2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The weight of evidence studies for in vitro genetic toxicity endpoints are as follows:

• Bacterial mutagenicity (no guideline, pre-GLP, RL2), negative ±S9 mix with S. typhimurium TA1535, TA1537, TA98 and TA100 (M- 087335-01-1, Herbold, 1979)


• Bacterial mutagenicity (OECD 471, GLP, RL2), negative ±S9 mix with S. typhimurium TA1535, TA1537, TA98 and TA100 (M- 086848-01-1-01-1, Herbold, 1991)


• Mammalian mutagenicity (EPA OPP 84-2, GLP, RL2), negative ±S9 mix in CHO cells (M- 068802-01-1, Harbell, 1988)


• Mammalian mutagenicity (OECD 476, GLP, RL2), negative ±S9 mix in CHO cells (M- 086712-01-1, Lehn, 1989)


• Cytogenicity (OECD 473, GLP, RL2), negative ±S9 mix in human lymphocytes (M-087510-01-1, Heidemann, 1992)


• Bacterial mutagenicity (OECD 471, GLP, RL2), negative ±S9 mix (M-068903-01-1, Laylor & Wagner, 1988)

No genetic toxicity of the test item was revealed in any of the above mentioned studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986-10-17 to 1988-05-17

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

100, 500, 2500, 5000, 10,000 µg per plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other:

Details on test system and experimental conditions:

Plating Procedures for the Mutagenicity Assay Serial dilutions of the test article will be prepared immediately prior to the mutagenicity assay. S9-mix will also be prepared immediately prior to its use in the mutagenicity assay.
At least five doses of the test article will be plated with the appropriate tester strains, both with, and without metabolic activation. Without metabolic activation, 100 µL of tester strain and 50 µL of vehicle or test article will be added to 2.5 mL of molten selective top agar at 45 ± 2°C.
With metabolic activation, 100 µL of tester strain, 50 µL of solvent or test article solution and 0.5 µL of S9-mix will be added to 2.0 mL of molten selective top agar at 45 ± 2°C. After vortexing, the mixture will be overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay has solidified, the plates will be inverted and incubated for approximately 48 hours at 37 ± 2°C. When necessary, aliquots of other than 50 µL of test article/vehicle will be plated.

Evaluation criteria:

For a test article to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase in the mean number, of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. If the study is to be judged positive solely on the basis of a dose-responsive, less than 3-fold increase on TA1537 or TA1538, the response must be confirmed in a repeat experiment.

Key result

Species / strain:

S. typhimurium TA 1538

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Triflumuron was non-mutagenic with and without metabolic activation in the Salmonella/microsome assay.

Executive summary:

The results of the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay indicate that under the conditions of this study, at 10000, 5000, 2500, 500 and 100 µg per plate, the test item did not cause a positive response on any of the tester strains with or without metabolic activation by Aroclor induced rat liver microsomes. There were no increases in the number of revertants per plate on any of the tester strains in either the presence or absence of metabolic activation.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

In-life initiated/completed: 1987-10-19 to 1988-12-02

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

K1 BH4

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix (derived from rat liver)

Test concentrations with justification for top dose:

100, 150, 200, 250 and 300 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Cells were exposed to the test article with and without metabolic activation (S9) for 5 hours at 37±1°C.

Evaluation criteria:

The assay will be considered positive in the event a dose-dependent increase in mutant frequency is observed with one or more of the five concentrations tested inducing a mutant frequency which is at least twice that of the solvent control and untreated control, and also is increased above that of the solvent control and the untreated control by at least 8.7 mutants/10^6 clonable cells. The assay will be considered suspect if there is no dose response but one or more doses induce a mutant frequency which is considered significant and is >20 mutants per 10^6 clonable cells. The assay will be considered negative if none of the doses tested induce a mutant frequency which is considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

K1 BH4

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material was partially soluble at 300, 250, 200, and 150 µg/mL, and soluble at 100 µg/mL in growth medium. The mutant frequencies of the test article groups were not significantly increased above the solvent control after treatment with and without metabolic activation. In contrast, the positive control substances induced distinctly increased mutant frequencies indicating the sensitivity of the test system.

Conclusions:

Triflumuron was negative in the CHO/HGPRT assay.

Executive summary:

The test article, triflumuron, was tested in the CHO/HGPRT mutation assay in the absence and presence of an Aroclor-induced rat liver S9 activation system. The assay was conducted at dose levels of 300, 250, 200, 150 and 100 µg/mL in the presence and absence of S9. Under the conditions of the assay, the test material was negative in the CHO/HGFRT mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Study initiated/completed: 1988-02-23 to 1988-03-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The study was carried out with a subclone of the CHO cell line designated CHO-K1-BH4

Metabolic activation:

with

Metabolic activation system:

Sprague-Dawley male rats, induced by Aroclor 1254, served as source of the S9 fraction. A rat liver S9 fraction buffered with 0.15 M KCl was purchased from Litton Bionetics, Inc., and used in this assay. Prior to use in the HGPRT test, the S9 fraction was tested for contamination and for cytotoxicity. Total Protein (mg/mL): 41.3

The S9 fraction was kept frozen at -80°C. Immediately prior to use, the S9-fraction was thawed at room temperature and mixed with the freshly prepared cofactor solution in a sodium phosphate buffer pH 7.4. The mixture of S9 fraction with cofactor solution is designated S9-mix. The S9-mix contained the following:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
1 mM NADP
40% S9 fraction

The S9 reaction mixture was kept on ice until use.

Test concentrations with justification for top dose:

Exponentially growing CHO cells were plated in culture medium. For each dose level, four cultures were tested (200 cells per tissue culture dish, respectively). After attachment (18-24 hours later), cells were exposed to vehicle alone and to 9 concentrations of the test article ranging from 0.39 µg/mL to 100 µg/mL for 5 hours in medium containing 5% PCS, both in the presence and absence of S-9 metabolic activation.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

Untreated cells were used as the negative control. In the activation assays, the cells were exposed to the S-9 activation mixture. This control assay is referred to as the negative control.

Negative solvent / vehicle controls:

yes

Remarks:

As second negative control, the cells were exposed to the same concentration of vehicle as the treated cultures. The final concentration of the vehicle in the medium was 1% (v/v) or less. This control is referred to as the vehicle control.

True negative controls:

no

Positive controls:

yes

Remarks:

Positive control without S-9 mix

Positive control substance:

9,10-dimethylbenzanthracene

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test material concentrations were limited by precipitation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

A negative result is reported for an HPRT assay in CHO cells (±S9). Test material concentrations were limited by precipitation.

Conclusions:

The test material, was assayed for mutagenic activity at the HGPRT locus in CHO cells from 50.0 µg/mL to 100.0 µg/mL both with and without metabolic activation.
Under both treatment conditions, no cytotoxic effects were induced. However, the test material was tested up to its limit of solubility under culture conditions. The absolute cloning efficiencies for the vehicle controls varied from 66.2% to 126.2% without activation and from 68.2% to 84.3% with activation demonstrating good cloning conditions for the assays. The vehicle control mutant frequencies were all in the normal range of background frequencies for the assay. In contrast, the positive controls EMS and DMBA induced a distinct mutagenic effect in mutant frequency, which was significantly increased over the negative controls demonstrating the sensitivity of the test system and the ability to detect known mutagens. From the lack of dose-related and reproducible increases in mutant frequency the test article is considered non-mutagenic in the CHO-HGPRT Forward Mutation Assay, both with and without metabolic activation, according to our evaluation criteria.

Executive summary:

Test substance was evaluated for mutagenic effects at the HGPRT locus (forward mutation assay) in CHO cell cultures after in vitro treatment at concentrations to 100 µg/mL, both with and without S9 mix. Under both treatment conditions, there was precipitation of test substance beginning with a test article concentration of 80 µg/mL. The test substance was therefore tested up to its limit of solubility under culture conditions test substance did not induce cytotoxic effects such as decreases in relative population growth or cloning efficiency under these test conditions. There were neither dose-related nor reproducible increases in mutant frequency which were significantly elevated over the negative controls. In contrast, the positive controls ethylmethanesulfonate (without S9 mix) and dimethylberizanthracene (with S9 mix) revealed a clear mutagenic effect in the assay. From these results, the test substance test substance can be considered as nonmutagenic in the CHO-HGPRT ForwardMutation Assay, both with and without metabolic activation.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

In-life initiated/completed: 1991-10-21 to 1992-01-03

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Previously known as EEC Directive 84/449, L 251, B 10

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity "In vitro mammalian cytogenetics"

Version / remarks:

July 1, 1986

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Dose levels (with and without metabolic activation (S9-mix)) were
- at 24 hours: 10, 30 and 80 mg/mL
- at 48 hours: 80 mg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

All incubations were done at 37°C in a humidified atmosphere with 15% CO2.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a concentration-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Key result

Species / strain:

lymphocytes: human

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no biologically relevant increase in the structural chromosomal aberration rate could be found when compared with the range of aberrations in the corresponding negative controls. These results were obtained at each fixation interval both in the absence and presence of S9-mix.

Summary of Results

Mutagenicity data

fixation interval: 24 h after start of the treatment

test group	number of cells analysed	conc. per mL	S9-mix	per cent incl. gaps	aberrant excl. gaps	cells exchanges
negative control	200	0.0 µg	-	0.5	0.0	0.0
solvent control	200	0.0 µg	-	1.0	0.5	0.0
positive control EMS	50	1.0 µg	-	20.0	20.0	6.0
test item	200	10.0 µg	-	3.5	0.5	0.0
test item	200	30.0 µg	-	2.0	1.0	0.0
test item	200	80.0 µg	-	1.5	1.0	0.0
negative control	200	0.0 µg	+	1.0	0.5	0.0
solvent control	200	0.0 µg	+	0.5	0.0	0.0
positive control CPA	50	30.0 µg	+	16.0	14.0	2.0
test item	200	10.0 µg	+	1.5	0.5	0.0
test item	200	30.0 µg	+	1.0	1.0	0.0
test item	200	80.0 µg	+	1.5	0.5	0.0

 

fixation interval: 48 h after start of the treatment

test group	number of cells analysed	conc. per mL	S9-mix	per cent incl. gaps	aberrant excl. gaps	cells exchanges
solvent control	200	0.0 µg	-	1.0	0.0	0.0
test item	200	80.0 µg	-	0.0	0.0	0.0
solvent control	200	0.0 µg	+	2.0	1.5	0.0
test item	200	80.0 µg	+	1.0	0.5	0.0

Conclusions:

Triflumuron is considered to be non-mutagenic in this chromosomal aberration test.

Executive summary:

The test article triflumuron was assessed for its potential to induce structural chromosome aberrations in human lymphocytes in vitro. Preparation of chromosomes was done 24 hours (low, medium and high concentration range), and 48 hours (high concentration range) after the start of treatment with the test article which was dissolved in DMSO. The treatment interval was 4 hours. A single experiment was performed. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control cultures where 25 metaphases were scored. The following concentrations were evaluated with and without S9-mix

24 hours: 10.0; 30.0; 80.0 µg/mL

48 hours: 80.0 µg/mL

The concentration of the test article applied had been determined in a pre-test phase using the mitotic index as indicator for toxicity response. Treatment of the cells with the highest concentration (80.0 µg/mL) did not reduce the mitotic index at any fixation intervals either in the presence or absence of S9 mix. With concentrations higher than 30.0 µg/mL the test article precipitated slightly and higher than 80.0 µg/mL strongly in the culture medium. There was no biologically relevant increase in cells with aberrations after treatment with the test article either with or without metabolic activation by S9-mix at both fixation intervals.  Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions of this study, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in human lymphocytes in vitro. Therefore, triflumuron is considered to be non-mutagenic in this chromosomal aberration test.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Previously EEC Directive 84/449/EEC B.14. Other Effects - Mutagenicity Salmonella typhimurium Reverse Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Revised Health Effects Test Guidelines October 1984 - Washington, DC (PB84-233295)

Version / remarks:

HG - Gene Muta - S. typhimurium, October 1984.

Deviations:

no

Principles of method if other than guideline:

Study was performed in accordance with the OECD Test Guideline 471 from May 1983 with four bacterial strains.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

1st test: Triflumuron: 8, 40, 200, 1000 and 5000 µg/plate.
2nd test: Triflumuron: 125, 250, 500, 1000, 2000 and 4000 µg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

sodium azide 10 µg/plate (TA 1535 only). nitrofurantoin 0.2 µg/plate (TA 100 only). 4-nitro-1,2-
phenylene diamine 10 µg/plate (TA 1537 only). 4-nitro-1,2-phenylene diamine 0.5 µg/plate (TA 98 only). 2-aminoanthracene 3 µg/plate.

Evaluation criteria:

- The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratories' own historical data.
- The positive controls had to show sufficient effects, as defined by the laboratories' experience.
- Titer determinations had to demonstrate sufficient bacterial density in the suspension.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and test material precipitation were observed at higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and test material precipitation were observed at higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and test material precipitation were observed at higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and test material precipitation were observed at higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the repeat tests. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

 

Tabulated Summary of Data

Table 1: Without S9 Mix

µg/plate	Strain
	TA 1535	TA 100	TA 1537	TA 98
Group 1-4
0 8 40 200 1000 5000 Na-azid NF 4-NPDA	12 12 14 14 15 10 730	89 100 110 105 97 81   354	13 10 12 11 13 P     60	32 35 35 32 30 29     80
Group 5-8
0 125 250 500 1000 2000 4000 Na-azid NF 4-NPDA	16 15 16 15 10 11 11 694	143 142 168 169 131 134 131   409	10 9 9 7 6 8 P     89	34 37 38 35 35 29 27     176

 

Table 2: With S9 Mix

µg/plate	Strain
	TA 1535	TA 100	TA 1537	TA 98
Group 1-4
30% S9 0 8 40 200 1000 5000 2-AA	18 21 23 17 16 14 149	152 155 146 135 151 117 710	16 19 19 19 17 P 85	39 43 41 40 46 33 508
Group 5-8
10% S9 0 125 250 500 1000 2000 4000 2-AA	17 14 12 12 13 11 9 176	149 159 150 155 156 164 139 1310	10 8 7 10 8 5 P 508	45 33 35 38 37 42 P 1087
Group 9-12
4% S9 0 125 250 500 1000 2000 4000 2-AA	13 13 10 11 11 11 12 235	145 149 149 157 130 143 150 1318	10 10 9 10 11 9 P 302	36 40 36 35 46 37 41 1646

Conclusions:

Triflumuron was non-mutagenic with and without metabolic activation in the Salmonella/microsome assay.

Executive summary:

Triflumuron was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 μg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 2000 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes. However, substance precipitation occurred at the dose 500 μg per plate and above, such that from 4000 μg per plate, this range could only be used to a limited extent up to 5000 μg per plate for assessment purposes. Evidence of mutagenic activity of triflumuron was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Therefore, triflumuron was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Study in four strains, not the guideline-recommended five strains

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was performed using the procedure of Ames et al. (1973a, 1975).This test represents a screening method to detect point-mutagenic effects caused by chemical agents in vitro. Auxotrophic mutants of Salmonella typhimurium are used to demonstrate such effects. This involves determining the rate of reverse mutation to prototroph in control and treatment groups. If this rate exhibits a sufficient increase in the treatment groups, a mutagenic effect is inferred.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-deficient mutants of Salmonella typhimurium LT2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix produced from the livers of at least six adult male Sprague-Dawley rats weighing about 200 - 300 grams each was used for metabolization. The animals were administered a single intraperitoneal injection of Aroclor® 1254 dissolved in peanut oil at a dose of 500 mg/kg body weight five days before workup in order to induce enzymes. The animals were worked up according to directions of Ames et al. (1975), and the S-9 fraction stored at -80°C in 10 mL portions. These portions were slowly thawed in ice water for use. The S9-mix was freshly prepared (Ames et al., 1973a) using an S-9 fraction derived from the workup of October 27, 1978.

Test concentrations with justification for top dose:

µg per plate
1. Negative control - 0
2.Triflumuron - 2500
3. Triflumuron - 500
4. Triflumuron -100
5. Triflumuron - 20
6. Triflumuron - 4
7. Positive control Endoxan® - *145 (only TA 1535 & TA 100)
8. Positive control trypaflavine - 200 (only TA 1537 & TA 98)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

no

Positive control substance:

other:

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The study was performed using S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. This Ames test reports a negative result (±S9); test material concentrations of up to 2500 µg/plate were tested; the concentrations tested were limited by precipitation.

Executive summary:

Triflumuron, was examined for mutagenic effects in the Salmonella/microsome test at doses of up to 2500 µg per plate using four Salmonella typhimurium LT2 mutants.
2. Doses of up to 2500 µg per plate did not lead to bacteriotoxic effects. The total bacteria counts remained unchanged. No growth inhibition could be observed.
3. Marked substance precipitation could be observed at the highest dose (2500 µg per plate).
4. No evidence was found for a mutagenic effect induced by triflumuron. Neither a dose-related increase nor doubling of the mutant counts were observed.
5. In contrast, the positive controls Endoxan® and trypaflavine exerted marked mutagenic effects, as is apparent from the increases in the numbers of mutant colonies to well above double those in the pertinent negative controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Two pre-guideline studies are available which are not reliable for a final assessment. A micronucleus study in mice (M-087291-01-1, Herbold, 1978) did not indicate a genotoxic effect however, the study is deficient compared to the current test guideline (OECD 474). Bone marrow was sampled at 6 hours following the second dose (the guideline states 18-24 hours); micronucleus incidence was assessed in 1000 PCEs (the guidelinje states 4000); there is no evidence of toxicity (clinical signs or effects on PCE:NCE ratio) and no direct evidence of bone marrow exposure. Therefore, this study is insufficient for assessment.

A second study, a dominant lethal assay (M-087331-01-1, Herbold, 1978) also pre-guideline, non-GLP and reported in limited detail. Reference is made to experience with reference mutagens, but no positive control data are included in the study report. Therefore, this study is also insufficient for assessment.

Both studies can only be used as supporting information showing no indication for a genotoxic potential of triflumuron.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

The study is pre-guideline, non-GLP and reported in limited detail.

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: A dominant lethal assay on male mice to determine mutagenic potential of a test substance.
- Short description of test conditions: Male test animals were given a single oral dose of the test substance and water and pelleted feed were provided ad libitum. Females remained untreated and were solely for reproductive purposes.
- Parameters analysed / observed: fertilisation quota, pre-implantation loss, post-implantation loss.

GLP compliance:

no

Remarks:

Test was conducted pre-GLP

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

At the start of the study, the male mice weighed 31 to 42 grams, and the females weighed 28 to 33 grams. The mice were between 8 and 12 weeks old. Each group consisted of 50 males and 600 females. The mice were housed in Type I Makrolon cages, as follows: a) During mating, each male was caged with one virgin female. b) After mating, the females were caged singly. Standardized housing conditions with 12 hours of electric light daily, room temperature of 20 to 26°C and average relative humidity of approx. 60%. The mice were fed pelleted ®Altromin Laboratory Small Animal Diet and tap water ad libitum.

Route of administration:

oral: unspecified

Vehicle:

yes - 0.5 % Cremophor emulsion used with test substance for oral dose.

Details on exposure:

All the females used in the study remained untreated. The males of the treated group received a single oral dose of 400 mg triflumuron /kg body weight in 0.5 % Cremophor emulsion, administered in a volume of 20 mL/kg body weight. The males of the control group received 0.5 % Cremophor emulsion containing no triflumuron , in a volume equivalent to that given to the treated mice.

Duration of treatment / exposure:

Not applicable for single dose study

Dose / conc.:

20 other: mL/kg bw

Remarks:

male mice only

Control animals:

yes, concurrent vehicle

Evaluation criteria:

After an interval of about 14 days counted from midway through a mating period, the uterus of each female was examined to determine pre-implantation and post-implantation losses, the criteria of the assessment. For this purpose, the total implants as well as the live and the dead implants (sum of the deciduomata, the resorptions and the dead embryos) and the Corpora lutea were counted.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

The dose tested was tolerated well. It did not lead to toxic signs or mortalities. The mice's fertility was not affected by the dose used. No treatment-related variations between control and treatment group were noted in respect to the parameters relevant to assessment of a mutagenic effect (dead implants, live implants, total implants, pre-implantation loss).

Conclusions:

Triflumuron was not mutagenic in the dominant lethal test on male mice.

Executive summary:

A dominant lethal assay was conducted on male mice to evaluate triflumuron for mutagenic potential. The male mice each received a single oral dose of triflumuron at a level of 400 mg/kg bw. The tested dose was well tolerated, and it did not cause any symptoms of poisoning or any mortalities. The fertility of the mice was not affected by the tested dose. No treatment-related differences were seen between the treated group and the controls with respect to those parameters of importance to the assessment of a mutagenic effect (dead implants, viable implants, total implants, pre-implantation loss). The dominant lethal assay on male mice thus provided no indication of triflumuron having a mutagenic effect at the oral dose level of 400 mg/kg body weight.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted in June/July 1978.

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline available

Principles of method if other than guideline:

The micronucleus test developed by SCHMED et al. (1, 2, 3» is recommended as a sensitive in vivo mutagenicity test on mammals. It is a test performed on a somatic tissue, the femoral marrow. The test is evaluated by scoring for micronucleated polychromatic erythrocytes. Normally, these erythrocytes are anucleate so that increased incidence of such cells with micronuclei, as compared with the negative control, is indicative of chromosome-breaking or spindle-active effects of the tested substance in the erythroblasts. Such effects simultaneously constitute a mutagenic effect. For comparison with test substance, a negative control group received the vehicle and a positive control group was treated with the known mutagen cyclophoshamide.
The study is deficient compared to the current test guideline (OECD 474). Bone marrow was sampled at 6 hours following the second dose (the guideline states 18-24 hours); micronucleus incidence was assessed in 1000 PCEs (the guidelinje states 4000); there is no evidence of toxicity (clinical signs or effects on PCE:NCE ratio) and no direct evidence of bone marrow exposure.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

The experimental animals used for the test were male and female mice of the NMRI strain, bred and supplied by S. Ivanovas GmbH, Kisslegg/Allgau. At the start of the study, the mice weighed between 26 and 40 grams, and were aged 8 to 12 weeks. They were housed in Type I Makrolon cages, in groups of no more than five per cage. The housing conditions were standardized, with 12 hours of electric light daily (7 a.m. to 7 p.m.), a room temperature of 23- 3°C, and an average relative humidity of 60 %. They received ®Altromin pelleted chow and tap water ad libitum.

Route of administration:

oral: gavage

Vehicle:

Endoxan (Asta), a known cytostatic drug and alkylating promutagen

Details on exposure:

The test substance was suspended in 0.5 % Cremophor emulsion and administered orally by stomach tube. The positive control substance was dissolved in demineralized water and applied by the same route as the test substance. The negative control group received the emulsifier (less test substance) likewise applied orally.

The volume applied was consistent at 10 mL/kg body weight in each group.

Duration of treatment / exposure:

They received two applications of the respective substance at an interval of 24 hours. Six hours after the second application, the mice were sacrificed by decapitation and the femoral marrow was prepared.

Frequency of treatment:

Two applications of the respected substance in interval of 24 hours

Post exposure period:

Six hours after the second application, the mice were sacrificed by decapitation and the femoral
marrow was prepared.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

The mice were randomly assigned to the test groups which each consisted of 5 males and 5 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control: known cytostatic drug and alkylating promutagen
- Route of administration: via gavage, two doses with an interval of 24 hours
- Doses / concentrations: 60 mg/kg bw at 10 mL/kg bw

Tissues and cell types examined:

1000 polychromatic erythrocytes per mouse were counted (prepared from bone marrow)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that the micronucleus test on the mouse, i.e. a somatic mutagenicity test system in vivo, provided no indication of Triflumuron having a mutagenic effect at the tested doses of up to and including 2 x 400 mg/kg body weight per os

Executive summary:

A micronucleus test was conducted on male and female mice to evaluate the test substance triflumuron for potential mutageniceffects on the chromosomes of bone marrow erythroblasts. The known mutagen cyclophosphamide was used as the reference substance. Two oral applications were made at an interval of 24 hours.  The femoral marrow was prepared 6 hours after the second application. The test substance doses were 2 x 200 mg/kg bw and 2 x 400 mg/kg bw, and the cyclophosphamide positive control doses were 2 x 60 mg/kg bw. The test provided no indication of triflumuron having a mutagenic effect at doses of up to and including 2 x 400 mg/kg bw/day. Erythrocyte production, measured against the ratio of polychromatic to normochromatic erythrocytes, also was not adversely affected.
Cyclophosphamide, the positive control, had a marked mutagenic effect manifested by the  ncrease in the incidence of polychromatic erythrocytes with micronuclei. Endoxan also slightly depressed erythropoiesis because the number of polychromatic cells decreased slightly in relation to normochromatic cells.

Justification for classification or non-classification

The mutagenic potential of triflumuron was tested in several in vitro studies (gene mutations in bacterial and mammalian cells and chromosome aberrations tests) and two in vivo tests (micronucleus assay and dominant lethal test in mice). Considering all information it is conlcuded that triflumuron has no potential for genotoxicity based on the negative results of the available studies.