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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

Referenceopen allclose all

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
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
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
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
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
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
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
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.
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.
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 106cells ± 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).
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)

Additional information

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.