1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Sep 2010 to 06 Oct 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

EC Number:

617-298-9

Cas Number:

82097-50-5

Molecular formula:

C14H16ClN5O5S

IUPAC Name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: mean value 34.4g (*SD ± 1.8g); 31.1 – 37.9 g
- Housing: single; in Makrolon Type II/III, with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water. Ad libitum
- Acclimatisation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45 - 71 %
- Photoperiod: Artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: 09 September 2010 to: 06 October 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

sterile water

Details on exposure:

FORMULATION:
On the day of the experiment, the test item was formulated in sterile water
Volume administered: 10 mL/kg body weight.

Duration of treatment / exposure:

Bone marrow samples were collected at the central sampling interval of 24 h after treatment.
For the highest dose level an additional bone marrow sample was taken at 48 h after treatment

Frequency of treatment:

Animals received a single dose of the test item

Post exposure period:

The animals of all dose groups, except the positive control were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and 48 h after administration of the test item or the vehicle controls. Sampling of the bone marrow was done 24 and 48 hours after treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 other: mg/kg bw

Remarks:

24 h preparation interval

Doses / concentrations 2

Dose / conc.:

1 000 other: mg/kg bw

Remarks:

24 h preparation interval

Doses / concentrations 3

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

24 h preparation interval

Doses / concentrations 4

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

48 h preparation interval

No. of animals per sex per dose:

14 males were assigned to the high dose group
7 males were assigned to the middle dose group
7 males were assigned to the low dose group
5 males were assigned to negative control groups
5 males were assigned to positive control groups

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide:
- Route of administration: oral
- Dose: 40 mg/kg b.w., once
-Volume: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:

Bone marrow; 2000 polychromatic erythrocytes (PCE) per animal were analysed for
micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed in both male and female mice (two animals per sex per dose level) under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item. The test dose levels were chosen according to the following scheme: 5 – 8 – 12.5 – 20 – 32 – 50 – 80 – 125 – 200 – 320 – 500 – 800 – 1250 – 2000 mg/kg b.w.. No substantial sex specific differences on toxic symptoms were observed, therefore, the main experiment was performed using male animals only. The treated animals did not express any clinical signs of toxicity. On the basis of these data 2000 mg/kg b.w., the maximum OECD Guideline recommended dose for this assay, was considered suitable. No gender specific differences in toxicity were observed, thus, the main study was performed using male animals only, as permitted by the Guideline.

TREATMENT AND SAMPLING TIMES:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle or the positive control substance once orally. Seven males were treated per dose group and sampling time. Five males each were treated for the vehicle and positive control group. The animals of all dose groups, except the positive control were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and 48 h after administration of the test item or the vehicle controls. Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 × g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear
increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
Statistical methods were used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of
micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
A test item failing to meet the criteria for a positive or negative response may be judged
equivocal in this assay and may be considered for further investigation.

ACCEPTANCE CRITERIA:
The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase
of micronucleated PCEs compared to the negative control.

Statistics:

Statistical methods were used as an aid in evaluating
the results: Statistical significance at the five per cent level (p < 0.05) for the incidence of micronuclei was evaluated by means of the non-parametric Mann-Whitney test

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals did not express any toxic reactions. The animals of the vehicle control groups (sterile water) for both sampling times did also not express any toxic reactions.
The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control, indicating that the test substance did not have any cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no biologically relevant enhancement and statistically significant increase in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item.
The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the respective vehicle control group and mainly within the historical vehicle control range. Additionally no clear dose dependence could be observed and furthermore the group of animals sampled 48 hours after treatment with the highest test item dose level did not show any statistically or biologically relevant enhancement of micronucleated PCEs.
A dose of 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control, which showed a substantial increase of induced micronucleus frequency.  The volume of the positive control administered was 10 mL/kg b.w.

Any other information on results incl. tables

Table 1. Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
vehicle	0	24	0.130	1 -4	1227
test item	500	24	0.086	0 -4	1220
test item	1000	24	0.071	0 -4	1172
test item	2000	24	0.086	0 -5	1217
positive control	40	24	2.760	42 -67	1068
vehicle	0	48	0.070	0 -3	1216
test item	2000	48	0.100	0 -4	1215

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Therefore, the test item is considered to be non-mutagenic in this bone marrow micronucleus assay.

Executive summary:

An OECD TG 474 genetic toxicity study was performed under GLP in order to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (NMRI).  The test item was formulated in sterile water, which was also used as vehicle control.  The volume administered orally was 10 mL/kg body weight (b.w.).  At 24 h and 48 h after a single administration of the test item, the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the control groups with 5 males only) were evaluated for the occurrence of micronuclei.  Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.  To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.  The following dose levels of the test item were investigated:  24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.  48 h preparation interval: 2000 mg/kg b.w.  The highest dose was estimated by a pre-experiment to be suitable. 

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control, thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.  In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.  The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group. 

A dose of 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control, which showed a substantial increase of induced micronucleus frequency.  The volume of the positive control administered was 10 mL/kg b.w.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.  Therefore, the test item is considered to be non-mutagenic in this bone marrow micronucleus assay.