N-ethyl-o(or p)-toluenesulphonamide

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:

16 November 2005 to 08 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N-ethyl-o(or p)-toluenesulphonamide

EC Number:

232-465-2

EC Name:

N-ethyl-o(or p)-toluenesulphonamide

Cas Number:

8047-99-2

Molecular formula:

C9H13NO2S

IUPAC Name:

N-ethyl-4-methylbenzene-1-sulfonamide

Test material form:

liquid: viscous

Remarks:

Colour: light yellow

Details on test material:

Ketjenflex 8 (NETSA)
Chemical name in report: N-ethyl-o/p-toluenesulfonamide (or N-substituted toluene sulphonamide)
Description: light yellow viscous liquid
Purity 100% (or 90%)

Test substance storage: at room temperature in the dark
Stability under storage conditions: stable

Test animals

Species:

rat

Strain:

Wistar

Remarks:

WI (SPF)

Details on species / strain selection:

Recommended by international guidelines (e.g. OECD, EEC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6 weeks old
- Weight at study initiation: The body weights of the rats at the start of the treatment were within 20% of the sex mean
- Assigned to test groups randomly: yes, the animals were allocated to treatment groups as they came to hand from the delivery boxes.
- Fasting period before study: In the dose range finding study and the micronucleus main test food was withheld overnight prior to dosing until 3 to 4 hours after administration of the test substance. In the additional dose range finding study no food was withheld prior to dosing.
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages (type MIV height: 18 em) containing Woody Clean bedding (Woody-Clean type 3/4; Technilab-BMI BV, Someren, The Netherlands). Paper bedding was provided as nest material (Technilab-BMI BV).
- Diet: standard pelleted laboratory animal diet (Aitromin (code VRF 1), Lage, Germany), ad libitum.
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range 18.6- 22.0)
- Humidity (%): 30-70 (actual range 25- 63).
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: propylene glycol (main study)
- Amount of vehicle: The dosing volume was 10 ml/kg body weight. NETSA was dosed undiluted in the additional range finding study at a dosing volume of 0.83 ml/kg body weight.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
NETSA was dissolved in propylene glycol (Merck, Darmstadt, Germany). In the additional dose range finding study the test substance was dosed undiluted. NETSA concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

one dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): According to Guideline
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg body weight.

Examinations

Tissues and cell types examined:

Bonemarrow, erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose range finding study
Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. In a dose range finding study 12 animals (group A, Band C: 1 male and 1 female, group D: 3 males and 3 females) were dosed orally with 2000, 1000, 500 and 400 mg/kg body weight (groups A, B, C and D, respectively). The group comprising 3 males and 3 females were dosed with the highest concentration that was used for the main study. The study duration per dosing was one to three days. During this period mortality and physical condition were recorded at least once a day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Bone marrow of the groups treated with Ketjenflex 8 (NETSA) was sampled 24 or 48 hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing.

DETAILS OF SLIDE PREPARATION:
- Isolation of bone marrow: Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 ml of fetal calf serum (Invitrogen). The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
- Preparation of bone marrow smears: The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether (Merck) and cleaned with a tissue). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.
- Staining of the bone marrow smears: The slides were automatically stained using the 'Wright-stain-procedure" in an "Ames" HEMA­ tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene (Kiinipath, Duiven, The Netherlands) before they were embedded in MicroMount (Kiinipath) and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1OOOx. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

OTHER:
Observations/measurements in the study were recorded electronically using the following programm: REES Monitoring system version 1.5 (REES Scientific, Trenton, NJ, USA).

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two­ sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

Statistical significance was determined with Wilcoxon Rank Sum Test; two­ sided test (P < 0.05)

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In a dose range finding study 12 animals (group A, Band C: 1 male and 1 female, group D: 3 males and 3 females) were dosed orally with 2000, 1000, 500 and 400 mg/kg body weight (groups A, B, C and D, respectively). Within 3 and 4 hours of dosing a male and female rat died in the 2000 and 1000 mg/kg bw dose group, respectively. Several systemic toxic signs were observed in the all dosed animals 25 min, 1, 3 and 4 hours after dosing. The surviving animals showed no abnormalities 2 days after dosing.

- Additional dose range finding study: In the additional dose range finding study 2 animals (group A: 1 male and 1 female) were dosed orally with 1000 mg undiluted NETSA/kg body weight.
The following clinical signs were observed during the first hour after dosing: lethargy, comatose, slow breathing, ventral recumbency and no reaction to a stimulus. Within 2 hours after dosing both animals still showed the following toxic signs: lethargy, comatose, ventral recumbency and no reaction to a stimulus. Both animals died within 18 hours after dosing. Based on the results of this additional dose range finding study no additional limit study was performed for the micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Micronucleated polychromatic erythrocytes: No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of NETSA treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met

- Ratio polychromatic to normochromatic erythrocytes: The animals of the groups, which were treated with NETSA showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Mortality and systemic toxic signs: The following clinical observations were made in the groups treated with 400, 200 and 100 mg NETSA/kg body weight:
During the first hour after dosing all animals of the groups treated with 400, 200 and 100 mg/kg body weight were lethargic. Eight males and seven females dosed with 400 mg/kg body weight also showed ventral recumbency. The other two males and three females dosed with 400 mg/kg body weight showed ataxia after dosing. All animals dosed with 200 and 100 mg/kg body weight also showed ataxia after dosing, except for one male animal dosed with 200 mg/kg body weight which showed ventral recumbency.
Within 18 hours after dosing all animals recovered from the treatment.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, it is concluded that this test is valid and that NETSA is not clastogenic in the micronucleus test.

Executive summary:

A Micronucleus Test in rats was performed to evaluate the genotoxic effect of NETSA on erythrocytes in bone marrow according to OECD TG 474.

Five male and five female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with NETSA at 400 (two groups), 200 (one group), and 100 (one group) mg/kg body weight. All animals of the dose levels of 400, 200 and 100 mg/kg body weight were lethargic after dosing. At the dose level of 400 mg/kg body weight eight males and seven females also showed ventral recumbency. The other two males and three females showed ataxia after dosing. All animals of the dose level of 200 and 100 mg/kg body weight also showed ataxia after dosing, except one male animal of the dose level of 200 mg/kg body weight which showed ventral recumbency. The animals of the control groups showed no abnormalities after dosing.

Bone marrow of the groups treated with NETSA was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with NETSA.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with NETSA showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

Under the conditions of the test, it is concluded that this test is valid and that NETSA is not clastogenic in the micronucleus test.