

(0-4)

Fluor area (%)2

 

(0-2)

Redness

(0-3)

Chemosis

(0-4)

Discharge

(0-3)

3241

 1 hour

0

2

n.a.

1

2

3

3

b, g

 

24 hours

1

2

35

1

3

2

1

-

 

48 hours

1

1

 n.a.

0

3

2

2

-

 

72 hours

0

1

10

0

3

1

1

g

 

 7 days

0

0

0

0

2

0

0

-

 

14 days

0

0

n.a.

0

1

0

0

-

 

21 days

0

0

n.a.

0

0

0

0

-

335

 1 hour

0

2

n.a.

1

2

2

2

b, g

 

24 hours

1

2

50

1

3

2

2

-

 

48 hours

1

2

 n.a.

1

3

3

3

-

 

72 hours

1

2

70

1

3

3

3

-

 

 7 days

1

2

70

1

2

2

2

f

 

14 days

0

1

10

0

1

0

0

f, g

 

21 days

0

1

3

0

1

0

0

f, g

336

 1 hour

0

2

n.a.

1

2

2

2

b, g

 

24 hours

1

2

50

1

3

2

2

-

 

48 hours

1

2

 n.a.

1

3

2

3

-

 

72 hours

1

1

25

1

2

1

2

f

 

 7 days

0

0

0

0

0

0

0

f

 

14 days

0

0

n.a.

0

0

0

0

-

 1Sentinel,2  Green staining after fluorescein treatment (percentage of total corneal area)

n.a.     Not applicable

Comments:

b          Remnants of the test item in the eye.

f          Reduced elasticity of the eyelids.

g          Slight dulling of the normal luster of the cornea.

TABLE 2: Mean Eye Irritation Scores

Animal

Mean 24, 48 and 72 hours

Corneal

opacity

Iris

 

Conjunctivae

Redness

 

Chemosis

324

0.7

0.3

3.0

1.7

335

1.0

1.0

3.0

2.7

336

1.0

1.0

2.7

1.7

 

 

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the not fully recovered effects on the cornea and conjunctivae of one animal after 21 days, dilithium tetraborate should be classified in accordance with the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) / Regulation (EC) No 1272/2008 (including all amendments) as Category 1 - causes serious eye damage.
Executive summary:

Single samples of approximately 61.6 mg of dilithium tetraborate (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made at 1, 24, 48 and 72 hours and 7 and 14 days, and for two animals, 21 days after instillation. This resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage which resolved within 7 days in two animals and persisted until termination in one animal. Iridial irritation was observed and resolved within 48 hours for one animal, 7 days for one animal and 14 days for the remaining animal. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in one animal, 21 days in one animal and persisted until termination in the remaining animal. Reduced elasticity of the eyelids was noted for one animal after 72 hours and 7 days and for one animal after 7, 14 and 21 days. Because the effects on the cornea and conjunctivae was not fully recovered after 21 days, dilithium tetraborate should be classified in accordance with the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) as Category 1 - causes serious eye damage.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In an OECD TG 405 study in rabbits, one of the test animals had a lack of recovered effects on the cornea and conjuctivae after 21 days. Therefore in accordance with the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) / Regulation (EC) No 1272/2008 (including all amendments) dilithium tetraborate is classified as Category 1- H318: Causes serious eye damage.