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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Ames, Krul (2002)

Under the conditions of this study, the test material was not mutagenic in bacterial reverse mutation tests conducted in four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA with or without metabolic activation.

In vitro: CHO chromosomal aberration, Strasser (1988)

Under the conditions of the tests conducted, the test material did not elicit mutagenic effects in this chromosome aberration assay with or without metabolic activation.

In vitro: HPRT, Dollenmeier (1988)

Under the conditions of the tests conducted, the test material did not elicit mutagenic effects in this mammalian cell forward mutation system with or without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 December 2001 to 10 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997 (Only a single experiment was conducted, which was clearly negative. In accordance with TG471 the lack of a repeat experiment is fully justified in the study report.)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Mutation in Genes for Histidine or Tryptophan amino acids:
TA 98: His D3052
TA 100: His G46
TA 1535: His G46
TA 1537: His C3076
WP 2 uvrA: Trp (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:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate (induced with Aroclor)
Test concentrations with justification for top dose:
0, 62, 185, 556, 1667, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water

- Additional information on solvent/vehicle: In vitro genotoxicity assays are aqueous based test systems. In this assay test substance is added to an aqueous mix of bacterial culture, buffer/S9 mix and molten soft agar, before pouring onto a solid (aqueous-based) agar plate. The study report indicates preparation of the MBTC formulations (in water) immediately prior to addition to the test system. Precipitation was recorded at the highest concentration but the lower concentrations remained fully soluble. It is known that an equilibrium exists between strong complexes of water with MBTC and chemical species where one or several chloride ions are abstracted, therefore it is considered highly unlikely that there would be any degradation of MBTC in the aqueous media during exposure of test systems. It can therefore be concluded that most of the examined concentrations appeared to be fully soluble in the assay system, and the test system was exposed to soluble MBTC.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: N-ethyl-N-nitrosourea (WP2 uvrA without S9) and 2-aminoanthracene (TA 98, TA 100, TA 1535 and WP2uvrA with S9)
Details on test system and experimental conditions:
SYSTEM OF TESTING - 
- Deficiences/Proficiences: TA 98, TA 100, TA 1535 and TA 1537 are his-deficient strains. WP2 uvrA is a trp-deficient strain.  
- Metabolic activation system: S9 supernatant of liver homogenate from male Wistar rats treated with Aroclor 1254. One dose of 500 mg/kg bw in soya bean oil (20% w/v) was injected intraperitoneally, and the rats were sacrificed five days later. The livers were homogenised in 0.15 M KCl for 10 minutes at 9,000 g and the supernatant (S9) collected and frozen before storing it at -60 °C. 

ADMINISTRATION: 
- Dosing: 62, 185, 556, 1667, 5000 µg/plate.
- Number of replicates: 3.
- Positive and negative control groups and treatment: The negative control groups were exposed to water. The positive control substances were sodium azide (TA 100, TA 1535), 9-aminoacridine (TA 1537),  2-nitrofluorene (TA 98) and N-ethyl-N-nitrosourea (WP2 uvrA) for assays without S9.  The positive control substances were benzo(a)pyrene (TA 1537) and 2-aminoanthracene (TA 98, TA 100, TA 1535, and WP2 uvrA) for assays with S9. All plates were incubated at 37 °C for 72 hrs.
Evaluation criteria:
The study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive  controls meet the criteria for a positive response, and if no more than 5 % of the plates are lost through contamination or other unforeseen events.

A test material is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates  is increased in a concentration-related manner, or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test material is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

In case of an inconclusive first assay, a second independent assay was conducted. The first mutagenicity assay was regarded as inconclusive if a positive or equivocal response at only one concentration is observed or if a positive or equivocal responses at several concentrations without a concentration-related increase is observed.

Omission of the second assay under these conditions is acceptable as a  single assay does not or hardly results in false negative conclusions. Positive results from the bacterial reverse mutation test indicate that a test material induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test material is not mutagenic in the tested strains.

Both numerical significance and biological relevance were considered in the evaluation.
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS:
- With metabolic activation: Negative.
- Without metabolic activation: Negative.

PRECIPITATION CONCENTRATION: 5000 µg/plate.

CYTOTOXIC CONCENTRATION:
- With metabolic activation: 1667 and 5000 µg/plate for TA 100 and WP2 uvrA. 556, 1667 and 5000 µg/plate for TA 1537.
- Without metabolic activation: 185, 556, 1667 and 5000 µg/plate for TA 100. 1667 and 5000 µg/plate for WP2 uvrA.

- The test material was toxic to the E. coli strain and to TA 100 at the two higher doses (1667 and 5000 µg/plate) in both the absence and presence of S9 mix, and to TA 100 also at 185 and 556 µg/plate in the absence of S9 mix, as was evidenced by a decrease in the mean number of revertant colonies. In addition, the test material was toxic to TA 1537 at 556, 1667 and 5000 µg/plate in the presence of S9 mix.
- In both the presence and the absence of S9 mix and in all strains the test material did not cause a more than two-fold or a dose-related increase in the mean number of revertant colonies appearing in the test plates, compared to the background spontaneous reversion rate observed with the negative control.
- The mean number of his+ and trp+ revertant colonies of the negative control were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.
- It is concluded that the test material was not mutagenic under the conditions employed in this study.

STATISTICAL RESULTS: No statistical analysis was performed.

Table 1: Summary of Experiment

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

62

185

556

1667

5000

158

138

100

117

69

83

22

21

18

13

19

20

34

26

23

23

21

19

25

19

19

19

21

18

9

13

8

3

6

5

+

Solvent

62

185

556

1667

5000

159

162

131

149

117

95

14

10

16

12

11

12

32

25

26

21

15

16

38

35

31

40

36

30

17

16

15

9

9

8

Positive Controls

-

Name

SA

SA

NENN

2NF

9AA

Mean no. colonies/plate

598

365

263

760

408

+

Name

2AA

2AA

2AA

2AA

BP

Mean no. colonies/plate

978

278

758

601

235

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

SA = Sodium azide

NENN = N-ethyl-N-nitrosourea

2NF = 2-Nitrofluorene

Conclusions:
Under the conditions of this study, the test material was not mutagenic in bacterial reverse mutation tests conducted in four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA with or without metabolic activation.
Executive summary:

The test material was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, the tryptophan-requiring Escherichia coli strain WP2 uvrA, and a liver fraction of Aroclor 1254 -induced rats for metabolic activation (S9 mix). The test was performed in accordance with the standardised guidelines OECD 471 and EPA OPPTS 870.5100, under GLP conditions.

Five different concentrations of the test material were used ranging from 62 - 5000 µg/plate. Vehicle and positive controls were run simultaneously.

The test material was toxic to E. coli WP2 uvrA strain and to TA 100 at the two highest doses, 1667 and 5000 ug/plate, in both the absence and presence of S9 mix, and also, to TA 100 at 185 and 556 ug/plate in the absence of S9 mix, evidenced by a decrease in the mean number of revertant colonies. It was also toxic to TA 1537 at 556, 1667, and 5000 µg/plate in the presence of the S9 mix. The test material did not cause a more than 2 -fold or a dose-related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate in the negative control. The mean number of his+ and trp+ revertant colonies in the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Under the conditions of this study, the test material was not mutagenic in bacterial reverse mutation tests conducted in four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA with or without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 1988 to 16 August 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Purity/composition of test material not provided.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Compared to the 2016 version of TG476
The deviation noted has the potential to reduce the sensitivity of the assay. However the test material has been tested to sufficiently high, toxic concentrations with no evidence of any increase in mutant frequency. All concentrations – even those at the limit of toxicity – have mutant frequencies that are highly comparable with the vehicle control.
Deviations:
yes
Remarks:
The number of cells initially treated and subsequently cultured throughout the study was lower than currently recommended.
Principles of method if other than guideline:
Deviation: The number of cells initially treated and subsequently cultured throughout the study was lower than currently recommended. This has the potential to reduce the sensitivity of the assay. However the test material has been tested to sufficiently high, toxic concentrations with no evidence of any increase in mutant frequency. All concentrations – even those at the limit of toxicity – have mutant frequencies that are highly comparable with the vehicle control.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Originally derived from embryonic lung tissue obtained from Dr. D. Wild, Freiburg, Germany
- clone 65/3
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver microsomes
Test concentrations with justification for top dose:
60, 120, 240, 480, 720, 960, and 1200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test material was dissolved in tissue culture medium - Ham's F10 culture medium and 3 % foetal calf serum.

- Additional information on solvent/vehicle: In vitro genotoxicity assays are aqueous based test systems. The study report indicates preparation of the MBTC formulations in tissue culture media immediately prior to addition to the test system. It is known that an equilibrium exists between strong complexes of water with MBTC and chemical species where one or several chloride ions are abstracted. In this study, test substance exposure occurred for 4 hours, therefore irreversible hydrolysis within the test system is highly unlikely to have occurred regardless of the vehicle used for testing. Precipitation was recorded at the highest concentration but the lower concentrations remained fully soluble, therefore the test systems was exposed to biologically available, soluble MBTC.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
two vehicle controls per experiment
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
- V79 Chinese hamster cells, originally derived from embryonic lung tissue, were obtained from Dr. D. Wild, Freiberg, FRG. The cells were cultured in 25 mL of growth medium (Ham's F10 plus 10 % pre-tested foetal calf serum, plus 100 U penicillin and 100 µg streptomycin per mL). Two tests were conducted, a cytotoxicity test and a mutagenicity test (with and without metabolic activation).

- The test material was filtered (0.22 µm) and added directly to the tissue culture/ treatment medium (97 % Ham's F10 plus 3 % foetal calf serum). The nominal stock test material concentrations were 11.11 mg/mL (cytotoxicity test) and 1.333 mg/mL (mutagenicity tests). Additional test concentrations were prepared by diluting the stock solutions with treatment medium.

- Aroclor 1254-induced male RAI rat liver homogenate (S9), mixed with a solution of co-factors, was used as the metabolic activation system at a concentration of 10 % of the original, and the final concentration was 2 % in the treatment medium.

- In the cytotoxicity test, V79 cells were exposed (in duplicate) to 16 concentrations of the test material ranging from 305 ng/mL to 9.8 mg/mL and two medium controls. V79 cells were seeded with growth medium. Eighteen hours later, cells were exposed to the test material for 5 hours in the presence of S9 or for 21 hours in the absence of S9. Treatment was terminated by washing the cultures with buffer, followed by re-incubation in fresh growth medium for six days. The cultures were then fixed and stained with Giemsa and the surviving colonies determined with an electronic colony counter. The concentrations to be selected as the highest for the mutagenicity assays were those causing 90 % reduction of viable cells in comparison with the medium controls.

- In the mutagenicity tests, 2.5 x 10e6 cells were plated in 25 mL growth medium for 42 hours prior to treatment. Growth medium was then replaced for 5 hours with 22.5 mL treatment medium + 2.5 mL S9 activation mixture, or for 21 hours with 25 mL treatment medium only. In the test without activation, 7 concentrations of test material were tested, plus 2 negative (medium) controls and 1 positive control [300 nL/mL ethyl methanesulphonate (EMS)]. In the test with activation, 7 concentrations were tested, plus 2 negative (medium) controls and 1 positive control [1 uL/mL N-nitrosodimethylamine (DMN)]. Treatment was terminated by washing the cells with phosphate-buffered saline. Cells were then suspended by trypsinisation, pelleted, re-suspended in fresh growth medium, counted with a haemocytometer, diluted with fresh growth medium, and re-plated into flasks at 10e6 cells. Six cultures, each containing 100 cells, were set up in parallel for each test material concentration. All cultures were incubated in growth medium for five days. The medium was replaced with fresh medium on day 2. Following the incubation period, the cultures were trypsinised and 18 x 100,000 cells from each culture were plated into 18 dishes for mutant selection.

- Cultures for the mutant selection procedure were supplemented with 8 µg/mL 6-thioguanine (6-TG). After 7-8 days, the cultures were fixed (methanol), stained (Giemsa), and counted with an electronic colony counter. Mutant clones were counted with the naked eye.
Evaluation criteria:
-For every concentration a mutant factor, which is defined as the ratio of the mutant frequencies of the treated and the negative control cultures, was calculated. The sensitivity of the test was restricted to a lower limit of mutant frequency of 4 x 10E-6. Test material is considered to be mutagenic if the mutant frequency of the treated culture exceeds that of the negative control by a mutant factor of 2.5 and there is a dose-dependent increase in the mutant frequency, or the mutancy frequency in a treated culture exceeds that of the negative control by a mutant factor of 3.0 at any concentration tested and the absolute number of clones in the treated and untreated cultures differ by more than 20 clones per 10E-6 cells plated.

The test substance was considered mutagenic if either:
- The mutant frequency of the treated culture exceeded that of the negative controls by a mutant factor of 2.5 and there was a dose-dependent increase of the mutant frequency with the test material.
or:
- The mutant frequency in a treated culture exceeded that of the negative control by a mutant factor of 3.0 at any concentration tested and reported, and the absolute number of clones in the treated and untreated cultures differed by more than 20 clones per million cells plated.
Statistics:
All mutant frequency values were determined to be below 24 x 10E-6 and there was no suspicion for a concentration-dependent change in mutant frequency. Therefore, statistical evaluation of the data was not indicated and not performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
90 % reduction in viability of cells observed between 625 and 1250 µg/mL both without and with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- The cytotoxicity tests with the test material (both with and without activation) demonstrated at least a 90 % reduction in cell viability at a concentration between 625 and 1250 µg/mL.
- Mutagenicity test without activation: After screening with 6-thioguanine, mutant-frequency values in the negative controls were <4 x 10E-6. The mean used for calculation was 4 x 10E-6. The 1200 µg/mL test material concentration was toxic. The calculated mutant-frequency values of the six lower concentrations (960, 720, 480, 240, 120, and 60 µg/mL) were all <4 x 10E-6. The ratio of the mutant frequency of test material to mutant frequency (controls) was 1.0. Results of this experiment were confirmed with an independent, confirmatory test (mutant factors: 1.0-1.1). The positive control (EMS) resulted in mutant factors of 435.6 (original test) and 331.7 (confirmatory test).
- Mutagenicity test with activation: After screening with 6-thioguanine, mutant-frequency values in the negative controls were <4 x 10E-6. The mean used for calculation was 4 x 10E-6. The two highest test concentrations (1200 and 960 µg/mL) were toxic. Mutant-frequency values (mutant factor) of the remaining test concentrations were: 720 µg/mL, <4 x 10E-6 (1.0); 480 µg/mL, 5.8 x 10E-6 (1.4); 240 µg/mL, 6.3 x 10E-6 (1.6); 120 µg/mL, <4 x 10E-6 (1.0); and 60 µg/mL, <4 x 10E-6 (1.0). Results of this experiment were confirmed with an independent, confirmatory test (mutant factors: 1.0-1.1). The positive control (DMN) resulted in mutant factors of 47.9 (original test) and 68.5 (confirmatory test).
- Under the conditions of the tests conducted, the test material did not elicit mutagenic effects in this forward mutation system.
Conclusions:
Under the conditions of the tests conducted, the test material did not elicit mutagenic effects in this mammalian cell forward mutation system with or without metabolic activation.
Executive summary:

A forward mutation assay was performed with the test material in V79 Chinese hamster cells in vitro with and without metabolic activation using S9 rat liver microsome fraction. The test was performed in accordance with the standardised guideline OECD 476, under GLP conditions.

Seven different concentrations of the test material (60 - 1200 µg/mL), two negative (culture medium) controls, and one positive control (ethylmethanesulphonate for without metabolic activation and N-nitrosodimethylamine for with metabolic activation) were used for each experiment. The results of each experiment were confirmed in a second and independent confirmatory experiment. No significant deviation in mutant frequencies (or mutant factors) was revealed in any experiment when cell cultures were challenged with 6 -thioguanine (6 -TG). The positive controls effectively caused mutations in the HGPRT gene locus and had high mutant factors.

Under the conditions of the tests conducted, the test material did not elicit mutagenic effects in this mammalian cell forward mutation system with or without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 March 1988 to 13 June 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Purity/composition of test material not provided.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Compared to 2016 version
Deviations not may reduce the sensitivity of the test. However, these are considered to not affect overall genotoxicity evaluation due to the availability of a complaint TG474 test, which confims that this end point has been adequately addressed
Deviations:
yes
Remarks:
100 not 300 metaphases scored. No continuous treatment in the absence of S9. Compliant short treatments (3 hours, with sampling 21 hours after treatment) with and without metabolic activation were conducted.
Principles of method if other than guideline:
This chromosome aberration test is deficient (vs 2016 TG473) as follows: only 100 (not 300) metaphases scored and no continuous treatment in the absence of S9 was conducted. However, given there is a compliant TG474 study, these deficiencies do not preclude a full assessment of genotoxicity.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cell line: ATCC (American Type Culture Collection), CCL 61 (ovary, Chinese hamster)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal fraction S9 prepared from Aroclor 1254-induced livers of male RAI rats
Test concentrations with justification for top dose:
0.98-500 µg/mL toxicity range test
62.5, 125.0, 250.0, and 500 µg/mL with and without activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water

- Additional information on solvent/vehicle: In vitro genotoxicity assays are aqueous based test systems. It is known that an equilibrium exists between strong complexes of water with MBTC and chemical species where one or several chloride ions are abstracted. In this assay test substance exposure occurred for 3 hours, therefore irreversible hydrolysis within the test system is highly unlikely to have occurred regardless of the vehicle used for testing. Precipitation was recorded at the highest concentration but the lower concentrations remained fully soluble, therefore the test system was exposed to biologically available, soluble MBTC.

Even if an organic vehicle (eg DMSO) had been used, degradation is likely to have occurred on contact with the test system. The study report indicates preparation of the MBTC formulations in water immediately prior to addition to the test system. Therefore, degradation prior to exposure to the test system was minimised as much as feasibly possible. In addition, it is considered highly unlikely that there would be any degradation of the actual MBT moiety itself in the aqueous media during exposure of test systems.

In this chromosome aberration test water was used as the vehicle and precipitation was recorded only at the highest final concentration tested (500 µg/mL – also the maximum recommended concentration). Therefore, in this study several of the examined concentrations appeared to be fully soluble in the assay system, suggesting insoluble MBT compounds were not formed, and the test system was exposed to soluble forms of the MBT moiety.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
- The cell line CCL 61 (Chinese hamster ovary [CHO] cells) was maintained in F12 Nutrient Mixture, supplemented with 10 % foetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL). Two tests were conducted, a preliminary cytotoxicity test and a mutagenicity test (with and without metabolic activation). The test material was dissolved in distilled water and filtered (0.2 µm). The stock solution concentration was 50.0 mg/mL. Additional test concentrations were prepared by diluting the stock solutions with distilled water. Aroclor 1254-induced male SAI rat liver homogenate (S9), mixed with a solution of co-factors, was used as the metabolic activation system.

- In the preliminary cytotoxicity tests, CHO cells were exposed to 10 concentrations of the test material in the presence and absence of S9. Concentrations of the test material ranged from 0.98 to 500.0 ug/mL. After 3 hours, the test material was removed and the cells were washed and incubated in culture medium for 15 hours. The percentage of mitotic suppression (compared with controls) was determined by counting at least 2000 cells/concentration tested. The highest concentration applicable or the concentration calculated to produce about a 50 % suppression of mitotic activity in comparison with control is used as the highest in the mutagenicity experiments. The highest concentration selected for the mutagenicity tests was 500 µg/mL. Three further concentrations were then selected, corresponding to factors of 0.5, 0.25, and 0.125. In the mutagenicity tests, cells were treated with the selected test material concentrations (62.5, 125.0, 250.0, and 500 µg/mL) both in the presence and absence of S9 (1 mL of activation mixture containing 0.15 mL S9 + 0.2 mL cofactors NADP and isocitric acid + 0.65 mL medium). Flasks seeded with CHO cells were incubated for 22 hours prior to exposure to the test material. Cells were then treated with the test material for 3 hours. In the test without activation, 4 concentrations were tested (62.5, 125, 250, and 500 µg/mL), plus a negative (distilled water vehicle) control and a positive control [1.0 µg/mL Mitomycin C]. Similarly, in the test with activation, 4 concentrations were also tested (62.5, 125, 250, and 500 µg/mL), plus a negative (distilled water vehicle) control and a positive control [40 µg/mL cyclophosphamide]. Treatment was terminated by washing the cells with 10 mL Hanks BSS. Fresh growth medium was then added and cells were allowed to grow for 4 and 21 hours. Two hours before harvest, cultures were treated with 0.4 µg/mL Colcemide. At the test termination, cells were treated with a hypotonic solution (0.075 M KCl), fixed with 3:1 methanol/acetic acid, and drop-prepared with the "air-drying technique."
Evaluation criteria:
- One hundred metaphase plates for the three highest test material concentrations (with and without activation), the negative (vehicle) controls, and the positive controls were examined for chromosomal aberrations.
- Slides were scored for specific aberrations, i.e., breaks, exchanges, deletions, fragments and minutes; unspecific aberrations, i.e., gaps, premature chromosome condensation, and chromosome decay; and numerical aberrations, i.e., metaphases with >21 chromosomes.
Statistics:
No details provided in the report.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
(CCL 61)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- At the 4-hour harvest time without activation, the percent of metaphases with specific aberrations was 9 % in the vehicle control, 5 % at 125 µg/mL, 6 % at 250 µg/mL, and 6 % at 500 µg/mL of the test material, and 5 % in the positive control.
- At the 4-hour harvest time with activation, the percent of metaphases with specific aberrations was 4 % in the vehicle control, and 2 % in each of the three highest test material concentrations (125, 250, and 500 µg/mL), and 3 % in the positive control.
- At the 21-hour harvest time without activation, the percent of metaphases with specific aberrations was 5 % in the negative control, 6 % at 125 µg/mL, 8 % at 250 µg/mL, and 6 % at 500 µg/mL of the test material, and 88 % in the positive control.
- At the 21-hour harvest time with activation, the percent of metaphases with specific aberrations was 5 % in the negative control, 6 % at 125 µg/mL, 3 % at 250 µg/mL, and 3 % at 500 µg/mL of the test material, and 90 % in the positive control.
- Under the conditions of the tests conducted, the test material did not exhibit mutagenic properties on Chinese hamster ovary cells in vitro.
Conclusions:
Under the conditions of the tests conducted, the test material did not provoke any effect interpretable as being suggestive of a mutagenic property on Chinese hamster ovary cells in vitro.
Executive summary:

Chromosomal aberration studies were conducted with the test material in Chinese hamster ovary cells (CCL61) in vitro.

First, a cytotoxicity test was conducted using 10 concentrations ranging between 0.98 and 500 µg/mL. Even at the highest dose tested, no suppression of mitotic activity was detected in the cells. This dose was selected as the highest test dose for the chromosomal aberration assay, along with three other doses: 62.5, 125, and 250 µg/mL. The chromosomal aberration assay was performed with and without metabolic activation using rat liver S9 fraction, along with a vehicle control and two positive controls, with harvesting of cells at 4 and 21 hours. Metaphase slides were scored for specific aberrations, unspecific aberrations, and numerical aberrations.

In all the experiments, the number of cells with chromosomal aberrations in the three highest treatment groups did not show any marked increase in comparison with the vehicle control. Positive controls showed a high incidence of specific chromosomal aberrations (88 and 90 %) in the experiments with harvesting time of 21 hours.

Under the conditions of the tests conducted, the test material did not provoke any effect interpretable as being suggestive of a mutagenic property on Chinese hamster ovary cells in vitro.

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

Genetic toxicity in vivo

Description of key information

In vivo: Micronucleus, Dance (1991)

Under the conditions of this study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January to 04 June 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for further information
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Principles of method if other than guideline:
The study is compliant with TG474 with just one exception: only 1000 and not 4000 PCE have been assessed for MN. The recent TG474 update increased the number of cells to be scored from 2000 PCE to 4000 PCE due to the low background MN frequency in the assay. This increase should assure that at least 1 MN per animal is identified in the vehicle controls and therefore increase the statistical power of the test. In this study the incidence of vehicle control animals with 0MN PCE is low so the study can be considered sufficiently sensitive to detect a biologically relevant increase in micronuclei.
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Dose preparations were freshly formulated in corn oil on the day of dosing, each concentration being individually formulated, and mixed prior to use.
- No allowance was made for purity or activity below 100 %. No determinations of homogeneity or concentration were performed on the dose preparations.
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 17.9 - 25.7 g
- Housing: Animals were in single-sex groups of 2 or 5 animals per cage in high density polypropylene cages with stainless steel tops. Cages housing animals of different treatment groups were distributed so that the effects of any spatially-variable components were equalised between groups as much as possible.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: 15 air changes per hour
- Photoperiod: 12-hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil was used as the vehicle.

- Justification for choice of solvent/vehicle: Test material was formulated in corn oil and therefore degradation due to hydrolysis prior to dose administration would not have occurred.

Clinical signs, especially those suggesting CNS effects, confirm systemic (and therefore bone marrow) exposure. In addition, weight loss was also seen in the main study and bone marrow toxicity in several animals in the MN DRF support that adequate systemic exposure was observed in the main study.
Details on exposure:
- A preliminary toxicity test was conducted. Test material dosages of 250, 500, 1000, and 2000 mg/kg were administered to groups of 4 test animals (2 males and 2 females). Volume of dosage was 10 mL/kg for both the preliminary test as well as the main micronucleus test.
- All animals dosed at 1000 and 2000 mg/kg, and 3 of the 4 dosed at 500 mg/kg, showed severe reactions to treatment and only three survived to the end of the 72-hour exposure period. All animals dosed at 250 mg/kg survived to termination. Slides were prepared and stained from all animals. Some bone marrow toxicity was apparent in individual animals dosed at 2000, 1000, and 500 mg/kg, despite the short time period between dosing and death or sacrifice. No real toxicity was apparent in the bone marrow of animals treated at 250 mg/kg. Therefore test material doses of 10, 50, and 250 mg/kg were chosen for subsequent testing. Concentrations of the test material were administered once orally (by gavage, in corn oil) to test animals (5 male/5 female in 10 and 50 mg/kg bw dose groups; 15 male/15 female in 250 mg/kg bw dose group). Control animals (15 male/15 female) were given corn oil at 10 ml/kg bw. Chlorambucil (30 mg/kg in aqueous 10 % ethanol; administered orally) served as the positive control (5 male/5 female). 5 males and 5 females per group were sacrificed at 24 hours following treatment. An additional 5 males and 5 females per test group in the vehicle control and the 250 mg/kg dose group, were sacrificed at 48 and 72 hours. Bone marrow smears were prepared from each animal, then stained and examined.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
single oral dose
Post exposure period:
Preliminary toxicity test: 72 hours after treatment
Main micronucleus test: 24, 48, and 72 hours
Dose / conc.:
10 mg/kg bw (total dose)
Dose / conc.:
50 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
No. of animals per sex per dose:
Preliminary toxicity test: 2 mice per sex per dose

Main micronucleus test: 5 mice per sex per dose per exposure duration
Control animals:
yes, concurrent vehicle
Positive control(s):
Main micronucleus test: Chlorambucil 30 mg/kg administered to 5 males and 5 females for 24 hours.
Tissues and cell types examined:
Bone marrow erythrocytes were examined.
Details of tissue and slide preparation:
After the animals were sacrificed, femurs from each animal were dissected out and their marrows extracted. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test and the main micronucleus test, respectively) were prepared, and the slides air-dried, fixed with methanol, and then stained using the Schmid (May-Grunwald and Giemsa) staining technique.
Evaluation criteria:
Slides were examined under the light microscope. A minimum of 2000 erythrocytes per animal were examined for the presence of micronuclei. The frequencies of micronucleated cells per 1000 erythrocytes were then calculated. The ratio of polychromatic to mature cells was also calculated for each animal as an indicator of gross toxicity - a decrease in this ratio may indicate inhibition of cell division following treatment. The incidence of micronuclei in the mature cell population 24 hours after treatment was also calculated and reflected the pretreatment situation, since most of these cells were produced before treatment.
Statistics:
Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling time (24, 48, or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No animals dosed at 10 or 50 mg/kg showed adverse reactions to treatment; however, some instances of weight loss were apparent in both groups. Only one animal treated at 50 mg/kg showed a marked weight loss. At 250 mg/kg, 6 of 15 males and one female showed reactions to treatment including rales, hunched posture, and/or piloerection on the day of dosing only. A single male showed these signs for the 24 hours until termination. At the 24 hour termination time, 6 of 10 animals had lost weight, and one failed to gain weight; at the 48 hour termination time, 5 of 10 animals lost weight; at the 72 hour termination, no animal showed weight loss. Of the 10 mice (5 males/5 females) given Chlorambucil (i.e., the positive control group), 7 lost weight during the 24-hour period before termination.
Among mice sacrificed at 24 hours following treatment, the mean incidence of micronucleated polychromatic erythrocytes (MPE) (per 1000 polychromatic cells scored) was 1.6 for the vehicle control, and 0.7, 1.1, and 1.0 for the 10, 50, and 250 mg/kg dose groups, respectively. The mean incidence of MPE for the positive control was 49.7. The ratio of polychromatic cells to mature cells was 1.0 for the vehicle control; 1.0,  1.1, and 0.9 for the 10, 50, and 250 mg/kg test material groups; and 0.8 for the positive control. 
Among mice sacrificed at 48 and 72 hours following treatment, the mean incidence of MPE was 0.6 and 1.4 for the vehicle control, and 1.3 and 1.8 for 250 mg/kg test material. The ratio of polychromatic cells to mature cells at 48 and 72 hours was 0.8 and 0.7 for the vehicle control, and 0.9 and 0.8 for 250 mg/kg test material.
A statistically significant increase in the incidence of micronucleated polychromatic cells was seen in mice in the 250 mg/kg dose group and sacrificed 48 hours later. However, the increase was not considered biologically relevant. The individual incidences of micronucleated  polychromatic cells in animals in this group were small and within the range observed in the vehicle control animals throughout the study. No biologically or statistically significant increase was recorded for animals treated with the test material and killed 24 or 72 hours later.
Under these experimental conditions, there is no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.
Conclusions:
Under the conditions of this study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.
Executive summary:

The effect of the test material on chromosome structure in bone marrow erythrocytes following acute oral administration in mice was investigated using the micronucleus assay. The test was performed in accordance with the standardised guidelines OECD 474 and EU Method B.12, under GLP conditions.

A preliminary toxicity test, using doses between 250 and 2000 mg/kg, guided the choice of doses for the main micronucleus test, which were 10, 50 and 250 mg/kg body weight, along with the vehicle (corn oil) control. Chlorambucil was administered at a dose of 30 mg/kg and served as the positive control. Animals were sacrificed 24 hours after treatment. In addition, some animals in the control and 250 mg/kg-dose groups were sacrificed at 48 and 72 hours.

Clinical signs observed at 250 mg/kg confirmed systemic exposure to MBTC following oral administration. Furthermore, evidence of bone marrow toxicity at doses >250 mg/kg was identified in the preliminary toxicity test. Exposure of the bone marrow is considered to have occurred in this study. Frequencies of micronucleated polychromatic erythrocytes in the treated groups were similar to those in control groups with all treatment durations. A statistically significant increase was observed in the 250 mg/kg dose group sacrificed at 48 hours. However, this increase was not considered to be biologically relevant. Statistically significant increases over controls were seen in positive controls given Chlorambucil.

Under the conditions of this study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.

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

Additional information

Hydrolysis data (Kleinmaier and Heitmann, 2018) confirm that irreversible hydrolysis does not occur over a timeframe of one to six days following dissolution of MBTC in neutral water, mildly or strongly acidic aqueous solutions of hydrochloric acid in water, thus supporting the assumption that in these solutions an equilibrium exists between strong complexes of water with MBTC and chemical species where one or several chloride ions are abstracted. Furthermore, in neutral water it takes at least seven days for any fully hydrolysed species to occur that would oligomerise and be removed from the equilibrium by precipitation. The position of this equilibrium is shifting continuously both with the concentration of MBTC in water and with the concentration of hydrochloric acid, i.e. the concentration of additional chloride ions.

The in vitro genotoxicity assays are aqueous based test systems. In all of the assays test substance exposure to the aqueous system occurred for a few minutes (Ames test) to 3 -4 hours (HPRT and chromosome aberration tests), therefore irreversible hydrolysis within the test system is highly unlikely to have occurred regardless of the vehicle used for testing. Precipitation was recorded at the highest concentrations but the lower concentrations remained fully soluble, therefore in each study the test system was exposed to biologically available, soluble MBTC.

In vitro: Ames, Krul (2002)

The test material was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, the tryptophan-requiring Escherichia coli strain WP2 uvrA, and a liver fraction of Aroclor 1254 -induced rats for metabolic activation (S9 mix). The test was performed in accordance with the standardised guidelines OECD 471 and EPA OPPTS 870.5100, under GLP conditions.

Five different concentrations of the test material were used ranging from 62 - 5000 µg/plate. Vehicle and positive controls were run simultaneously.

The test material was toxic to E. coli WP2 uvrA strain and to TA 100 at the two highest doses, 1667 and 5000 ug/plate, in both the absence and presence of S9 mix, and also, to TA 100 at 185 and 556 ug/plate in the absence of S9 mix, evidenced by a decrease in the mean number of revertant colonies. It was also toxic to TA 1537 at 556, 1667, and 5000 µg/plate in the presence of the S9 mix. The test material did not cause a more than 2 -fold or a dose-related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate in the negative control. The mean number of his+ and trp+ revertant colonies in the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Under the conditions of this study, the test material was not mutagenic in bacterial reverse mutation tests conducted in four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA with or without metabolic activation.

In vitro: HPRT, Dollenmeier (1988)

A forward mutation assay was performed with the test material in V79 Chinese hamster cells in vitro with and without metabolic activation using S9 rat liver microsome fraction. The test was performed in accordance with the standardised guideline OECD 476, under GLP conditions.

Seven different concentrations of the test material (60 - 1200 µg/mL), two negative (culture medium) controls, and one positive control (ethylmethanesulphonate for without metabolic activation and N-nitrosodimethylamine for with metabolic activation) were used for each experiment. The results of each experiment were confirmed in a second and independent confirmatory experiment. No significant deviation in mutant frequencies (or mutant factors) was revealed in any experiment when cell cultures were challenged with 6 -thioguanine (6 -TG). The positive controls effectively caused mutations in the HGPRT gene locus and had high mutant factors.

Under the conditions of the tests conducted, the test material did not elicit mutagenic effects in this mammalian cell forward mutation system with or without metabolic activation.

In vitro: CHO chromosomal aberration, Strasser (1988)

A chromsoome aberration assay was performed with the test material in Chinese hamster ovary cells (CCL61) in vitro with and without metabolic activation using S9 rat liver microsome fraction. The test was performed in according to the general principles of the standardised guideline OECD 476, under GLP conditions.

Four different concentrations of the test material (62.5 - 500 µg/mL), together with vehicle (water) and positive controls (mitomycin C for without metabolic activation and cyclophosphamide for with metabolic activation) were used. Cell were exposed for 3 hours and then harvested 4 and 21 hours after treatment. Metaphase slides were scored for specific aberrations, unspecific aberrations, and numerical aberrations.

The number of cells with chromosomal aberrations in the treated groups did not show any marked increase in comparison with the vehicle control. Positive controls showed a high incidence of specific chromosomal aberrations (88 and 90 %) in the experiments with harvesting time of 21 hours.

Under the conditions of the tests conducted, the test material did not provoke any effect interpretable as being suggestive of a mutagenic property on Chinese hamster ovary cells in vitro.

In vivo: Micronucleus, Dance (1991)

The effect of the test material on chromosome structure in bone marrow erythrocytes following acute oral administration in mice was investigated using the micronucleus assay. The test was performed in accordance with the standardised guidelines OECD 474 and EU Method B.12, under GLP conditions.

A preliminary toxicity test, using doses between 250 and 2000 mg/kg, guided the choice of doses for the main micronucleus test, which were 10, 50 and 250 mg/kg body weight, along with the vehicle (corn oil) control. Chlorambucil was administered at a dose of 30 mg/kg and served as the positive control. Animals were sacrificed 24 hours after treatment. In addition, some animals in the control and 250 mg/kg-dose groups were sacrificed at 48 and 72 hours.

No real indication of bone marrow toxicity was noted in any group treated. Frequencies of micronucleated polychromatic erythrocytes in the treated groups were similar to those in control groups with all treatment durations. A statistically significant increase was observed in the 250 mg/kg dose group sacrificed at 48 hours. However, this increase was not considered to be biologically relevant. Statistically significant increases over controls were seen in positive controls given Chlorambucil.

Under the conditions of this study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.

Short description of key information:

A complete data set of mutagenicity studies is available for MBTC, evaluating all the usual in vitro end points (i.e. gene mutation in bacterial cells, gene mutation in mammalian cells, and clastogenic potential) as well as in vivo clastogenic potential.

Justification for classification or non-classification

According to Directive 67/548/EEC and to regulation EC no.1272/2008 (CLP), the test material is not classified for genetic toxicity endpoint.

Justification : Negative results in Ames test, in the in vitro gene mutation, in the in vitro mammalian chromosome aberration test and negative results in the in vivo micronucleus test.