N-ethyl-o(or p)-toluenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames) (OECDTG 471): negative

Chromosome abberation in human lymphocytes (OECDTG 473): negative

Gene mutation in mammalian cells (Mouse Lymphoma Assay) (OECDTG 476): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 September 1983 - 30 October 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Pre-GLP period

Principles of method if other than guideline:

Test procedures based on the work of Ames et al (1975), "Methods for detecting carcinogens and mutagens with the salmonella mammalian-microsome test"

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Test Material: Santicizer 8
- Purity: 89%
- Storage condition of test material: stored in the dark at ambient temperature
- Stability under test conditions: indicated not to be unstable to heat, light, and water by the sample submitter

Target gene:

Histidine gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix ( as described by ames at el.(1975)

Test concentrations with justification for top dose:

TA 1535, TA 1537, TA 98, and TA 100 (without and with S9): 0.01, 0.04, 0.20, 1.00, 3.00 and 10.00 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Solutions of the test material were prepared with ACS grade dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

benzo(a)pyrene

other: NaNO2 and 2-aminoanthracene

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

METHOD OF APPLICATION:
- Method: plate incorporation method

DURATION
- Exposure duration: 72 hours at 37 degrees celsius

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain

Evaluation criteria:

For the test substance to be considered mutagenic, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. This was done using statistical methods to evaluate the test results ( see below).

Statistics:

Statistical analysis was performed on the plate incorporation assay results after transforming revertant/ plate values as log 10 (revertant/plate). Analysis included Bartlett’s test for homogeneity of variance and comparison of treatments with controls using within-levels pooled variance and a one-sided t-test. Grubb’s test was performed to determine if outliers were present. Dose response was evaluated with regression analysis for log 10 transformed doses and revertants/ plate. Significance of dose-response was evaluated by a t-tester.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only the maximum concentration of 10 mg per plate was toxic in the test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

Only the maximum concentration of 10 mg per plate was toxic in the test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

Only the maximum concentration of 10 mg per plate was toxic in the test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

Only the maximum concentration of 10 mg per plate was toxic in the test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In all strains toxicity was observed at the higher doses (at 10 mg /plate), both in the absence and/or presence of S9, with the exception of TA98 (with the absence of S9).

Conclusions:

Under the conditions of this study, Santicizer 8 was determined to be not mutagenic and does not need to be classified for mutagenicity in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The mutagenic activity of Santicizer 8 was conducted based on test procedures described by Ames et al (1975) according to OECDTG 471 . Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with the test item using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of S9-mix. Adequate negative and positive controls were included. Only four strains of bacteria were used, none of which would be able to detect certain oxidizing mutagens, cross- linking agents and hydrazines as detected by E.Coli WP2 strains or s.typhimurium TA102.

The test material, Santicizer 8, was not mutagenic towards salmonella typhimurium test strains TA98, TA100, TA1535 or TA1537 in the plate incorporation assays conducted with and without a rat microsomal activation system. A maximum of 10 mg per plate was used in the plate incorporation tests. Levels of 10 mg per plate were toxic in the plate incooperation test in the presence and absence of a rat microsomal activation system, whereas the lower concentrations tested were not. The plate incorporation test results indicated no significant mutagenic activity for Santicizer 8, and was therefore considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2005 to 27 September 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n.a.

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Healthy adult
- Suitability of cells: Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD and EEC).
- Sex, age and number of blood donors if applicable: Blood was collected from healthy adult, non-smoking male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2004) are presented below:
Dose range finding study: age 29, AGT = 14.4 h
First cytogenetic assay: age 35, AGT = 14.5 h
Second cytogenetic assay: age 39, AGT = 14.4 h
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood {0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium
- Methods for maintenance in cell culture if applicable: All incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% C02 in air in the dark at 37 ± 1°C. The temperature, humidity and C02-percentage were monitored throughout the experiment.

MEDIA USED
Culture medium consisted of RPMI 1640 medium {Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum {Invitrogen Corporation), L-glutamine (2 mM) (Merck), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) {Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijndrecht, the Netherlands).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

colchicine

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

First assay:
3h exposure, 24h fixation: 333, 1000, 1200, 1400, 1600 µg/mL (With and Without metabolic activation)
Second assay:
24h exposure, 24h fixation: 100, 200, 300, 400, 500, 600 µg/mL (Without metabolic activation)
48h exposure, 48h fixation: 100, 200, 300, 400, 500, 600, 700 µg/mL (Without metabolic activation)
3h exposure, 48h fixation: 300, 1000, 1200, 1300, 1400, 1500, 1600 µg/mL (With metabolic activation)

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First cytogenetic assay
- Preincubation period: 48 h cultured with mitogen phytohaemagglutinin.
- Exposure duration: 3 h in the absence and presence of S9-mix
- Expression time (cells in growth medium): 20-22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

Second cytogenetic assay
- Preincubation period: 48 h cultured with mitogen phytohaemagglutinin.
- Exposure duration: 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
- Expression time (cells in growth medium): 44-46 h in the presence of S9-mix, in te absence of the S9-mix the cells were not rinsed after exposure.
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h in the presence of S9-mix, and 24 h and 48 h in te absence of the S9-mix.

SPINDLE INHIBITOR: colchicine

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate and in two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene (Kiinipath, Duiven, The Netherlands) before they were embedded in MicroMount (Kiinipath) and mounted with a coverslip.

NUMBER OF CELLS EVALUATED :
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. In case the test substance was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture medium. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 h continuous exposure times was based on toxicity irrespective of the solubility of test substance in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.


DETERMINATION OF CYTOTOXICITY
- Method: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Cytotoxicity of the substance in the lymphocyte cultures was determined using the mitotic index.
The test substance was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.The highest tested concentration was 1993 µg/mL

Rationale for test conditions:

Test guideline OECD 473

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

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

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.
If P< 0.05, the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Species / strain:

primary culture, other: peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN STUDY
- First cytogenetic assay
Further investigation showed that a concentration of 1600 µg/mL already precipitated in the culture medium. Therefore, a concentration of 1600 µg/mLwas used as the highest concentration.
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
With and without S9-mix: 333, 1000, 1200, 1400 and 1600 µg test substance/mL culture medium (3 h exposure time, 24 h fixation time).
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix: 1200, 1400 and 1600 µg test substance/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 1000, 1200 and 1400 µg/mL culture medium (3 h exposure time, 24 h fixation time).
Note: In the presence of S9-mix, a concentration of 1400 µg/mL resulted in an inhibition of the mitotic index of 49% (MI =51). A quality control performed by a second scorer (who scored the blank and the concentration of 1400 µg/mL) showed that this concentration inhibited the mitotic index by 52% (MI = 48). Therefore, the concentration of 1400 µg/mL was chosen as the highest concentration, since this concentration was toxic enough.
In the absence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations but the number of polyploid cells was increased.
In the presence of S9-mix the test substance induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration of 1400 µg/mL both when gaps were included and excluded.

- Second cytogenetic assay
The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 100, 200, 300, 400, 500 and 600 µg test substance/mL culture medium (24 h exposure time, 24 h fixation time).
100, 200, 300, 400, 500, 600 and 700 µg test substance /mL culture medium (48 h exposure time, 48 h fixation time).
With S9-mix 300, 1000, 1200, 1300, 1400, 1500 and 1600 µg/ test substance /mL culture medium (3 h exposure time, 48 h fixation time).
The pH and osmolarity of a concentration of 1500 µg/mL were 7.30 and 416 mOsm/kg respectively (compared to 7.29 and 426 mOsm/kg in the solvent control).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix 200, 400 and 500 µg/test substance /ml culture medium (24 h exposure time, 24 h fixation time).
100, 300 and 500 µg test substance/mL culture medium (48 h exposure time, 48 h fixation time).
With 89-mix 1000, 1300 and 1600 µg test substance/mL culture medium (3 h exposure time, 48 h fixation time).
Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Both in the absence and presence of S9-mix the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

RANGE-FINDING/SCREENING STUDIES:
A concentration of 1993 µg/ml (= 0.01 M) the test substance precipitated in the culture medium and was used as the highest concentration. The pH and osmolarity of a concentration of 1000 µg/mL were 7.29 and 387 mOsm/kg respectively (compared to 7.30 and 389 mOsm/kg in the solvent control).

In the dose range finding test blood cultures were treated with 33, 100, 333, 1000 and 1993 µg test substance /mL culture medium with and without S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Remarks on result:

other: In the first cytogenetic assay in the presence of S9-mix, the test substance induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested cytotoxic concentration.

Conclusions:

Under the conditions of the test NETSA is not clastogenic in human lymphocytes in the absence of S9-mix. NETSA was found to be clastogenic in human lymphocytes in the presence of S9-mix in one of the two experiments. The clastogenic activity is confined only to high and cytotoxic concentrations.

Executive summary:

The ability of NETSA to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment) was examined according to OECD TG 473 In the first cytogenetic assay, NETSA was tested up to 1600 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence of S9-mix. NETSA precipitated in the culture medium at this dose level and appropriate toxicity was reached. In the presence of 1.8% (v/v) S9-fraction, NETSA was tested up to 1400 µg/mL for a 3 h exposure time with a 24 h fixation time. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, NETSA was tested up to 500 µg/mL for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. In the presence of S9-mix NETSA was tested up to 1600 µg/mL for a 3 h exposure time with a 48 h fixation time. NETSA precipitated in the culture medium at this dose level. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the first cytogenetic assay in the absence of S9-mix and in the second cytogenetic assay in the absence and presence of S9-mix NETSA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. In the first cytogenetic assay in the presence of S9-mix, no increase in the number of cells with chromosome aberrations was observed at concentration levels up to and including 1200 µg/mL. Only at the highest tested cytotoxic concentration of 1400 µg/mL, a statistically significant relevant increase in the number of cells with chromosome aberrations was observed, both when gaps were included and excluded. Although the number of cells with chromosome aberrations was just above the historical control data range pronounced exchange figures were observed at this concentration and the increase was considered biologically relevant. It was noted that NETSA increased the number of polyploid cells in the absence of S9-mix in the first cytogenetic assay. This may indicate that NETSA has the potential to disturb mitotic processes and cell cycle progression.

Finally, it is concluded that this test is valid and that NETSA is not clastogenic in human lymphocytes in the absence of S9-mix.

NETSA was found to be clastogenic in human lymphocytes in the presence of S9-mix in one of the two experiments. The clastogenic activity is confined only to high and cytotoxic concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-06-2005 to 26-09-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, {ATCC, Manassas, USA) (2001).
- Periodically checked for Mycoplasma contamination: yes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 Hepes buffered medium (Dutch modification) {Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 J.Jg/ml, respectively) (Sigma), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Merck).
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10).
- Exposure medium
For 3 hour exposure: in basic medium supplemented with 5% (v/v) heat­ inactivated horse serum (R5-medium).
For 24 hour exposure:in basic medium supplemented with 10% (v/v) heat­ inactivated horse serum (R10-medium).
- Selective medium: growth medium (R10) supplemented with 10% (v/v) heat-inactivated horse serum (total amount of serum = 20%) and 5 J.Jg/ml TFT (Sigma).
- Non-selective medium: growth medium (R10) supplemented with 10% (v/v) heat­ inactivated horse serum (total amount of serum= 20%).

- Periodically 'cleansed' against high spontaneous background: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4M hypoxanthine (Merck), 2 x 10^-7 M aminopterin (Fiuka Chemie AG, Buchs, Switzerland) and 1.6 x 10^-5 M thymidine (Merck) (HAT-medium) to reduce
the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and Beta-naphthoflavone induced Rat S9-mix

Test concentrations with justification for top dose:

Eight doses of the test substance were selected for the mutation assays, both in the absence and presence of S9-mix. Except in the first experiment in which seven dose levels were tested.
Experiment 1:
100, 250, 400, 500, 600, 700, 800 µg/mL (without metabolic activation)
250, 500, 600, 700, 800, 850, 900 µg/mL (with metabolic activation)
Experiment 2:
100, 250, 400, 500, 550, 600, 650, 700 µg/mL (without metabolic activation)
400, 500, 550, 600, 650, 700, 750, 850 µg/mL (with metabolic activation)
Experiment 3:
500, 600, 700, 735, 770, 800, 835, 870 µg/mL (with metabolic activation)

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 8 x 10^6 cells (10^6 cells/ml for 3 hours treatment; 0.25 x 10^6 cells/ml for 24 hours treatment) were used. If test substance concentrations were expected to be toxic, 12 to 16 x 10^6 cells were used per culture.

DURATION
- Exposure duration: In the first experiment, cell cultures were exposed for 3 hours to Ketjenflex 8 (NETSA) in exposure medium. In the second experiment, cell cultures were exposed to Ketjenflex 8 (NETSA) in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. In the third experiment, cell cultures were exposed to Ketjenflex 8 (NETSA) in exposure medium for 3 hours in the presence of S9-mix. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.5 °C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 25 cm2 culture flasks at 37.0 ± 1.5 °C. Solvent and positive controls were included.
- Expression time: 2 days

SELECTION AGENT: trifluorothymidine

NUMBER OF REPLICATIONS: two independent studies were performed. 5 replicate plates were used per study.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, cloning efficiency

Rationale for test conditions:

According to test guideline

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induced at least a three-fold increase in the mutation frequency compared to the solvent control in a dose-dependent manner; or
b) In case a positive result was repeated, the positive response should be reproducible in at least one repeated experiment
c) In addition, the observed increase should be biologically relevant and was compared with the

historical control data range.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutation frequency of at least three-fold compared to the solvent control.
b) The results were confirmed in an independently repeated test.

A test substance is considered equivocal (questionable) in the mutation assay if:
No clear conclusion for positive or negative result could be made after an additional confirmation study.

Statistics:

The experimental results were not subjected to statistical analysis.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN STUDY
- First mutagenicity test
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 850 µg/mL and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 950 µg/mL and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-Iocus were:
Without S9-mix: 100, 250, 400, 500, 600, 700 and 800 µg/mL exposure medium.
With 8% (v/v) S9-mix: 250, 500, 600, 700, 800, 850 and 900 µg/mL exposure medium.
In the absence of 89-mix, the relative total growth of the highest test substance concentration was reduced by 84% compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 92% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable
to the numbers of small and large colonies of the solvent controls. Fluctuations in the mutation frequency above the laboratory historical control data range were observed. However, since no dose-relationship was observed, the increases are not considered to be biologically relevant.

- Second mutagenicity test
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 750 µg/mL and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 100 to 600 µg/mL showed no cytotoxicity. Therefore, the dose levels of 100 and 250 µg/mL were not regarded relevant for mutation frequency measurement. The dose level of 800 µg/mL showed an unexplainable high relative survival growth compared to the dose levels of 750 and 850 µg/mL and was not regarded relevant for mutation frequency measurement. The dose levels of 900 µg/mL and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-Iocus were:
Without S9-mix: 100, 250, 400, 500, 550, 600, 650 and 700 µg/mL exposure medium.
With S9-mix: 400, 500, 550, 600, 650, 700, 750 and 850 µg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89% compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 90% compared to the total growth of the solvent controls.

Evaluation of mutagenicity
In the absence of S9-mix, the test substance did not induce an increase in the mutation frequency at the TK locus.
In the presence of S9-mix, the test substance induced an up to 3.2-fold increase in the mutation frequency at the TK locus. The test substance showed up to 3.0- and 2.9-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

-Third mutagenicity test
Evaluation of toxicity
The dose levels of 400 to 600 µg/mL showed no cytotoxicity. Therefore, the dose level of 400 µg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 900 µg/mL and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-Iocus were: 500, 600, 700, 735, 770, 800, 835 and 870 µg/mL exposure medium.
The relative total growth of the highest test substance concentration was reduced by 88% compared to the total growth of the solvent controls.

Evaluation of mutagenicity
The test substance induced an up to 2.0-fold increase in the mutation frequency at the TK locus.
The test substance showed up to 2.0- and 1.6-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

TEST-SPECIFIC CONFOUNDING FACTORS
The test substance precipitated in the exposure medium at the concentration of 2000 µg/mL. Since a concentration of 2000 µg/mL was equal to 0.01 M, this concentration was used as the highest test substance concentration for the dose range finding test.
The highest concentration, which did not precipitate in the exposure medium, was 1400 µg/mL. The pH and osmolarity of a concentration of 1400 µg/mL were 7.71 and 0.421 Osm/kg respectively (compared to 7.72 and 0.438 Osm/kg in the solvent control).

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 33 to 2000 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
In the absence and presence of S9-mix, no toxicity in the suspension growth was observed up to and including the test substance concentration of 333 µg/mL compared to the suspension growth of the solvent controls. Hardly or no cell survival was observed at test substance concentrations of 1000 and 2000 µg/mL.
The toxicity in the suspension growth was 21% at the test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentrations of 1000 and 2000 µg/mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Mutation frequencies in cultures treated with positive control chemicals were increased by 18- and 20-fold for MMS in the absence of S9-mix in the first and second experiment,
respectively, and by 27-, 19- and 20-fold for CP in the presence of S9-mix, in the first, second and third experiment, respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay

- Negative (solvent/vehicle) historical control data: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

Conclusions:

Under the conditions of the test , it is concluded that NETSA is not mutagenic in the mouse lymphoma L5178Y test system in the absence of S9-mix and equivocally mutagenic in the presence of S9-mix.

Executive summary:

Evaluation of the mutagenic activity of NETSA in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat) according to OECD TG 476. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone). To obtain more information about the possible mutagenicity of the test substance, an additional experiment was performed in the presence of 12% (v/v) S9-mix.

In the first experiment, the test substance was tested up to concentrations of 800 and 900 µg/mL in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. The test substance was tested up to cytotoxic levels of 84% in the absence of 89-mix and up to 92% in the presence of 89-mix.

In the second experiment, the test substance was tested up to concentrations of 700 and 850 µg/mL in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9 metabolic activation, respectively. The test substance was tested up to cytotoxic levels of 89% in the absence of 89-mix and up to 90% in the presence of S9-mix.

To verify the results obtained in the second experiment in the presence of S9-mix, a third experiment was performed. In this third experiment The test substance was tested up to concentrations of 870 µg/mL in the presence of 12% (v/v) S9-mix. The test substance was tested up to the cytotoxic level of 88%.

Mutation frequencies in cultures treated with positive control chemicals were increased by 18- and 20-fold for MMS in the absence of S9-mix in the first and second experiment, respectively, and by 27-, 19- and 20-fold for CP in the presence of S9-mix, in the first, second and third experiment, respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, The test substance showed fluctuations in the mutation frequency above the laboratory historical control data range in the first experiment. However, since no dose­ response relationship was observed and the mutation frequency was not above a factor 3 compared to the solvent control (up to 1.8-fold), a second experiment was performed with modifications in the concentration of the S9 for metabolic activation. The test substance induced an up to 3.2-fold increase in the mutation frequency at the TK locus. Verification of this result was performed in a third experiment, in which The test substance showed an up to 2.0-fold increase in the mutation frequency at the TK locus.

Although the test substance induced a more than three-fold increase in the mutation frequency in the second experiment in the presence of 12% (v/v) S9-mix, this increase was not observed in the first experiment in the presence of 8% (v/v) S9-mix (up to 1.8-fold) nor in the verification of this result in the third experiment (up to 2-fold). However, since the increases observed in the second and third experiment were outside our historical control data range but with no clear dose­ response relationship, the results obtained in this study were considered equivocal and the test substance is considered equivocally mutagenic in the presence of S9-mix.

Under the conditions of the test , it is concluded that NETSA is not mutagenic in the mouse lymphoma L5178Y test system in the absence of S9-mix and equivocally mutagenic in the presence of S9-mix. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Chromosomal aberrations in mammalian cells (Micronucleus test) ( OECD TG 474): negative

DNA repair assay rat hepatocyte (OECDTG 486): negative

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

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

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

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

Dose / conc.:

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

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:

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.

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)

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.

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.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 November 2009 to 12 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

WI (SPF)

Details on species / strain selection:

These rats are recommended by international guidelines (e.g. OECD, EC)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany.
- Age at study initiation: 7 or 8 weeks old at the start of treatment for the 12 - 16 hours and 2 - 4 hours sampling time respectively
- Weight at study initiation: 208 ± 21 g, the range: 177 - 236 g
- Assigned to test groups randomly: The animals were allocated at random to the treatment groups.
- Fasting period before study: For the animals of the 12 - 16 hours sampling time, feed was withheld 4 hours prior to dosing (late in the afternoon) until perfusion. The animals had free access to tap water. For the animals of the 2 - 4 hours sampling time, a limited quantity of food was supplied during the night before dosing (approximately 7 g/rat). The animals had free access to tap water.
- Housing:The animals were group housed (5 animals per cage) in labelled polycarbonate cages (type MIV height: 18 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). The animals were housed in individual treatment groups comprising 2 to 3 animals per group the day before dosing.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual range: 19.5 - 21.9)
- Humidity (%): 40 - 70 (actual range: 45 - 66)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s): Propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
NETSA was dissolved in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of 1.04 is g/mL. NETSA concentrations were dosed within 2.5 hours after preparation. Animals received a single dose. The dosing volume was 5 ml/kg body weight.

Duration of treatment / exposure:

2-4 hours and 12 -16 hours

Frequency of treatment:

Single dose

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

3 male rats were used per treatment time per sampling time, except for the vehicle and positive control groups. These groups consisted of 2 animals, since a significant historical database is available.

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

The positive control for the 2 - 4 h treatment time was dimethylnitrosamine (DMN; CAS-no. 62-75-9, purity 99%, Sigma- Aldrich Chemie GmbH); 10 mg/kg body weight, administered in milli-Q water (Millipore Corp., Bedford, MA, USA).
The positive control for the 12 - 16 h treatment time was 2-Acetylaminofluorene (2-AAF; CAS-no. 53-96-3, purity >99%, Acros Organics, Belgium); 50 mg/kg body weight, administered in corn oil (Roth, Karlsruhe, Germany).

Tissues and cell types examined:

liver, hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose range for the in vivo rat hepatocyte DNA-repair assay was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test. Two dose groups, one comprising of 1 male and the other comprising of 3 males received a single dose of NETSA. The group comprised of 3 males were dosed with the highest concentration that was used for the main study. The study duration was 3 (for the 500 mg/kg body weight group) or 2 days (for the 750 mg/kg body weight group). During this period mortality and physical condition were recorded.

TREATMENT AND SAMPLING TIMES:
Sampling time was 2-4 or 12-16 hours after dosing of the vehicle control or test substance. The sampling time for dimethylnitrosamine was 2-4 hours and for 2-Acetylaminofluorene 12-16 hours. Three male rats were used per sampling time in each treatment group, except for the vehicle and positive control group in which one animal was used per sampling time. Additional rats were exposed to vehicle and positive control for potential replacement in case the viability of the hepatocytes of these control rats would be below 50% (one rat per solvent or positive control group). In case the animals were not used for perfusion they were euthanized

DETAILS OF SLIDE PREPARATION:
- Liver perfusion: The liver was excised and transferred to a sterile petri dish and hepatocytes were isolated in a flow cabinet by cutting the Glisson capsules and shaking the liver in HBSS supplemented with 0.09% (w/v) NaHCO3 solution, 2.5x10^-3 mol.l-1 CaCl2 and 2.5% (w/v) Bovine Serum Albumin. In this way the tissue was dissociated into single cells. The obtained cell suspension was incubated at 37°C under 95 ± 0.5% O2 and 5 ± 0.5% CO2, in a humid atmosphere for 10 minutes. Dead cells, endothelial cells and Kupffer cells were removed by centrifugation (78 g), whereas living parenchymic liver cells, the hepatocytes, were retained.
- Cell culture
Primary cell culture conditions: Viability of the hepatocytes was determined using the trypan blue dye exclusion method. The hepatocytes were counted by using a microscope with an "Improved Neubauer" haemocytometer (Merck, The Netherlands) and 10^5 cells were seeded on 1 cm2 cover slips in wells of a 24-well dish. Four cover slips were prepared per animal.
Culture medium: The culture medium consisted of Williams E culture medium (Invitrogen Corporation) supplemented with fetal calf serum (10%, Invitrogen Corporation), L-glutamine (2 x 10^-3 mol.l-1, Invitrogen Corporation) and gentamycin (50 μg/mL, Sigma).
Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity. The deviation exceeding 1 hour is described in protocol deviation 1.
- Labelling of the cells in culture: The cultures were washed once with culture medium to remove unattached cells and debris 1 - 2 hours after seeding of the cells. Subsequently, 0.5 ml of culture medium supplemented with tritiated thymidine solution (3H-TdR; 370 KBq/mL (10 μCi/ml); specific activity 666 - 1110 GBq/mol (18 - 30 x 10^3 Ci/mol), Amersham Biosciences Benelux, Roosendaal, The Netherlands) was added to the cells. The cultures were incubated for 4 hours at 37°C (5.0 ± 0.5% CO2 in air). After incubation the cultures were washed once with culture medium and 1.0 ml of culture medium containing 0.25 x 10^-3 M unlabelled thymidine (Sigma, Zwijndrecht, The Netherlands) solution was added to each well to diminish unincorporated radioactivity. Cultures were incubated overnight (14 - 18 h). Thereafter the cultures were washed with HBSS and fixed with methanol-acetic acid (3:1 v/v, Merck).
- Autoradiographic procedure: After fixation of the cells the coverslips were mounted on microscopic slides. These slides were dipped in Ilford K5D emulsion (Ilford Imaging, Cheshire, UK) at 40 - 45°C and dried for 2 h at room temperature in the dark. After drying, the slides were placed in light tight boxes in the presence of silica gel. The photographic emulsion was exposed for 7 days at 4°C. The emulsion was developed for 4 min in Kodak D19 developer (Kodak, Rochester, USA) at 15°C, rinsed in Milli-RO water (Millipore Corp., Bedford, Mass., USA) and fixed for 5 min in Kodak fixative (Kodak). The slides were rinsed with running tap water and the cells were stained with haematoxylin/eosin (both Klinipath, Duiven, The Netherlands). At least 3 scorable coverslips per animal were subjected to the autoradiographic procedure.

METHOD OF ANALYSIS:
- Scoring: The slides were checked for the presence of sufficient cells of normal morphology to permit a meaningful assessment of UDS. Preparations were examined microscopically for signs of overt cytotoxicity (e.g. pyknosis). Slide evaluation was performed without knowledge of treatment schedule. Grain counts were determined over the nuclei (nuclear grains, N) and the nucleus-equivalent areas over the cytoplasm (cytoplasmic grains, C) manually on the coverslips. Fifty cells were counted on each coverslip, 2 coverslips per animal (in total 100 cells per animal) and 3 animals per treatment group were examined (except for the negative and positive controls where 1 animal was examined). The following criteria were used to determine if a cell was countable.
1) Cells with abnormal morphology, such as those with pyknotic or lysed nuclei, were not counted.
2) Isolated nuclei not surrounded by cytoplasm were not counted.
3) Cells with unusual staining artifacts or in the presence of debris were not counted.
4) Heavily labelled cells in S-phase were not counted.
The normal cells observed while moving the microscope stage were counted. Averages and standard deviations were calculated. Grain counts over nuclear areas (N) were compared to grain counts over the most heavily labelled adjacent cytoplasm area (C) of the same size as the corresponding nuclear area (Lonati-Galliani et al., 1983). The net nuclear grain count (NNG) was calculated for each cell by subtracting cytoplasmic grain counts from nuclear grain counts. The background counts of a single area of the same size as the corresponding nuclear area was recorded per coverslip.

OTHER:
Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA), Temperature and humidity.

Evaluation criteria:

A test substance is considered positive in the DNA-repair assay if it yields greater than or equal to 5 net nuclear grain (NNG) counts per group average, and greater than or equal to 20% of the cells responding (i.e. with NNG values of 5 or more). These values are based on the laboratory historical data. A test substance is considered negative in the DNA-repair assay if it does not induce a net nuclear grain (NNG) count ≥ 5 and ≥ 20% of the cells responding. In addition to the above-mentioned criteria, biological relevance of the data was also considered, i.e. parameters such as inter-animal variation, dose response relationship and cytotoxicity were taken into account. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

slide preparations showed no or slight overt cytotoxicity.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a dose range finding study 4 animals (group A: 1 male, group B: 3 males) were dosed orally with 500 and 750 mg/kg body weight (groups A and B, respectively) of NETSA. The animal dosed with 500 mg/kg body weight was lethargic within 1 hour after dosing. The animal was still lethargic within 4 hours after dosing. The animal recovered from the treatment within 21 hours after dosing. Three animals dosed with 750 mg/kg body weight showed the following clinical signs within 1 hour after dosing: ataxia (2 animals), lethargy (3 animals), no reaction to a stimulus (1 animal) and ventro-lateral recumbency (1 animal). Within 5 hours after dosing the animals showed the following toxic signs: ataxia (2 animals), lethargy (3 animals), no reaction to a stimulus (1 animal), ventro-lateral recumbency (1 animal) and rales (1 animal). All animals recovered within 26 hours after dosing. One day after exposure a pilot perfusion was performed in two of the animals dosed with 750 mg NETSA/kg body weight to identify any adverse effects on the liver at this dose level that could interfere with a good performance of the main experiment. No macroscopic effects in the liver were observed and the viability of the liver cells was 82 and 78%, indicating that hepatotoxicity would not adversely influence the performance of the in vivo rat hepatocyte DNA-repair assay.

RESULTS OF DEFINITIVE STUDY
Based on the results of the dose range finding study and the pilot perfusion dose levels of 750 and 375 mg/kg body weight were selected as appropriate doses for the in vivo rat hepatocyte DNA-repair assay. Three male rats were used per sampling time in each treatment group, except for the vehicle and positive control group in which one rat was used per sampling time.
- Mortality / signs of toxicity: The animals of the negative and positive control groups showed no treatment related clinical signs or mortality. The following clinical observations were made in the groups treated with 750 and 375 mg NETSA/kg body weight (2 - 4 hours treatment time): Within 1.5 hour after dosing all animals dosed with 750 and 375 mg/kg body weight were lethargic, 5 animals showed ataxia and the other animal dosed with 750 mg/kg body weight showed ventro-lateral recumbency. Just prior to perfusion all animals still showed ataxia, two animals dosed with 750 mg/kg body weight and two animals dosed with 375 mg/kg body weight also were lethargic. The following clinical observations were made in the groups treated with 750 and 375 mg NETSA/kg body weight (12 - 16 hours treatment time): Within 10 minutes after dosing all animals treated with 750 and 375 mg/kg body weight showed ataxia and one animal dosed with 750 mg/kg body weight was also lethargic. Just prior to perfusion no treatment related clinical signs were observed.
- Viability of the hepatocytes: At the 2 - 4 hour sampling time, the viability of the hepatocytes, used for the evaluation of DNA repair inducing ability of NETSA was at least 75% indicating no direct liver toxicity. A viability of 75% was found for the vehicle control culture. At the 12 - 16 hour sampling time, the viability of the hepatocytes, used for the evaluation of DNA repair inducing ability of NETSA was at least 76% indicating no direct liver toxicity. A viability of 82% was found for the vehicle control culture.
- Results DNA repair assay: In all slides used for grain counts sufficient cells of normal morphology to permit a meaningful assessment of unscheduled DNA-synthesis were present. Preparations showed no or slight overt cytotoxicity (e.g. pyknosis ≤ 50%).
As a result of oral dosing with NETSA the NNG per coverslip and per animal, as well as the group average revealed no positive response in this assay at any of the dose levels. The percentage of cells in repair (repair taken as NNG > 5), both per individual animal and for the group average, revealed no increase at any dose.

Acceptability:
The NNG in the solvent-treated control cultures was within the historical control data range.
Oral dosing of a male rat with dimethylnitrosamine (DMN) resulted in a NNG of 32.2 with 100% of the cells in repair (NNG > 5). Oral dosing of a male rat with 2-acetylaminofluorene (2-AAF) resulted in a net nuclear grain count (NNG) of 26.6 with 100% of the cells in repair (NNG > 5). In the scored coverslips the mean background of a single area of the same size as the corresponding nuclear area, located outside the cytoplasm, was always less than 11 grains, indicating that the autoradiographic procedure functioned adequately. It can be concluded that the test system was functioning correctly.

Conclusions:

When treated orally with NETSA at doses up to 750 mg/kg body weight (maximum tolerated dose) male Wistar rats showed no induction of DNA repair in hepatocytes isolated 2 - 4 or 12 - 16 hours after dosing, respectively. Under the conditions of the test, NETSA was not genotoxic in the DNA repair assay using hepatocytes obtained from male rats following in vivo exposure to the test substance.

Executive summary:

The DNA repair inducing ability of NETSA was investigated in male Wistar rat hepatocytes, measured as unscheduled DNA synthesis (UDS) according to OECD TG 486. In the dose range finding study 1 male was dosed by oral gavage with 500 mg NETSA per kg body weight. The animal was only lethargic after dosing. Three males were dosed by oral gavage with 750 mg NETSA per kg body weight. The animals showed the following toxic signs after dosing: lethargy, ataxia (2 animals), ventro-lateral recumbency (1 animal), no reaction to a stimulus (1 animal) and rales (1 animal). One day after exposure a pilot perfusion was performed with two animals dosed with 750 mg/kg body weight. The viability of the hepatocytes was within the normal range (78% and 82%). Two groups of 3 male Wistar rats received a single oral dose of 750 or 375 mg NETSA per kg body weight. At the 2 - 4 hours treatment time, the animals treated with 750 and 375 mg/kg body weight were lethargic, showed ataxia and one animal dosed with 750 mg/kg body weight showed ventro-lateral recumbency. At the 12 - 16 hours treatment time, the animals treated with 750 and 375 mg/kg body weight showed ataxia and one of the animals dosed with 750 mg/kg body weight was also lethargic. Hepatocytes from 3 rats per group were sampled 2 - 4 or 12 - 16 hours after dosing, and evaluated for the occurrence of unscheduled DNA synthesis, as measured by nuclear grains. Two slides per animal and 3 animals for each treatment group were examined for the presence of nuclear grains in the hepatocytes. Corresponding vehicle treated groups served as negative controls (vehicle was propylene glycol).

Hepatocytes of positive control animals treated with single oral doses of dimethylnitrosamine (DMN, 10 mg/kg body weight) or 2-acetylaminofluorene (2-AAF, 50 mg/kg body weight) were harvested 2 - 4 or 12 - 16 hours after dosing respectively. No treatment related clinical signs or mortality were noted in control animals. Two slides per animal and one animal for each control group were examined. As a result of oral dosing with NETSA the net nuclear grain count (NNG) per slide and per animal, as well as the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as NNG > 5), both per individual animal and for the group average, revealed no significant increase at any dose. The results of the negative and positive controls were within the expected range. Therefore, it can be concluded that the test system functioned properly. When treated orally with NETSA at doses up to 750 mg/kg body weight (maximum tolerated dose) male Wistar rats showed no induction of DNA repair in hepatocytes isolated 2 - 4 or 12 - 16 hours after dosing, respectively.

Under the conditions of the test, NETSA was not genotoxic in the DNA repair assay using hepatocytes obtained from male rats following in vivo exposure to the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in bacteria (Ames) (OECDTG 471)

The mutagenic activity of Santicizer 8 was conducted based on test procedures described by Ames et al (1975) according to OECDTG 471 . Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with the test item using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of S9-mix. Adequate negative and positive controls were included. Only four strains of bacteria were used, none of which would be able to detect certain oxidizing mutagens, cross- linking agents and hydrazines as detected by E.Coli WP2 strains or s.typhimurium TA102.

The test material, Santicizer 8, was not mutagenic towards salmonella typhimurium test strains TA98, TA100, TA1535 or TA1537 in the plate incorporation assays conducted with and without a rat microsomal activation system. A maximum of 10 mg per plate was used in the plate incorporation tests. Levels of 10 mg per plate were toxic in the plate incooperation test in the presence and absence of a rat microsomal activation system, whereas the lower concentrations tested were not. The plate incorporation test results indicated no significant mutagenic activity for Santicizer 8, and was therefore considered to be non-mutagenic under the conditions of this test.

Chromosome abberation in human lymphocytes (OECDTG 473)

The ability of NETSA to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment) was examined according to OECD TG 473 In the first cytogenetic assay, NETSA was tested up to 1600 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence of S9-mix. NETSA precipitated in the culture medium at this dose level and appropriate toxicity was reached. In the presence of 1.8% (v/v) S9-fraction, NETSA was tested up to 1400 µg/mL for a 3 h exposure time with a 24 h fixation time. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, NETSA was tested up to 500 µg/mL for a 24 h and 48 h continuous exposure time with a 24 hand 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. In the presence of S9-mix NETSA was tested up to 1600 µg/mL for a 3 h exposure time with a 48 h fixation time. NETSA precipitated in the culture medium at this dose level. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the first cytogenetic assay in the absence of S9-mix and in the second cytogenetic assay in the absence and presence of S9-mix NETSA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. In the first cytogenetic assay in the presence of S9-mix, no increase in the number of cells with chromosome aberrations was observed at concentration levels up to and including 1200 µg/mL. Only at the highest tested cytotoxic concentration of 1400 µg/mL, a statistically significant relevant increase in the number of cells with chromosome aberrations was observed, both when gaps were included and excluded. Although the number of cells with chromosome aberrations was just above the historical control data range pronounced exchange figures were observed at this concentration and the increase was considered biologically relevant. It was noted that NETSA increased the number of polyploid cells in the absence of S9-mix in the first cytogenetic assay. This may indicate that NETSA has the potential to disturb mitotic processes and cell cycle progression.

Finally, it is concluded that this test is valid and that NETSA is not clastogenic in human lymphocytes in the absence of S9-mix.

NETSA was found to be clastogenic in human lymphocytes in the presence of S9-mix in one of the two experiments. The clastogenic activity is confined only to high and cytotoxic concentrations.

Gene mutation in mammalian cells (Mouse Lymphoma Assay) (OECDTG 476)

Evaluation of the mutagenic activity of NETSA in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat) according to OECDTG 476. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone). To obtain more information about the possible mutagenicity of the test substance, an additional experiment was performed in the presence of 12% (v/v) S9-mix.

In the first experiment, the test substance was tested up to concentrations of 800 and 900 µg/mL in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. The test substance was tested up to cytotoxic levels of 84% in the absence of 89-mix and up to 92% in the presence of 89-mix.

In the second experiment, the test substance was tested up to concentrations of 700 and 850 µg/mL in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9 metabolic activation, respectively. The test substance was tested up to cytotoxic levels of 89% in the absence of 89-mix and up to 90% in the presence of S9-mix.

To verify the results obtained in the second experiment in the presence of S9-mix, a third experiment was performed. In this third experiment The test substance was tested up to concentrations of 870 µg/mL in the presence of 12% (v/v) S9-mix. The test substance was tested up to the cytotoxic level of 88%.

Mutation frequencies in cultures treated with positive control chemicals were increased by 18- and 20-fold for MMS in the absence of S9-mix in the first and second experiment, respectively, and by 27-, 19- and 20-fold for CP in the presence of S9-mix, in the first, second and third experiment, respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, The test substance showed fluctuations in the mutation frequency above the laboratory historical control data range in the first experiment. However, since no dose­ response relationship was observed and the mutation frequency was not above a factor 3 compared to the solvent control (up to 1.8-fold), a second experiment was performed with modifications in the concentration of the S9 for metabolic activation. The test substance induced an up to 3.2-fold increase in the mutation frequency at the TK locus. Verification of this result was performed in a third experiment, in which The test substance showed an up to 2.0-fold increase in the mutation frequency at the TK locus.

Although the test substance induced a more than three-fold increase in the mutation frequency in the second experiment in the presence of 12% (v/v) S9-mix, this increase was not observed in the first experiment in the presence of 8% (v/v) S9-mix (up to 1.8-fold) nor in the verification of this result in the third experiment (up to 2-fold). However, since the increases observed in the second and third experiment were outside our historical control data range but with no clear dose­ response relationship, the results obtained in this study were considered equivocal and the test substance is considered equivocally mutagenic in the presence of S9-mix.

Under the conditions of the test , it is concluded that NETSA is not mutagenic in the mouse lymphoma L5178Y test system in the absence of S9-mix and equivocally mutagenic in the presence of S9-mix. 

Chromosomal aberrations in mammalian cells (Micronucleus test) ( OECD TG 474)

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.

DNA repair assay rat hepatocyte (OECDTG 486)

The DNA repair inducing ability of NETSA was investigated in male Wistar rat hepatocytes, measured as unscheduled DNA synthesis (UDS) according to OECD TG 486. In the dose range finding study 1 male was dosed by oral gavage with 500 mg NETSA per kg body weight. The animal was only lethargic after dosing. Three males were dosed by oral gavage with 750 mg NETSA per kg body weight. The animals showed the following toxic signs after dosing: lethargy, ataxia (2 animals), ventro-lateral recumbency (1 animal), no reaction to a stimulus (1 animal) and rales (1 animal). One day after exposure a pilot perfusion was performed with two animals dosed with 750 mg/kg body weight. The viability of the hepatocytes was within the normal range (78% and 82%). Two groups of 3 male Wistar rats received a single oral dose of 750 or 375 mg NETSA per kg body weight. At the 2 - 4 hours treatment time, the animals treated with 750 and 375 mg/kg body weight were lethargic, showed ataxia and one animal dosed with 750 mg/kg body weight showed ventro-lateral recumbency. At the 12 - 16 hours treatment time, the animals treated with 750 and 375 mg/kg body weight showed ataxia and one of the animals dosed with 750 mg/kg body weight was also lethargic. Hepatocytes from 3 rats per group were sampled 2 - 4 or 12 - 16 hours after dosing, and evaluated for the occurrence of unscheduled DNA synthesis, as measured by nuclear grains. Two slides per animal and 3 animals for each treatment group were examined for the presence of nuclear grains in the hepatocytes. Corresponding vehicle treated groups served as negative controls (vehicle was propylene glycol). Hepatocytes of positive control animals treated with single oral doses of dimethylnitrosamine (DMN, 10 mg/kg body weight) or 2-acetylaminofluorene (2-AAF, 50 mg/kg body weight) were harvested 2 - 4 or 12 - 16 hours after dosing respectively. No treatment related clinical signs or mortality were noted in control animals. Two slides per animal and one animal for each control group were examined. As a result of oral dosing with NETSA the net nuclear grain count (NNG) per slide and per animal, as well as the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as NNG > 5), both per individual animal and for the group average, revealed no significant increase at any dose. The results of the negative and positive controls were within the expected range. Therefore, it can be concluded that the test system functioned properly. When treated orally with NETSA at doses up to 750 mg/kg body weight (maximum tolerated dose) male Wistar rats showed no induction of DNA repair in hepatocytes isolated 2 - 4 or 12 - 16 hours after dosing, respectively. Under the conditions of the test, NETSA was not genotoxic in the DNA repair assay using hepatocytes obtained from male rats following in vivo exposure to the test substance.

Justification for classification or non-classification

Based on the available data, substance N-ethyl-o (or p)-toluenesulfonamide (NETSA) does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).