Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria

A bacterial reverse mutation test (Ames test) was conducted on the test material to assess its potential to cause gene mutation. The study was conducted according to official Japanese Ministry of Labour test guidelines, and in compliance with GLP.

The test material at concentrations 0 (solvent control), 313, 625, 1250, 2500, and 5000 µg/plate was exposed to four strains of Salmonella Typhimurium (TA98, TA100, TA1535, and TA1537) and one strain of Escherichia Coli (WP2 uvr A), both in the presence and in the absence of metabolic activation (achieved by the addition of S9 mix).

The test material did not induce more than twice increase in the revertant colony count compared with the count of the negative control in any of the tester strains, either with or without metabolic activation.

Therefore, it was not considered that the test material is mutagenic.

Chromosomal aberration

A chromosomal aberration test was conducted on the test material to assess its potential to cause chromosome aberrations in mammalian cells. The study was conducted according to OECD test guidelines and in compliance with GLP.

The study was conducted in Chinese hamster lung fibroblasts; metabolic activation (S9-mix) was used in short-term tests, only. A cell-growth inhibition test was conducted at 13.3, 26.6, 53.1, 106, 213, 425, 850, and 1700 µg/mL. The subsequent chromosome aberration test was conducted at 425, 850, and 1700 µg/mL.

Neither incidence of structural aberrations nor the number of polyploid cells in the cultures treated with the test material were significantly greater than that seen in the negative controls.

It was concluded that the test material induced neither structural chromosome aberration nor numerical chromosome aberration under the conditions of this study.

Gene mutation in mammalian cells

An in-vivo cell gene mutation assay was performed in mouse lymphoma L5178Y cells to assess the cytogenetic activity of the test material. The study was conducted according to several international test guidelines including OECD and EC guidelines, and in compliance with GLP.

The mutation test was conducted at concentrations 50.07, 100.13, 200.26, 400.53, 801.05, and 1602.1 µg/mL, with and without metabolic activation (S9-mix).

The treated cells did not show an increased mutant frequency when compared to the negative controls.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described. 

Short description of key information:

Bacterial reverse mutation (Ames test) - no evidence of mutagenic potential.

Chromosome aberration assay in Chinese Hamster Lung cells (in-vitro) - no evidence of mutagenic potential.

Gene mutation assay in Mouse Lymphoma cells (in-vitro) - no evidence of mutagenic activity.

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

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:
1992-01-09 to 1992-02-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to official test guidelines (Japanese Ministry of Labour), and in compliance with GLP.
Qualifier:
according to
Guideline:
other: Guidelines of the Ministry of Labour (Notification No. 77, 1988)
Deviations:
no
GLP compliance:
yes
Remarks:
The study report cites GLP according to Ministry of Labour guidelines, and includes a statement of Quality Assurance.
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
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:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (prepared in-house from SD rat livers after treatment at PB and BF ip)
Test concentrations with justification for top dose:
0 (Solvent control), 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Material is soluble in DMSO, and soluble at 6.8% in water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-AA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 Minutes
- Exposure duration: 48 Hours

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Not reported
Evaluation criteria:
The increase in the number of revertant colonies reletive to the negative control was determined for each concentration level. A two-fold increase was considered to show positive evidence of mutagenicity.
Statistics:
None
Key result
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 applicable
Positive controls validity:
valid
Key result
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 applicable
Positive controls validity:
valid
Additional information on results:
The test material did not induce more than twice increase in the revertant colony count compared with the count of the negative control in any of the tester strains, either with or without metabolic activation.
Remarks on result:
other: all strains/cell types tested

Test Results: main Study Test Material Name: Solfit-AC 
 With or Without S9-Mix   Test substance concentration (µg/plate)   Number of revertants (mean number of colonies perplate) 
 Base-pair substitution type   Frameshift type  
 TA100   TA1535   WP2uvrA-   TA98   TA1537 
 -   0 (Solvent control)  140 146 (143)  26 34 (30)  34 41 (38)  20 30 (25)  4 8 (6)
 313  135 132 (134)  24 25 (25)  38 38 (38)  29 22 (26)  10 3 (7)
 625  122 116 (119)  19 21 (20)  45 32 (39)  37 18 (28)  10 2 (6)
 1250  132 144 (138)  18 32 (25)  49 32 (41)  21 34 (28)  10 7 (9)
 2500  141 145 (143)  21 24 (23)  37 29 (33)  30 33 (32)  9 9 (9)
 5000  154 143 (149)  28 29 (29)  36 25 (31)  33 24 (29)  4 8 (6)
 +   0 (Solvent control)  145 149 (147)  30 32 (31)  33 38 (36)  34 42 (38)  12 11 (12)
 313   153 160 (157)  20 30 (25)  38 58 (48)  43 34 (39)  9 7 (8)
 625   153 113 (133)  27 23 (25)  46 37 (42)  36 44 (40)  7 9 (8)
 1250   143 157 (150)  25 20 (23)  50 46 (48)  42 41 (42)  14 10 (12)
 2500   168 164 (166)  20 22 (21)  41 35 (38)  45 54 (50)  10 12 (11)
 5000   177 165 (171)  19 21 (20)  49 40 (45)  58 59 (59)  6 10 (8)
 Positive controls   S-9 Mix -   Name   AF-2   NaN3   ENNG   AF-2   9-AA 
 Concentration (µg/plate)   0.01   0.5   2   0.1   80 
 No. colonies per plate   403 390 (397)   221 240 (231)   466 400 (433)   367 310 (339)   74 70 (72) 
 S-9 Mix +   Name   BP   2-AA   2-AA   BP   BP 
 Concentration (µg/plate)   5   2   10   5   5 
 No. colonies per plate   470 600 (535)   177 146 (162)   425 462 (444)   244 259 (252)   106 116 (111) 
Conclusions:
Interpretation of results (migrated information):
negative With and without metabolic activation

The test material did not induce more than twice increase in the revertant colony count compared with the count of the negative control in any of the tester strains, either with or without metabolic activation.
Therefore, it was not considered that the test material is mutagenic.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-08-27 to 2007-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung fibroblast (CHL/IU)
Details on mammalian cell type (if applicable):
The passage number of the cells at the time of use was 9 for the cell-growth inhibition test, 13 for the short-term treatment in the chromosome aberration test, 17 for the continuous treatment in the chromosome aberration test and 21 for the continuous treatment in the chromosome aberration test (retest).

Culturing Conditions
The cells were cultured in a 5% CO2 incubator at 37°C at a high humidity. Subcultivation was carried out every 1 to 4 days.

Characteristics Test of Cells
Cell characteristics test revealed that the modal number of chromosomes was 25 and the doubling time was 18.7 hours (at the 3rd subcultivation) and there was no problem in cell morphology. The cells were verified to be mycoplasma negative.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (S9 purchased from Oriental Yeast Co., Ltd.)
Test concentrations with justification for top dose:
Cell-Growth Inhibition test: 1700, 850, 425, 213, 106, 53.1, 26.6, and 13.3 µg/mL.
Chromosome aberration test: 1700, 850, and 425 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The information supplied indicated that the test material was soluble in DMSO (at least 500 g/L), and DMSO is widely used in chromosome aberration tests.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used for test system with metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Used for test systems without metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 Hours (Short-term treatment), 24 or 48 hours (Continuous treatment)
- Expression time (cells in growth medium): 18 Hours (Short-term treatment, only)

STAIN (for cytogenetic assays): 2% Giemsa solution

NUMBER OF REPLICATIONS: 4 (2 plates in each test had colcemid added 2 hours before the end of the incubation period in order to prepare slides for chromosome examination).

NUMBER OF CELLS EVALUATED: 200 well-spread metaphases.

DETERMINATION OF CYTOTOXICITY
- Method: A cell-growth inhibition test was performed prior to the main chromosomal aberration test - cell density was counted after incubation and the cell growth relative to the negative control was assessed.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
The incidence of cells with structural and numerical (cells with polyploidy and endoreduplication) aberrations was used as a measure of clastogenicity. The test was considered negative if there were fewer than 5% of cell with aberrations, equivocal if there were between 5 and 10% cells with aberrations, and positive if the incidence of cells with aberrations was 10% or greater.
Clastogenicity of the test article was judged positive if the incidence of aberrant cells showed a dose-related increase or the results were reproduced.
Statistics:
Statistical analysis was not applied for the judgment.
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung fibroblast (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No evidence of test material precipitation was seen.
Remarks on result:
other: all strains/cell types tested

In the chromosome aberration test of the test material, the incidence of cells with structural chromosome aberrations and the incidence of polyploid cells were less than 5% in both short-term and continuous treatments, and thus the test article was judged negative. In each negative control group, the incidence of cells with structural chromosome aberration and the incidence of polyploid cells were less than 5%, which was within the negative range in the judging criteria. In contrast, chromosome aberrations were remarkably induced in each positive control group. Since the incidence of cells with chromosomal aberrations was comparable between the two plates at the same dose level in each group, the study was judged to be conducted appropriately.

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that 3-methoxy-3-methylbutyl acetate induced neither structural chromosome aberration nor numerical chromosome aberration under the conditions of this study.
Executive summary:

A chromosome aberration study was conducted using cultured Chinese hamster lung fibroblast (CHL/IU) cells to examine whether 3-methoxy-3-methylbutyl acetate has the potential to induce chromosomal aberrations.

In the cell-growth inhibition test, no cell-growth inhibition was observed even at the highest dose level, 1700 µg/mL (equivalent to 10 mM) in any treatments. Hence, the highest dose level of 3-methoxy-3-methylbutyl acetate in the chromosome aberration test was set at 1700 µg/mL for all treatments.

In the chromosome aberration test, the incidence of cells with chromosome aberrations excluding gaps (TA value), an index of structural chromosome aberrations, was less than 5% at all treatments with the test article. Therefore, inducibility of structural chromosome aberration by 3-methoxy-3-methylbutyl acetate was judged to be negative. Since the incidence of polyploid cells was less than 5% in all the test article treatment groups, inducibility of numerical chromosome aberration by 3-methoxy-3-methylbutyl acetate was judged to be negative. In each negative control group, the incidence of cells with structural chromosome aberration and the incidence of polyploid cells were less than 5%, which was within the negative range. In contrast, structural chromosome aberrations were remarkably induced in each positive control group.

Based on the results described above, it was concluded that 3-methoxy-3-methylbutyl acetate induced neither structural chromosome aberration nor numerical chromosome aberration under the conditions of this study.

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:
2010-01-14 to 2010-02-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with several international test guidelines, including OECD guidelines, and complied with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996 and 1998) Guidelines S2A and S2B.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The study report includes a certificate of GLP compliance issued by the MHRA.
Type of assay:
mammalian cell gene mutation assay
Target gene:
Detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: R10p media
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 3.13, 6.26, 12.52, 25.03, 50.07, 100.13, 200.26, 400.53, 801.05 and 1602.1 µg/mL.
Mutation tests: 50.07, 100.13, 200.26, 400.53, 801.05 and 1602.1 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was found to be soluble at 160.21 mg/mL in DMSO. When this solution was dosed at 1% (i.e. test material at 1602.1 µg/mL in medium) into medium, no test material precipitate was observed. Solutions prepared using DMSO at 1% did not cause excessive changes to the osmolarlity or the pH of the medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Used in the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Used in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 Hours
- Selection time (if incubation with a selection agent): 7 days for viability plates, approximately 10 to 14 days for mutant plates.

SELECTION AGENT (mutation assays): R20p media

NUMBER OF REPLICATIONS: Duplicate cultures for each test substance concentration and positive control; quadruplicate cultures for vehicle controls.

NUMBER OF CELLS EVALUATED: 2 x 10^3 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF (mutant frequency per 10^6 survivors), and percent small colonies in positive control cultures.

Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6 (Moore et al., 2006).

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of test material was observed by eye.


Preliminary toxicity test
No precipitate (observed by eye at the end of treatment) was observed at any concentration of MMB-AC tested in either the absence or presence of S9 mix, following a 3 hour exposure.
Exposure to MMB-AC at concentrations from 3.13 to 1602.1 µg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 157 to 99% and from 112 to 89% respectively.
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentration of MMB-AC tested. Exposure to MMB-AC at concentrations from 3.13 to 1602.1 µg/mL resulted in RSG values from 106 to 50%.
Concentrations used in the main test were based upon these data.

Main mutation test 1 - 3 hour treatment in the absence of S9 mix
Cultures were exposed to MMB-AC at concentrations from 50.07 to 1602.1 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to MMB-AC at concentrations from 50.07 to 1602.1 µg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 133 to 96% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test 1 - 3 hour treatment in the presence of S9 mix
Cultures were exposed to MMB-AC at concentrations from 50.07 to 1602.1 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to MMB-AC at concentrations from 50.07 to 1602.1 µg/mL were assessed for determination of mutation frequency. RTG values from 153 to 122% were obtained relative to the vehicle control.
There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test 2 - 24 hour treatment in the absence of S9 mix
Cultures were exposed to MMB-AC at concentrations from 50.07 to 1602.1 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to MMB-AC at concentrations from 50.07 to 1602.1 µg/mL were assessed for determination of mutation frequency. RTG values from 124 to 41% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

MMB-AC was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

MMB-AC was found to be soluble at 160.21 mg/mL in DMSO. A final concentration of 1602.1 µg/mL, dosed at 1%v/v, (10 mM) was used as the maximum concentration in the preliminary toxicity test.

Following a 3 hour exposure to MMB-AC at concentrations from 3.13 to 1602.1 µg/mL, relative suspension growth (RSG) was reduced from 157 to 99% and from 112 to 89% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 106 to 50%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective to assess exposure up to 10mM.

Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix was 1602.1 µg/mL. In the absence and presence of S9 mix RTG was 96 and 122% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 1602.1 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 1602.1 µg/mL, where RTG was reduced to 41%.

In all tests the concurrent vehicle and positive control were within acceptable ranges. It was concluded that MMB-AC did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Results are available for a bacterial reverse mutation assay, an in-vitro chromosome aberration test in mammalian cells, and an in-vitro cytogenicity study in mammalian cells. As all of these studies were negative for mutagenic potential, there is therefore no evidence to suggest that classification as dangerous based on mutagenic properties is required.