Triboron trimethyl hexaoxide

Endpoint summary

Description of key information

TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found in the body fluids and tissues following absorption by any route of administration. Therefore, an assessment of repeat dose toxicity was conducted taking account of the hydrolysis breakdown products of TMBX.

A number of sub-chronic and chronic studies on boric acid and disodium tetraborate decahydrate were carried out in rats, mice and dogs. Most support that boron can cause adverse haematological effects and that the main target organ of boron toxicity is the testis. The NOAEL for fertility effects is equivalent to 17.5 mg B/kg bw/day that corresponds to NOAEL of 100 mg Boric Acid/kg bw (Weir, 1966a, b).

A reliable oral NOAEL for methanol toxicity following repeated exposure is not available. A LOAEL of 2340 mg/kg bw/day resulted from a study in monkeys where all the subjects died within 3 days of the initial dosing (Rao et al, 1977).A further oral repeat dose study in monkeys (Martin-Amat et al, 1977), which examined the ocular toxicity following an initial dose of 2000 mg/kg bw on day 1 followed by 500 mg/kg at varying intervals did not allow a NOAEL to be derived, despite producing ocular lesions over the course of the study.

The most appropriate point of departure following repeated oral exposure is based on the NOAEL for Boric acid of 17.5 mg B/kg bw day, an equivalent NOAEL for TMBX of 93.73 mg/kg bw/day can be derived.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedures in accordance with accepted standard methods, sufficiently documented.

Principles of method if other than guideline:

Test model in monkeys for methanol-induced occular toxicity after short-term exposure to characterize the toxicity syndrome and histological manifestations.

GLP compliance:

no

Limit test:

no

Species:

monkey

Strain:

other: Macaca mulatta

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.6-4.4 kg

Route of administration:

other: nasogastric tube

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20 % (w/v)
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

approx. 1.5 to 6 days

Frequency of treatment:

variable

Remarks:

Doses / Concentrations:
initially 2000 mg/kg, thereafter 500 mg/kg at variable frequencies and time points (exception: one animal 1000 mg/kg at 44 and 72 h and 2000 mg/kg at 144h)
Basis:
other: nominal

No. of animals per sex per dose:

6 males in total

Control animals:

other: internal/same animal prior to treatment

Details on study design:

- Dose selection rationale: A high initial dose (2000 mg/kg) was followed by lower doses depending on the animal´s acidotic response in blood. Experience had told (McMartin et al., 1975) that after a single dose of 3000 mg/kg bw, in general, the animals died within 20 to 30 h without demonstrating ocular abnormalities. (Martin-Amat et al., 1977).

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (McMartin 1975, 3000 mg/kg)
- Time schedule: Continuously from application until death (approx. 33 h after application
- Cage side observations included: clinical signs, mortality


DETAILED CLINICAL OBSERVATIONS: Yes: cerebrospinal fluid pressure in cerebellomedullary cistern by cisternal puncture (2 animals)
- Time schedule: not specified


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: one week before and during the course of intoxication, not further specified
- Dose groups that were examined: all animals: stereoscopic color fundus photography, fluorescein fundus angiography, pupillary light reflex,


HAEMATOLOGY: No


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment and throughout the course of the study
- Animals fasted: No data
- How many animals: all
- Parameters examined: blood pH, bicarbonate levels, methyl alcohol, blood formate (cerebrospinal fluid: 2 animals), pO2, pCO2


URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes (light and electron microscopic studies of neuronal tissues and nerve fibres associated with the eyes: retina, optic nerve heads, optic nerves of 2 control and 3 treated animals)

Details on results:

OPHTHALMOSCOPIC EXAMINATION
The only detectable ocular change was optic disc edema (of the optic papilla). The primary sites of ocular injury were the optic nerve heads and the anterior segment of the optic nerve rather than the retinal ganglion cells themselves. In all eyes with optic disc changes, pupils were dilated and reacted poorly to light.

CLINICAL CHEMISTRY
Under methanol treatment acc. to this test design, formate levels were between min. 7.2 and max. 14.4 mEq/L in blood and 7.9 to 13.9 mEq/L in cerebrospinal fluid, blood bicarbonate min. 4.0 and max. 10.2 mEq/L, and blood pH min. 7.13 and max. 7.28. Methanol levels ranged from 1540 to 2840 mg/L (Martin-Amat et al., 1977).

HISTOPATHOLOGY: NON-NEOPLASTIC
All six animals developed fundus changes at the head of the optic nerve (optic disc) within 43 to 171 h after methanol ingestion, expressed as intraaxonal swellings (Hayreh et al, 1977). Electronmicroscopic studies revealed swelling of the nerve fibers with an accumulation/clustering of swollen mitochondria in the optic nerve head being maximally in the lamina cribrosa region. Furthermore, in the retrolaminar and intraorbital optic nerve, swelling of astrocytes was prominent as well as swelling of the cytoplasm of the oligodendroglial cytoplasm in contact with the axons (Baumbach et al., 1977). Alterations were not observed in the retina itself: the ganglion cells of the retina were intact with only minimal swellings of the mitochondria and loss of cristae. But these findings were also present in the control tissue (Baumbach et al., 1977).

Dose descriptor:

NOAEL

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

While acute methanol toxicity in monkeys (after a single dose) does not yield ocular signs, repeated dosing succeeded in producing ocular lesions (Martin-Amat et al, 1977).

The only detectable ocular change was optic disc edema (of the optic papilla) which was similar to that seen in raised intracranial pressure in humans, but without this pressure after methanol (Hayreh et al, 1977). The primary sites of ocular injury were the optic nerve heads and the anterior segment of the optic nerve rather than the retinal ganglion cells themselves. It appears that interference with oxidative phosphorylation causes mitochondrial damage, thus disruption of active axoplasmic flow in the retrolaminar optic nerve (Baumbach et al., 1977; Hayreh et al., 1977). [note: In humans it has been hypothesized that optic atrophy, which often follows acute methanol intoxication, is secondary to injury of the retinal ganglion cells.]. Mechanistically, there is a close causal relationship between the prolonged increase in formic acid from methanol and the development of optic edema. Similar effects can be produced by intravenous administration of formate without acidosis (Martin-Amat et al., 1978).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, sufficiently documented.

Principles of method if other than guideline:

Daily application of a single dose of methanol to monkeys by gavage over a period of 3 days.

GLP compliance:

not specified

Limit test:

no

Species:

monkey

Strain:

not specified

Sex:

male

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30 % solution
- Amount of vehicle (if gavage): no data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

3 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
2340 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

7 males

Control animals:

not specified

Dose descriptor:

LOAEL

Effect level:

2 340 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: mortality; 2340 mg/kg bw was the lethal dose for all 7 animals under test after 3 days.

Critical effects observed:

not specified

2340 mg/kg bw was the lethal dose for all 7 animals under test after 3 days.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

other: no data

Deviations:

not specified

Principles of method if other than guideline:

2 year dietary feeding study in Sprague Dawley rats, 35 per sex per treated group and 70 controls per sex with interim kills of 5/sex/group at 6 and 12 months at 0; 670 (117); 2000 (350); 6690 (1170) ppm boric acid (ppm as boron equivalents) equivalent to 0, 33 (5.9), 100 (17.5), 334 (58.5) mg boric acid (B)/kg bw per day.

GLP compliance:

no

Remarks:

Study pre-dates GLP

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Males 93 - 129 g; females 86 - 128 g

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

2 years

Frequency of treatment:

Daily; ad libitum.

Remarks:

Doses / Concentrations:
0; 670 (117); 2000 (350); 6690 (1170) ppm boric acid (ppm as boron equivalents) equivalent to 0, 33 (5.9), 100 (17.5), 334 (58.5) mg boric acid (B)/kg bw per day
Basis:
nominal in diet

No. of animals per sex per dose:

35/sex/group

Control animals:

yes, plain diet

Details on study design:

No data

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: recorded weekly for the first 52 weeks, then 4 weekly


BODY WEIGHT: Yes
- Time schedule for examinations: recorded weekly for the first 52 weeks, then 4 weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): recorded weekly for the first 52 weeks, then 4 weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood:at 1, 2, 3, 6 ,12, 18 and end of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: on 5/sex/group
- Parameters examined: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at interim sacrifice at 6, 18 and 24 months for blood pH, sodium, potassium, chloride and carbon dioxide combining power; and at 6, 12 and 24 months for SGOT and SGPT
- Animals fasted: No data
- How many animals: 2/sex/group except SGOT and SGPT which were in 5/sex/group in the hihg and control dose groups
- Parameters: blood pH, sodium, potassium, chloride, carbon dioxide combining power, SGOT and SGPT


URINALYSIS: Yes
- Time schedule for collection of urine: at 6 months
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: appearance, volume, osmolality, specific gravity, pH, protein, glucose, blood, acetone, bilirubin and microscopy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes at 6 and 12 months 5 rats per sex per group, all interim deaths and at termination in 10 per sex per group in controls and high dose surviving animals.
Organs: Brain, pituitary, thyroid, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, heart, lungs, gonads, urinary bladder, sternum, rib junction and all unusual lesions.

HISTOPATHOLOGY: Yes 10 rats per sex per group from the mid and low dose groups had gonads examined histologically

Other examinations:

Samples of blood, brain, liver and kidney were taken at 6, 12 and 24 months and frozen for boron analysis.

Statistics:

As appropriate.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No signs in the low and mid dose groups. Coarse hair coats, hunched position, swollen pads and inflamed bleeding eyes were observed in animals receiving the highest dose of boric acid.
Survival at 6, 12 and 24 months was comparable in all groups including controls.


BODY WEIGHT AND WEIGHT GAIN
No difference from controls in the low and mid dose group. Retarded body weight gain in animals receiving the highest dose of boric acid.


FOOD CONSUMPTION AND COMPOUND INTAKE
No difference from controls in the low and mid dose group. Reduced food intake in the highest dose group during weeks 1-13 in males, and in weeks 1-13 and 42-52 in females.


HAEMATOLOGY
No difference from controls in the low and mid dose groups. Significantly decreased red cell volume and haemoglobin were observed in the high dose group males at 3, 6, 12, 18 and 24 months. Hemoglobin values for the males in the high level test group were consistently below the normal range for adult male rats. Cell volume values for this group were, at most periods of determination, also below normal or within low normal range. The total leukocyte counts for the high level males were lower than those for the male controls at each determination but generally within normal limits. The hematological values determined during the first year for the low and intermediate level males and the females at all three test levels were generally within normal limits and comparable with the control values.


CLINICAL CHEMISTRY
No significant differences between groups.

URINALYSIS
No significant differences between groups.


ORGAN WEIGHTS
The testes weights and the testes/bodyweight ratios were significantly lower in the high dose group than those of control animals. The brain- and thyroid-to-bodyweight ratios in the high dose females were significantly higher than those of controls. This was thought to relate to the reduced bodyweight of the animals.

GROSS PATHOLOGYAND HISTOPATHOLOGY
Atrophic testes were found in all males exposed to the high dose 334 (58.5) mg boric acid (B)/kg bw) of boric acid at 6, 12 and 24 months. Microscopic examination of the tissue revealed atrophied seminiferous epithelium and decreased tubular size in the testes. Cysts in the eyelids, probably in the Meiobomian glands were observed in 4 high dose females, probably related to treatment. There was no treatment related increase in tissue masses.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

334 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on testicular atrophy in males and reduced body weight in females

Dose descriptor:

NOAEL

Effect level:

17.5 mg/kg bw/day (nominal)

Based on:

element

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

58.5 mg/kg bw/day (nominal)

Based on:

element

Sex:

male/female

Basis for effect level:

other: Based on testicular atrophy in males and reduced body weight in females.

Critical effects observed:

not specified

 

Parameter	Control		Low dose		Medium dose		High dose		Dose- response +/-
	m^a	f^a	m^a	f^a	m^a	f^a	m^a	f^a	m	f
number of animals examined	70	70	35	35	35	35	35	35
Mortality at 104 weeks	25/60	20/60	6/25	8/25	9/25	10/24	7/25	5/25	N	N
clinical signs*
body weight gain 0-104 weeks (g)	557	405	546	318	499	359	449	238	Y	Y
food consumption at week 52 (g/kg/day)	33.3	43.7	35.4	42.9	35.3	44.6	39.7	52.7
clinical chemistry*	no differences
haematology*	see separate  table
urinalysis*	No differences
testes weight*(g) at 26 weeks	3.76+0.29		3.67+0.29		3.81+0.14		0.95+0.06 sig low
testes weight (g) at 104 weeks	3.65+0.84		3.65+0.63		3.30+0.60		0.99+0.24 sig low
microscopic pathology* Testes atrophy at 24 months	3/10		1/10		4/10		10/10

 

 

Summary of haematological data from 2 year rat study boric acid:

Months	Cell Volume (%)
	Male
	Control	0.067%	0.2%	0.67%
	0	5.9 mg B/kg	17.5 mg B/kg	58.5 mg B/kg
1	42.6	45.3	42.7	39.0
2	44.1	44.9	45.5	40.8*
3	45.9	46.7	45.7	39.7*
6	45.4	45.9	46.5	44.6
12	47.3	45.5	44.8	41.4*
18	47.8	43.2*	42.8*	39.2*
24	46.4	36.4*	43.8	41.68
	Female
1	42.1	44.5	42.4	43.3
2	41.7	43.7	43.0	40.8
3	44.2	47.2	45.1	42.0
6	43.3	44.7	Data missing
12	42.8	43.9	41.8	40.6
18	43.0	43.0	42.8	39.3*
24	46.2	45.6	44.4	41.6

 

 

Months	Hb Value (g/100 mL)
	Male
	Control	0.067%	0.2%	0.67%
	0	5.9 mg B/kg	17.5 mg B/kg	58.5 mg B/kg
1	14.5	14.2	14.2	12.6*
2	14.7	14.1	14.4	13.2
3	15.7	15.2	14.9	13.3*
6	15.4	15.0	14.2	13.7*
12	14.1	13.2	13.4	12.6*
18	15.6	14.9	13.8*	12.7*
24	14.7	11.9	13.6*	12.8*
	Female
1	14.6	15.3	14.3	14.0
2	14.9	15.2	14.4	14.7
3	14.9	15.7	14.0	14.2
6	14.5	14.8	Data missing
12	12.9	13.2	13.2	12.6
18	14.8	13.9	14.6	13.6
24	14.4	13.2*	13.0*	12.5*

 

 

Months	WBC Count (x10^3/cm^2)
	Male
	Control	0.067%	0.2%	0.67%
	0	5.9 mg B/kg	17.5 mg B/kg	58.5 mg B/kg
1	18.1	13.6	15.3	8.0*
2	19.3	18.4	16.8	14.7
3	20.9	23.4	19.4	16.7
6	19.4	15.6	14.3	15.3
12	10.9	10.9	10.9	10.5
18	23.4	22.9	19.5	18.4
24	19.8	18.1	14.3	13.2*
	Female
1	19.8	20.9	17.3	14.7
2	16.6	28.9	17.1	17.4
3	26.6	19.0	18.6	21.1
6	14.6	14.1	Data missing
12	9.5	13.5	7.3	11.4
18	10.9	11.5	16.4	11.6
24	17.6	12.8	11.3	10.5

 

 

Months	RBC Count (x10^3/cm^2)
	Male
	Control	0.067%	0.2%	0.67%
	0	5.9 mg B/kg	17.5 mg B/kg	58.5 mg B/kg
1
2	8.2	7.68	7.98	7.00*
3	7.14	6.72	7.47	6.47
6
12
18	5.16	5.46	5.55	4.92
24	7.09	5.72	7.35	7.90
	Female
1
2	7.36	7.44	7.46	7.57
3	5.64	7.03	6.47	6.52
6
12
18	6.58	6.11	5.69	5.73
24	6.22	6.24	6.22	5.92

* Significantly different from controls

Missing data not thought to be significant according to the summary of the study

 

Conclusions:

Endpoint Effect level
NOAEL 17.5 mg Boron/kg bw/day (nominal)
LOAEL 58.5 mg Boron/kg bw/day (nominal)

Testicular atrophy and seminiferous tubule degeneration was observed at 6, 12 and 24 months at the highest dose level only. No treatment related effects were observed in the mid and low dose groups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

93.7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The study meets generally accepted scientific standards with acceptable restrictions.

System:

male reproductive system

Organ:

gonad

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Boric acid is classified under the 1^stATP to CLP as Repr. 1B; H360FD, consequently TMBX also warrants a similar classificaiton based upon the hydrolysis products previously described.

Regarding systemic target organ toxicity after repeated exposures (STOT-RE), boric acid , and hence TMBX, does not meet criteria for classification and labelling according to EU CLP Regulation (EC) No. 1272/2008) because no other than testis target organs were identified during the study (Weir, 1966).

Chronic studies in monkeys clearly demonstrate the potential of the hydrolysis product, methanol to cause neurological and

myocardial effects, but these studies were conducted at a much longer daily exposure time than ususal.

Therefore it is conceivable, that the observed effects were more severe than in comparable studies

with shorter daily exposure times because the biologically available dose of methanol was much higher.

Inhalation of TMBX is also unlikely based upon its anticipated use patterns.

Furthermore, the different susceptibilities of primates and rodents have to be considered.

Although there is a clear potential of methanol, and consequently TMBX, to cause adverse health effects especially in primates,

the experimental studies in non-primates do not provide clear evidence for the necessity for classification.

Methanol, and by extension TMBX, is classified as acute toxic by oral, dermal and inhalative exposure, and as capable

of inducing serious irreversible effects upon single exposure by the oral, dermal and inhalation route thus making classification for repeat dose toxicity redundant.