Triboron trimethyl hexaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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 genotoxic potential was conducted taking account of the hydrolysis breakdown products of TMBX.

In vitro gene mutation studies in bacteria (Stewart, 1991) in vitro gene mutation studies in mammalian cells (Rudd, 1991) and in vitro cytogenicity studies (NTP, 1987) concluded that boric acid is not genotoxic under the conditions of the studies.

Methanol has been examined in numerous tests including bacterial, mammalian and fungal test systems. Most studies failed to demonstrate mutagenic activity, with four exceptions: 1) in Salmonella tester strain TA 102, a questionable positive response was observed (DeFlora et al., 1984); 2) in a DNA damage and repair assay with E. coli WP strains (repair proficient and deficient types), the result was considered ambiguous (DeFlora et al. 1984b); 3) in a mouse-lymphoma assay, a significant increase in the mutation rate was reported at 7.9 mg/mL methanol (McGregor et al., 1985); and 4) in a test of mitotic chromosomal segregation in Aspergillus nidulans, a positive result was observed (Crebelli et al., 1989). All other studies produced consistently negative results (Shimizu et al., 1985; DeFlora, 1981; NEDO, 1987; Lasne et al., 1984).

Overall from the weight of evidence presented, the in vitro genotoxic potential of TMBX is negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Principles of method if other than guideline:

Determination of chromosomal malsegregation in ascomycetes during mitosis induced by test substance application.

GLP compliance:

not specified

Type of assay:

other: Mitotic recombination in ascomycetes

Target gene:

not applicable

Species / strain / cell type:

other: Aspergillus nidulans diploid strain P1

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

5.2, 5.6, 6.0, 7.0 % (v/v)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Species / strain:

other: Aspergillus nidulans diploid strain P1

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

7.0 % (lowest concentration to arrest conidial germination)

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

There was a concentration-related increase in non-disjunctions (chromosomal malsegregation) with a maximum of about 3 %. No aneuploidies were noted at the lowest test concentration. The result was statistically significant at two concentrations and a dose-response relationship was evident (IPCS/WHO 1997).

No other forms of malsegregation were statistically significant (1/265 cross-over seen at 6.0 %). The survival was reduced in dose-related manner.

Survival and amount of aneuploidy in Aspergillus nidulans after methanol-treatment:

Concentration [%]	Survival [%]	Scored colonies	Aneuploids (yellow segregants)
			counts	[%]
5.2	26	268	0	0
5.6	19	407	6	1.47*
6.0	10	265	8	3.02**
7.0	5	176	0	0
Spontaneous control
0	100	2673	7	0.26

* P<0.01; ** P<0.001

Note: Similar results were obtained with ethanol and other primary aliphatic alcohols. The aneuploidy rate of ethanol reached a maximum of about 10 %.

Conclusions:

Interpretation of results (migrated information):
positive

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comprehensive test programme using test procedures in accordance with accepted standard methods, but limited documentation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only strains TA97 and TA102 tested

Principles of method if other than guideline:

According to Maron and Ames, 1983.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with

Metabolic activation system:

S9 mix from Aroclor-induced SD rat livers

Test concentrations with justification for top dose:

<= 7.5 mg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

Positive response: dose response relationship, revertant ratio >=2.

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

slightly positive trend

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

In TA102, a slightly positive trend was indicated [(±) ambiguous], reproducible in 5 parallel independent experiments, but never exceeding the revertant ratio of 2: Spontaneous mutation rate 200 - 300/plate, while in the presence of high methanol doses, an increase in the mutation frequency above background of 140 revertants was found.

The methanol-related increase in the mutation frequency in TA102 did not fulfil the accepted criteria for mutagenic activity. Along with the high methanol doses required to induce such a weak effect and the negative results observed in all other Ames tests, the overall evidence clearly demonstrates that methanol is not mutagenic in bacterial reverse-mutation systems.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

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, well documented with methodological restrictions.

Principles of method if other than guideline:

The endpoint of the test was cytotoxicity due to chromosome damage. The ratio of the minimal inhibitory concentration (MIC) of repair proficient to the MIC of repair-deficient E. coli strains was taken as an indicator for genotoxicity.

GLP compliance:

not specified

Type of assay:

other: DNA damage and repair assay in bacteria

Target gene:

not applicable

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

other: wild-type, repair proficient

Species / strain / cell type:

E. coli, other: WP67

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

other: repair deficient: uvrA-, polA-

Species / strain / cell type:

E. coli, other: CM871

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

other: repair deficient: uvrA-, recA-, lexA-

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

No details: the initial concentration was governed by the solubility or by the toxicity of the TS as inferred from preliminary testing. Starting from this, eight 2-fold dilution steps followed in general.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium and 2-h preincubation:
Trials were conducted in miniature form (liquid micromethod procedure: 350 μl) using microwell plates that contained nutrient broth, and as 2-h preincubation assay ("treat-and-plate method" on nutrient broth agar).

DURATION
- Preincubation period: 2 h
- Exposure duration: 16 h (liquid micromethod procedure)

DETERMINATION OF CYTOTOXICITY
- Method: other: determination of the Minimal Inhibitory Concentration (MIC): growth retardation

Evaluation criteria:

For each tester strain, the Minimal Inhibitory Concentration (MIC) was determined. The endpoint was cytotoxicity due to chromosome damage. The ratio of the MIC of the repair proficient to the MIC of the repair deficient strains was taken as indicator for genotoxicity. A ratio of greater than 2 was accepted as significant only if reproducible in 5 parallel independent experiments.

Species / strain:

E. coli, other: WP2, WP67, CM871

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

40 mg/well

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli, other: WP67, CM871

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

20 mg/well

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

40 mg/well

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli, other: not specified

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli, other: not specified

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: other: microwell method

Remarks:

Migrated from field 'Test system'.

The microwell method resulted in a negative result in the presence of S9 and in a positive result in the absence of S9 at 20 mg/well (Flora et al., 1990, 1984). But the preincubation procedure was negative without S9, but ambiguous with S9 (Flora et al. 1984).

Note: The MIC concentrations were very high (40 and 20 mg/well = about 120 g/L and 60 g/l).

Given the high concentrations and the conflicting findings, it is concluded that observations made at the margin of significance (ratio = 2) are of low reliability and biological relevance. The weak relative increase of toxicity in repair-deficient strains may be an increase in unspecific cytotoxicity rather than solely "genotoxicity". (Note: A similar result was obtained with ethanol at somewhat lower concentrations).

Conclusions:

Interpretation of results (migrated information):
ambiguous

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comprehensive test programme using test procedures in accordance with accepted standard methods, but limited documentation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Salmonella TA102 and/or E.coli WP2 missing

Principles of method if other than guideline:

according to Ames et al., 1975.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor-induced SD rat livers

Test concentrations with justification for top dose:

<= 2.5 mg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

non-toxic within the range of testing

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

non-toxic within the range of testing

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Interpretation of results (migrated information):
negative

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with accepted standard methods, sufficiently documented, acceptable for assessment.

Principles of method if other than guideline:

In vitro micronucleus test with V79 cells comparing alcohols, acetone and various alkylating agents.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

50 µL/mL (approx. 40 mg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium (Eagle's MEM + 10 % FCS), acetone for positive controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

0, 0.02, 0.04, 0.08 µg/mL; solvent acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

0, 0.4, 2, 10, 50, 100 µg/mL; solvent acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0, 25, 50, 100 µg/mL; solvent acetone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 48 h

STAIN (for cytogenetic assays): 4% Giemsa

NUMBER OF REPLICATIONS: not reported

NUMBER OF CELLS EVALUATED: 7000 interphase cells at each concentration used

OTHER
Cells were plated at a density of 13000 cells/cm², incubated for 15-18 h, then treated with the test substance. After treatment, the cells were incubated for 48 h, then collected, subjected to hypotonic treatment with KCl, fixed with acetic acid-methanol, and then stained with 4% Giemsa.

Evaluation criteria:

Criteria used to score MN: (1) staining intensity equal to that of the nucleus, (2) diameter less than one-fifth that of the nucleus, (3) location in cytoplasm, (4) no contact with nucleus to distinguish from nuclear blebs.

Statistics:

The dose response was estimated by linearr regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

No MN increases induced by any alcohol or acetone, whereas the alkylating agents produced significant MN frequencies above medium controls.

	Max. number of MN/1000 cells [mean±S.E.]
	Control (solvent dose)	Chemical (dose)
Methanol	4.00±0.71 (50 µL/mL)	3.50±1.19 (50 µL/mL)
MNNG	3.2±0.40 (5 µL/mL)	8.2±0.83 (0.08 µg/mL)
MMS	3.9±0.59 (5 µL/mL)	16.5±0.50 (100 µg/mL)
EMS	2.2±0.40 (5 µL/mL)	7.0±0.69 (100 µg/mL)

Solvent for methanol: medium

Solvent for MNNG, MMS, EMS: acetone

Conclusions:

Interpretation of results (migrated information):
negative

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only abstract, no detailed data.

Principles of method if other than guideline:

Optimisation of the amount of S9-mix to be used to obtain maximum yield of mutations. Comparative study including typical mutagens. According to Clive and Spector (1975), Mutat Res 31: 17-29.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidin-kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix

Test concentrations with justification for top dose:

7.9 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Details on test system and experimental conditions:

DURATION
- Exposure duration: at least 4 h

SELECTION AGENT (mutation assays): TFT (trifluorothymidine)

Evaluation criteria:

Significant increases in mutation frequency with the test substance

Statistics:

no data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In the presence of 10 - 15 µL/mL S9 and methanol (7.9 mg/mL), there was a significant increase in mutation frequency. No detailed results presented.

Conclusions:

Interpretation of results (migrated information):
positive

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, also largely meeting current standards, sufficient documentation, acceptable for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Exposure and expression period combined

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

15.8, 31.7, 47.4, 63.3 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (Eagle's MEM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S9

Migrated to IUCLID6: 1 and 2 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: MNNG, 0.29 and 0.59 µg/mL

Remarks:

without S9

Details on test system and experimental conditions:

DURATION
- Exposure duration: 6 days
- Expression time (cells in growth medium): combined with 6 days exposure
- Selection time (if incubation with a selection agent): after 6 days

SELECTION AGENT (mutation assays): 8-Azaguanin, 6-Thioguanin, Ouabain

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (colony formation)

Evaluation criteria:

Significant increase in V79 cells resistant to 8-Azaguanine, 6-Thioguanine or Ouabain.

Statistics:

Calculation of mean mutation frequency±S.D. per 10e6 surviving cells.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

63.3 mg/ml (approx. 70 % inhibition of colony formation)

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test substance induced no increases in mutant frequency in gene mutation to drug resistance vs. negative control, whereas the positive control DMN produced increases in dose-related manner in the presence of metabolic activation (S9 mix), and MNNG in the absence of metabolic activation.

Maximum mutation frequency of V79 cells (per 10e6 survival cells, mean±SD) for resistance towards 6-TG, 8-AG and Ouabain after methanol treatment:

	Control		Test substance (mg/mL)
Selectant	-S9	+S9	-S9	+S9
6-Thioguanine	0.70±1.79	0.94±2.18	1.55±3.08 (47.4 mg/mL)	0.84±2.21 (31.7 mg/mL)
8-Azaguanine	23.42±8.70	24.29±7.30	22.34±7.39 (31.7 mg/mL)	20.05±13.01 (31.7 mg/mL)
Ouabain	1.23±2.23	0	2.64±2.79 (47.7 mg/mL)	0.11±0.36 (47.7 mg/mL)

Conclusions:

Interpretation of results (migrated information):
negative

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-05-91 to 12-08-91

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevent results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

There is a failure to justify the maximum concentration of 2500 ug/plate

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR Part 158; FIFRA, Section 158.340

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 at 4 % and 10 %

Test concentrations with justification for top dose:

10; 50; 100; 1000; 2500 μg/plate

Vehicle / solvent:

Water

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: In water

DURATION
- Preincubation period: None

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The study was performed according to Guideline 84-2 and is comparable to OECD 471. The test substance was not mutagenic in any of the strains tested with or without metabolic activation.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevent results.

Qualifier:

equivalent or similar to guideline

Guideline:

other: Carried out to an internal NTP protocol, which generally complies with OECD guidelines.

Deviations:

not applicable

Principles of method if other than guideline:

Although this was carried out to an internal NTP protocol, it generally complies with OECD guidelines. Other literature data confirm the results. Also based on Galloway et al; 1985.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix - Aroclor 1254 induced rat (Sprague-Dawley) liver S9 fraction

Test concentrations with justification for top dose:

With S-9: 500, 1000, 1500 and 2000 μg/mL.
Without S-9: 1000, 1600, 2000 and 2500 μg.

Vehicle / solvent:

Water

Positive controls:

yes

Positive control substance:

mitomycin C

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: In water
NUMBER OF CELLS EVALUATED: Not stated

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Test substance is non genotoxic.

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

other: 40 CFR Part 158 US-EPA-FIFRA, Section 156.340

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix or other (Aroclor 1254 induced rat (Fischer 344) liver S9 fraction used at 1 %).

Test concentrations with justification for top dose:

0, 1.2, 1.7, 2.45, 3.5 and 5.0 mg/mL boric acid.

Vehicle / solvent:

No data

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Positive controls:

yes

Positive control substance:

other: Hycanthone methylsulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: ROP plus 5 % heat treated horse serum

NUMBER OF CELLS EVALUATED: Approximately 600/dose

Evaluation criteria:

Mutations at the TK locus

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentration related cytoxicity (60 % reduction over controls at 5 mg/mL)

Additional information on results:

Concentration related cytotoxicity (60 % reduction over controls at 5 mg/mL).
Increase in ouabain resistance seen (not significant).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Gene mutation assay results:

Concentration mg/mL	Number of mutant cells per 10^6cells ± SD				Comments give information on cytotoxicity or other
	Exp 1	Exp 2	Exp 1	Exp 2
	-S9	-S9	+S9	+S9
0	54 ± 10	42 ± 1	29 ± 10	36 ± 7
1.2	46 ± 28	38 ± 15	34 ± 0	36 ± 7
1.7	39 ± 17	31 ± 9	41 ± 7	49 ± 4
2.45	27 ± 3	32 ± 9	40 ± 16	36 ± 6	Minor cytotoxicity seen
3.5	31 ± 18	46 ± 1	41 ± 13	41 ± 6	Cytotoxicity seen
5	50 ± 22	41 ± 5	53 ± 2	47 ± 3	Cytotoxicity seen. Increase in + S9 in first study not reproduced.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was not mutagenic but cytotoxicity observed at 5 mg/mL (maximum dose level).

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comprehensive test programme using test procedures in accordance with accepted standard methods, sufficiently documented.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon, Trp-operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of KC500-pretreated rats

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.01 and 5.0 µg/plate, respectively

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

both 5.0 µg/plate, respectively

Positive control substance:

other: N-ethyl -N'-nitro-N-nitrosoguanidine (ENNG) without S9, 2-aminoanthracene (2AA) with S9

Remarks:

TA1535both 5.0 µg/plate, respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 and 5.0 µg/plate, respectively

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, 2-aminoanthracene (2AA) with S9

Remarks:

WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 and 5.0 µg/plate, respectively

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

80.0 and 5.0 µg/plate, respectively

Positive control substance:

other: 9-aminoacridine (9AC) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.25 and 5.0 µg/plate, respectively

Positive control substance:

other: 4-nitroquinoline-1-oxide (4NQO) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA1538

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition of revertant clones

Evaluation criteria:

Doubling of revertant numbers in comparison to control and dose-response correlation.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Maximum number of revertants:

	Control (water) [mean±SD]		Test substance (µg/plate)
Strain	-S9	+S9	-S9	+S9
TA100	149±17.1	161±16.2	175 (100)	180 (50)
TA1535	28±6.9	15±3.6	35 (50)	17 (5000)
TA98	29±6.2	39±8.6	42 (50)	39 (1000)
TA1537	16±6.4	21±8.1	22 (5000)	35 (5)
TA1538	21±5.5	28±7.0	18 (500)	31 (5)
E.coli WP2 uvrA	32±7.3	33±10.3	36 (5000)	43 (10)

The test substance was not mutagenic in the tested strains up to 5000 µg/plate.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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 genotoxic potential was conducted taking account of the hydrolysis breakdown products of TMBX.

The results of an in vivo bone marrow cytogenetic assay (chromosome aberration assay, O’Loughlin 1991) also showed boric acid to be non genotoxic. 

The available in vivo assays for methanol genotoxicity are negative for mutagenicity and clastogenicity. Some of these assays were conducted under specific stress conditions using folate-deficient mice (Am. Petrol. Inst., 1991; Fu, 1996). Specifically methanol was found to have no genotoxic potential in erthyrocytes (micronucleus), somatic epithelial lung cells(micronucleus and chromosome aberration) and reproductive tissues (pacytene spermatocytes) (Campell et al 1991)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based upon the lack of genotoxic potential for the hydrolysis products in in vivo test systems including chromosome aberration, micronucleus formation and germ cell mutation tests, the classificaiton criteria for germ cell mutation set out in 1272/2008/EC are not met.