Triboron trimethyl hexaoxide

Endpoint summary

Description of key information

Skin irritation

An in vitro study using the EpiDermTM human skin model showed the non-corrosivity of

Trimethoxyboroxin . In order to further assess the acute skin irritation potential of the test

substance, a dermal irritation/corrosion test in White New Zealand rabbits was performed

according to the method described in OECD guideline 404 .

An amount of 0 .5 mL of the test substance was applied for 4 hours to the intact skin of three

rabbits, using a patch of 2 .5 cm x 2 .5 cm, covered with semiocclusive dressing . After removal

of the patch the application area was washed off .

The cutaneous reactions were assessed immediately after removal of the patch,

approximately 1, 24, 48 and 72 hours after removal of the patch and then in weekly intervals

until day 14 . Slight to marked erythema and slight to moderate edema were observed in the animals

during the course of the study. In addition scaling was noticed in one animal on day 7 .

The cutaneous reactions were reversible in all animals within 14 days after removal of the

patch . The average score (24 to 72 hours) for irritation was calculated to be 1 .8 for erythema and

0.2 for edema. Considering the described cutaneous reactions as well as the average score for irritation,

Trimethoxyboroxin shows a skin irritation potential under the test conditions chosen , however the criteria for classificaiton as a skin irritant under 1272/2008/EC were not met.

The eye irritancy potential of TMBX was examined in the HET-CAM using the ICCVAM protocol 2006. When using the IS analysis method, the severe irritancy classification for a test substance is used when the value is greater than nine. The IS obtained was 7.38, however the study director assigned Category 1 Causes Severe eye damage to the substance. A further in BCOP study was conducted to validate the HET-CAM result. The outcome was inconclusive, with one of the three repeats resulting in no classification and two further repeats being inconclusive (IVIS > 3 ≤ 55)

All together the test item resulted in equivocal results and no prediction of the category can be made based on these experiments.  

Consequently the precautionary principle was applied and the original study directors conclusion of Category 1 H318 Causes severe eye damage was adopted.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Qualifier:

according to guideline

Guideline:

other: Japan MAFF Testing Guideline of 12 Nousan No. 8147

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre Lago S.A., 01540 Vonnas, France
- Age at study initiation: 7 - 8 months
- Weight at study initiation: 3.62 - 3 .88 kg
- Housing: Single housing in stainless steel wire mesh cages with grating, floor area: 3000 cm2
- Diet: Kliba-Labordiät (Kaninchen & Meerschweinchenhaltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland (about 130 g/animal per day)
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes: The animals were housed in fully air-conditioned rooms.
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated skin of the same animal

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

up to 14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: flank: 2.5 x 2.5 cm
- Type of wrap if used: a test patch (Idealbinde, Pfälzische Verbandstoff-Fabrik, Kaiserslautern) and Fixomull® stretch (adhesive fleece), Beiersdorf AG

REMOVAL OF TEST SUBSTANCE
- Washing: with Lutrol® and Lutrol® / water (1 : 1)
- Time after start of exposure: 4 hours

SCORING SYSTEM: according the quoted guidelines

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

1.8

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0.2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Moderate or marked erythema (grade 2 or 3), observed in all animals immediately after removal of the patch up to 24 hours, persisted in two animals up to 72 hours. Moderate erythema decreased to slight (grade 1) in one animal after 48 hours and in two animals on day 7. Slight erythema persisted in one animal from 48 hours up to day 7.
Moderate edema (grade 2), noted in all animals immediately after removal of the patch up to 1 hour, decreased to slight (grade 1) in two animals after the 24-hour reading.
In addition, scaling was noticed in one animal on day 7.
The cutaneous reactions were reversible in all animals within 14 days after removal of the patch.
Mean scores over 24, 48 and 72 hours for each animal were 1.3, 2.0 and 2.0 for erythema and 0.3, 0.3 and 0.0 for edema.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the criteria as laid down in GHS-UN, the substance can be considered as slightly irritating.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICCVAM HET-CAM

Principles of method if other than guideline:

HET-CAM in vitro corrosion test (alternative method to study the potential of serious damage to the eyes/mucous membranes in incubated hen eggs).

GLP compliance:

not specified

Species:

other: chorionallantoic membrane of fertilized hen eggs

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.3 mL

Duration of treatment / exposure:

single application

Observation period (in vivo):

3.5 minutes

Number of animals or in vitro replicates:

3 eggs

Details on study design:

SCORING SYSTEM:
After application of the test substance the chorionallantoic membrane was observed by means of a stereomicroscope until unambiguous irritation
reactions were detected or up to a maximum time period of 3.5 minutes, respectively.
The time of appearance (in seconds after application) of intravascular resp. extravascular coagulation and, if applicable, other reactions
(haemorrhagia, vessel lysis) were determined.

The evaluation of the reactions was performed according to the following grading:
0 = no visible change
1 = slight reaction
2 = moderate reaction
3 = severe reaction

TOOL USED TO ASSESS SCORE: stereomicroscope

Irritation parameter:

other: time until appearance of coagulation

Run / experiment:

1

Value:

ca. 64

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

positive indication of irritation

Remarks:

intravascular coagulation

Irritation parameter:

other: time until appearance of coagulation

Run / experiment:

2

Value:

ca. 81

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

positive indication of irritation

Remarks:

intravascualr coagulation

Irritation parameter:

other: time until appearance of coagulation

Run / experiment:

3

Value:

ca. 49

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

positive indication of irritation

Remarks:

intravascular coagulation

Irritation parameter:

other: time until appearance of coagulation

Run / experiment:

mean n=3

Value:

ca. 64

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

positive indication of irritation

Remarks:

intravascular coagulation

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

ca. 7.38

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The formula used to generate the IS is {(301-hemorrhage time)/300}*5 + {(301-lysis time)/300}*7 + {(301-coagulation time)/300}*9

CRITERIA FOR AN ACCEPTABLE TEST
A test is considered acceptable if the negative and positive controls each induce a response
that falls within the classification of nonirritating and severely irritating, respectively.
Historical control studies indicate that using 0.9% NaCl, as a negative control, the IS value
was 0.0. Historical control studies indicate that using 1% SDS and 0.1 N NaOH, as positive
controls, the IS values ranged between 10 and 19, respectively.
9.0 DATA INTERPRETATION
When using the IS analysis method, the severe irritancy classification for a test substance is
used when the value is greater than nine.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Conclusions:

The eye irritancy potential of TMBX was examined in the HET-CAM using the ICCVAM protocol 2006. When using the IS analysis method, the severe irritancy classification for a test substance is used when the value is greater than nine. The IS obtained was 7.38, however the study director assigned Category 1 Causes Severe eye damage to the substance.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch 10037-115-5
Purity 99%

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands),
- Number of animals: not determined
- Characteristics of donor animals (e.g. age, sex, weight): young cattle, sex/weight not determined
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples: 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.
- Indication of any antibiotics used: not determined

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750µL of test item

Duration of treatment / exposure:

10 +/- 1 minutes

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

1 hour pre-exposure in cMEM; 120 +/- 10 minutes post exposure

Number of animals or in vitro replicates:

3 (three) corneas per treatment

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1C. The corneas were incubated for the minimum of 1 hour at 32 +/-1C.
Cornea Selection and Opacity Reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES
three corneas per treatment
NEGATIVE CONTROL USED
yes, physiological saline
SOLVENT CONTROL USED (if applicable)
N/A
POSITIVE CONTROL USED
Ethanol
APPLICATION DOSE AND EXPOSURE TIME
750µL for 10+/-1 minutes
TREATMENT METHOD: [closed chamber / open chamber]
closed chamber
POST-INCUBATION PERIOD: yes. If YES please specify duration
at least 1 hour
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION:
120 +/-10 minutes
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured using OP-KIT
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): none specified
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.- Yes

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

>= 3 - <= 7.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Run / experiment:

1

Value:

>= 0.032 - <= 0.202

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

>= 5.6 - <= 8.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.2-0.7

Positive controls validity:

valid

Remarks:

55-59

Remarks on result:

not determinable

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

>= -1.8 - <= -0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Run / experiment:

2

Value:

>= 0.005 - <= 0.015

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

>= -1.5 - <= -0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.3-2.3

Positive controls validity:

valid

Remarks:

37-53

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

>= -0.8 - <= 0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Run / experiment:

3

Value:

>= 0.021 - <= 0.657

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

>= -0.5 - <= 10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

-0.5-1.3

Positive controls validity:

valid

Remarks:

28-61

Remarks on result:

not determinable

In the first test, the individual in vitro irritancy scores for the negative controls ranged from 0.2 to 0.7. The individual positive controlin vitroirritancy scores ranged from 55 to 59. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Trimethoxyboroxine (TMBX) showed opacity values ranging from 3.0 to 7.9 and permeability values ranging from 0.032 to 0.202. The corneas were translucent after the 10 minutes of treatment with Trimethoxyboroxine (TMBX). A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, thein vitroirritancy scores ranged from 5.6 to 8.7 after 10 minutes of treatment with Trimethoxyboroxine (TMBX).

In the repeat test, the individual in vitro irritancy scores for the negative controls ranged from 0.3 to 2.3. The individual positive controlin vitroirritancy scores ranged from 37 to 53. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Trimethoxyboroxine (TMBX) showed opacity values ranging from -1.8 to -0.2 and permeability values ranging from 0.005 to 0.0015. The corneas were clear after the 10 minutes of treatment with Trimethoxyboroxine (TMBX). A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, thein vitroirritancy scores ranged from -1.5 to -0.1 after 10 minutes of treatment with Trimethoxyboroxine (TMBX).

In the second repeat test, the individual in vitro irritancy scores for the negative controls ranged from -0.5 to 1.3. The individual positive controlin vitroirritancy scores ranged from 28 to 61. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Trimethoxyboroxine (TMBX) showed opacity values ranging from -0.8 to 0.2 and permeability values ranging from 0.021 to 0.657. The corneas showed a spot in the middle after the 10 minutes of treatment with Trimethoxyboroxine (TMBX). A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, thein vitroirritancy scores ranged from -0.5 to 10 after 10 minutes of treatment with Trimethoxyboroxine (TMBX).

Interpretation of results:

study cannot be used for classification

Conclusions:

In the first test, since Trimethoxyboroxine (TMBX) induced an IVIS > 3 ≤ 55, no prediction on the classification can be made in this experiment.
In the repeat test, since Trimethoxyboroxine (TMBX) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. Since this outcome was different from the initial test a second repeat test was performed.
In this second repeat test, since Trimethoxyboroxine (TMBX) induced an IVIS > 3 ≤ 55, no prediction on the classification can be made in this experiment.
All together the test item resulted in equivocal results and no prediction of the category can be made based on these experiments.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin irritaiton potential of TMBX was tested in an in vivo assay in acordance with OECD 404.

Slight to marked erythema and slight to moderate edema were observed in the animals

during the course of the study. In addition scaling was noticed in one animal on day 7 .

The cutaneous reactions were reversible in all animals within 14 days after removal of the

patch . The average score (24 to 72 hours) for irritation was calculated to be 1 .8 for erythema and

0.2 for edema. Considering the described cutaneous reactions as well as the average score for irritation,

Trimethoxyboroxin shows a skin irritation potential under the test conditions chosen , however the criteria for classification as a skin irritant under 1272/2008/EC were not met.

The eye irritancy potential of TMBX was examined in the HET-CAM using the ICCVAM protocol 2006. When using the IS analysis method, the severe irritancy classification for a test substance is used when the value is greater than nine. The IS obtained was 7.38, however the study director assigned Category 1 Causes Severe eye damage to the substance. A further in BCOP study was conducted to validate the HET-CAM result. The outcome was inconclusive, with one of the three repeats resulting in no classification and two further repeats being inconclusive (IVIS > 3 ≤ 55)

All together the test item resulted in equivocal results and no prediction of the category can be made based on these experiments.  

Consequently the precautionary principle was applied and the original study directors conclusion of Category 1 H318 Causes severe eye damage was adopted.