1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was found to cause reverse mutations in bacteria.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 1999 to 25 February 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: FAT 93460/A
- Source and lot/batch No.of test material: EN-No.: 037098 A7
- Expiration date of the lot/batch: November, 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test substance in the solvent/vehicle: at least 24 hours in water, PEG 400, saline, FCA/NaCl-solution, and CMC-solution

OTHER SPECIFICS:
- Aggregate state at room temperature: Solid
- Colour: Yellow
- Purity: 23 % active ingredient

Target gene:

Histidine requiring genes of Salmonella strains

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
Precultures; From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µL ampicillin (25 µg/ml) was added to the strains TA 98, and TA 100.
This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 8 hours at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Füllinsdorf; weight approx. 220 - 320 g)
- method of preparation of S9 mix: Rats received daily applications of 80 mg/kg b.w. Phenobarbital i.p. dissolved in deionised water (Desitin; D-22335 Hamburg) and ß-Naphthoflavone orally dissolved in corn oil (Aldrich, D-89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment. After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use. The protein content was determined using an analysis kit of Bio-Rad Laboratories, D-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 28.3 mg/ml (lot no. 271198) in the preexperiment and in experiment I, and 50.4 mg/ml (lot no. 181298) in experiment I and II.

- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the cultures. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix:
8mM MgCl2
33 mM KCl
5mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient)

Vehicle / solvent:

DMSO (purity >99 %, MERCK, D-64293 Darmstadt)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolism: TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolism: TA 1537 and TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation: TA 1535, TA 1537, TA 98, TA 100

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration Triplicate
- Number of independent experiments
: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium, Plate incorporation assay.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
The bacterial cultures were incubated in a shaking water bath for 8 hours at 37 °C.

FOR GENE MUTATION:
- Method used: Agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, and TA 100 or thrice in strains TA 1535 and TA 1537 (3, 4). Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless of whether the highest dose induced the criteria described above or not.

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test article producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system. A biologically relevant response is described as follows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, and TA 100 or thrice in strains TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Test article concentration per plate µg: 3, 9, 31, 92, 307, 920, 2300, 4600.
The plates with the test article showed normal background growth up to 4600 µg/plate in strain TA 98 and TA 100.

- No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

- The plates incubated with the test article showed normal background growth up to 4600 µg/plate with and without S9 mix in all strains used.

- Substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and thrice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the following concentrations:

Strain	Experiment I (μg/plate)		Experiment II (μg/plate)
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	>307	> 307	>307	>307
TA 1537	>307	> 920	>307	> 92
TA 98	>920	> 31	> 92	> 31
TA 100	>920	>2300	>920	>920

Positive control substances showed a distinct increase in induced revertant colonies, thereby validating the test.

Conclusions:

FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was performed according to OECD test guideline 471 and EU Method B.13/14 to investigate the potential of FAT 93460/A to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 4600 μg/plate with and without S9 mix in all strains used. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and trice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the concentrations. Based on the findings of the study, it can be concluded that the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance did not cause clastogenic effects in a micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test start date: March 27, 2001; Test end date: 24 April 2001; Study completion date: July 17, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material identified as: FAT 93529/A
- Source and batch No.of test material: 1-5 / 2000 laborgetrocknet
- Expiration date of the batch: March 2006

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: 24 hours in water

OTHER SPECIFICS:
- Aggregate state at room temperature: solid
- Colour: yellow
- Purity: technical purity

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: NMRI
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Number of animals: 72 (36 males/36 females)
- Initial age at start of acclimatization: 8-10 weeks
- Acclimatization: minimum 5 days
- Initial body weight at start of treatment: males mean value 33.6 g (SD ± 2.3 g); females mean value 25.5 g (SD ± 2.3 g)

Husbandry:
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Bedding: Granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Feed: Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Environment: temperature 21 ± 4 °C
- Relative humidity 30-70 %
- Artificial light: 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in corn oil.

- All animals received a single standard volume of 20 mL/kg bw orally.

Duration of treatment / exposure:

The animals received the test article, the vehicle or the positive control substance once.

Frequency of treatment:

Single treatment

Post exposure period:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

24 hour interval only

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

24 hour interval only

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 and 48 hour interval

No. of animals per sex per dose:

6 male and 6 female rats

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Justification for choice of positive control(s): Recommended by the guidelines
- Route of administration: Oral
- Doses : 40 mg/kg b.w.
- Frequency: Once

Tissues and cell types examined:

The animals were sacrificed by cervical dislocation: The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Details of tissue and slide preparation:

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated.

Evaluation criteria:

- A test article is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
- A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
- Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results.
- However, the primary point of consideration is the biological relevance of the results.

Statistics:

Non parametric Mann-Whitney test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: A single dose of 2000 mg/kg bw was used
- Clinical signs of toxicity in test animals: The treated animals expressed no toxic reactions. The urine color was orange at 2-4 hours after treatment, while 6 hours after treatment, it was yellow orange.
- Rationale for exposure: Highest (limit dose) was used as recommended by the guideline.


RESULTS OF DEFINITIVE STUDY
- The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 93'529/A had no cytotoxic properties in the bone marrow. However, the urine colour of the treated animals had taken on the colour of the test item indicating the systemic distribution of the test item and its bioavailability in the target organ.

- In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 93'529/A were below or near to the value of the vehicle control group.

- 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusions:

The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The test substance FAT 93529/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The test substance was formulated in corn oil. Corn oil was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw, 48 h preparation interval: 2000 mg/kg bw. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 93529/A had no cytotoxic effectiveness in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating the systemic distribution and the bioavailability of the test item in the target organ. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity (in vitro): bacetrial reverse mutation assay

A study was performed to investigate the potential of FAT 93460/A to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 31; 92; 307; 920; 2300; and 4600 μg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revenants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 4600 μg/plate with and without S9 mix in all strains used. Substantial and dose dependent increases in revenant colony numbers were observed following treatment with FAT 93460/A in all strains with and without metabolic activation in experiment I and II. The required threshold of twice (strains TA 98 and TA 100) and trice (TA 1535 and TA 1537) the number of the corresponding solvent control was reached or exceeded at the concentrations. Based on the findings of the study, it can be concluded that the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, FAT 93460/A is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

 

Genetic toxicity (in vivo): Micronucleus assay

The test substance FAT 93529/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 and EU Method B.12. The test substance was formulated in corn oil. Corn oil was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw, 48 h preparation interval: 2000 mg/kg bw. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 93529/A had no cytotoxic effectiveness in the bone marrow. However, the urine of the treated animals had taken over the colour of the test item indicating the systemic distribution and the bioavailability of the test item in the target organ. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Based on the above data, it can be concluded that the substance induced reverse mutations in bacteria, while it had no clastogenic effect in an in vivo micronucleus assay.

Justification for classification or non-classification

Based on the available database of genetic toxicity studies, the substance does not warrant classification according to CLP (Regulation 1272/2008) criteria.