Chlorothalonil

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +S9 negative , -S9 negative, S. typhimurium: TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA pKM101 and WP2 pKM101, OECD 471, Chang 2017

- in vitro mammalian cell gene mutation test, +S9 negative, -S9 negative, Chinese hamster lung fibroblasts (V79) and Mouse BALB/3T3 cells, No guideline, Kouri 1977

- in vitro chromosome aberration test, +S9 negative, -S9 positive, Chinese hamster ovary cells, OECD 473, Mizens 1988

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mammalian cell mutagenesis tests with Chinese hamster cells and/or mouse fibroblasts are performed according to the method of Schechtman et al. Ouabain is used as the selective agent to detect point mutations in treated cells. This mutant cell is resistant to ouabain and hence forms mutant colonies in the presence of ouabain. Plates, with treated cells are incubated in ouabain containing medium for 14 days for Chinese hamster cells and 25 days for mouse fibroblasts. Ouabain resistant mutant colonies are counted and compared with controls.

GLP compliance:

no

Type of assay:

other: In vitro mammalian cell gene mutation test using the ouabain resistant locus

Target gene:

Ouabain resistant locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The growth medium (Microbiological Associates) was Eagle’s minimal essential medium (MEM) supplemented with L-glutamine (2 mM), nonessential amino acids (0.1 mM), penicillin (100 units/mL), streptomycin (100 mg/mL) and 5% heat-inactivated fetal bovine serum.

Species / strain / cell type:

other: Mouse BALB/3T3

Details on mammalian cell type (if applicable):

The growth medium (Microbiological Associates) was Eagle’s minimal essential medium (MEM) supplemented with L-glutamine (2 mM), nonessential amino acids (0.1 mM), penicillin (100 units/mL), streptomycin (100 mg/mL) and 10% heat-inactivated fetal bovine serum.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- method of preparation of S9 mix: Aroclor 1254 (500 mg/kg body weight) was inoculated intraperitoneally (IP) in male Fischer 344 rats and 48 hours later animals were sacrificed, livers were excised, maintained sterile at 4°C, rinsed free of excess blood with 0.15 M KCl buffer, and minced and homogenized at 30% w/v in 0.15 M KCl. The homogenate was centrifuged at 9000 x g for 20 minutes at 4°C. This post-mitochondrial enzyme preparation (S-9) was stored frozen in aliquots at 80°C.
- Biochemical properties of subcellular enzyme fractions (S-9) were determined as follows. Aryl hydrocarbon hydroxylase (AHH) activity which follows the conversion of BP to the phenol 3-OH BP was measured according to the procedures of Nebert and Gelboin (2). Protein determination was made according to the procedure of Lowry.

Test concentrations with justification for top dose:

Main test in presence of S9: 0.3 µg/mL
Main test in absence of S9: 0.03 and 0.3 µg/mL

Vehicle / solvent:

- Solvent used: Acetone
- Final concentration: 2.5 µL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: MNNG

Details on test system and experimental conditions:

Exponentially growing Chinese hamster lung fibroblasts (V-79) and mouse BALB/3T3 clone A31-1 were grown in 25 cm2 flasks and cells were collected by trypsinization. One to 2 x 10^6 cells were suspended in 4 mL whole medium and various concentrations of test chemical were added to the tube. Cell suspensions in the presence of test chemical were incubated with continuous gentle agitation at 37°C for 2 hours in a humidified atmosphere of 5% CO2 in air. The reaction was stopped by centrifugation (1000 x g, 4°C, 10 minutes). Cells were fixed and stained 8-10 days later with Giemsa stain.

When no activation was required, cells were collected in 4 mL whole medium and approximately LD50 concentrations of test chemical were added. MNNG was used as the positive control. Cell suspension was incubated at 37°C for 2 hours in a humidified atmosphere of 5% CO2 in air. The reaction was stopped by centrifugation (100 x g, 4°C, 10 minutes). Cells were seeded in 75 cm2 Falcon flasks and grown for 48 hours (V-79 cells), or 96 hours (3T3 cells) to allow for expression of the mutagenic event. Cells were then collected by trypsinization and plated at a density of 1 x 10^5 cells/dish (for V-79) or 2 x 10^5 cells/dish (for 3T3) for selection of mutant colonies resistant to 1 mM ouabain according to the procedure of Huberman. Plating efficiency assays were run concomitantly in order to determine the number of cells at risk.

For in vitro metabolic activation, 1-2 x 10^6 cells were suspended in a 4 mL reaction pool consisting of phosphate-buffered saline, pH 7.6, 1.8 mM NADPH , 1.4 mM NADH, 7.4 mM glucose-6-phosphate and 1.9 mM NADP. To this was added 100 µL S-9 + test chemical at a dose determined from LD50 cytotoxicity. The reaction mixture was incubated with continuous gentle agitation at 37°C for 2 hours in humidified atmosphere of %. CO2 in air. Positive control was + BP (12.5 µL/mL) and 100 µL S-9. The reaction was stopped by centrifugation (1000 x g,4°C, 10 minutes). AHH activity was measured in the supernatant fluids. Cells were seeded for expression time as previously described. Selection of mutant colonies resistant to 1 mM ouabain was according to the procedure described above. Cells were fixed and stained after 7-9 days for plating efficiency (cells at risk) or 14 days (V-79 mutagenesis ) and 25 days (3T3 mutagenesis), and the number of surviving. colonies in each dish determined .

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

other: BALB/3T3, Clone A31

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The cytotoxicity in the preliminary test in V79 cells was determined at concentrations of 30, 10, 3, 1 and 0.3 µg/mL. The test substance is toxic, even at 0.3 µg/mL. Mutation frequency in the presence of test chemical is not significant even though the spontaneous mutation frequency is ten times higher than it should be. Addition of an exogenous source of liver enzyme (i.e., S-9) did not affect the ability of the test agent to increase the frequency of ouabain resistant mutation. As mentioned in an earlier report, the spontaneous mutation frequency is much too high in this specific clone of V-79, the studies were repeated using a different cell line, namely BALB/ 3T3 clone A31-1.

It was observed that even at 0.03 µg/mL with 2 hours exposure there was approximately 33% death. This indicates that BALB/ 3T3 was about 10 times more sensitive to the cytotoxic effects of the test substance than were the V-79 cells.

At 0.03 µg/mL, the test substance was not effective in inducing ouabain resistant mutants without activation using BALB/3T3 as the target cells. BP was used as the positive control. BP, in the presence of exogenous mammalian S-9 enzyme fraction was converted to 11.5 nM of 3-OH BP in 2 hours incubation. This activating system was effective in inducing mutation frequency of 17.1 x 10^-6. The test chemical failed to increase the mutation frequency above the spontaneous level.

It must be pointed out that the presence of the S-9 preparation allowed us to increase the concentration of the test substance that could be evaluated. A concentration of 0.3 µg/mL (10 times higher than could be used without activation or in the presence of heat inactivated S- 9) was evaluated and yet this higher dose still failed to significantly increase the mutation frequency at the ouabain resistant locus.

Conclusions:

Under the conditions of this bioassay, the test substance did not induce point mutations at the ouabain resistant locus in both V-79 clone 8 Chinese hamster cells or BALB/3T3 clone A subline 1 cells either with or without the addition of an exogenous source of liver enzyme (S-9) for metabolic activation of the test agent.

Executive summary:

Mammalian cell mutagenesis tests with Chinese hamster cells and/or mouse fibroblasts are performed according to the method of Schechtman et al. Ouabain is used as the selective agent to detect point mutations in treated cells. This mutant cell is resistant to ouabain and hence forms mutant colonies in the presence of ouabain. Plates, with treated cells are incubated in ouabain containing medium for 14 days for Chinese hamster cells and 25 days for mouse fibroblasts. Ouabain resistant mutant colonies are counted and compared with controls. Positive controls and negative controls are included in each experiment. N-methyl-N-nitro-N-nitrosoguanidine (MNNG) serves as the positive control without activation and Benzo (a) pyrene (BP) serves as the positive control with activation. Acetone is the negative control without activation and S-9 alone, heat-inactivated S-9 + BP, are the negative controls without activation.

The cytotoxicity in the preliminary test in V79 cells was determined at concentrations of 30, 10, 3, 1 and 0.3 µg/mL. The test substance is toxic, even at 0.3 µg/mL. Mutation frequency in the presence of test chemical is not significant even though the spontaneous mutation frequency is ten times higher than it should be. Addition of an exogenous source of liver enzyme (i.e., S-9) did not affect the ability of the test agent to increase the frequency of ouabain resistant mutation. As mentioned in an earlier report, the spontaneous mutation frequency is much too high in this specific clone of V-79, the studies were repeated using a different cell line, namely BALB/ 3T3 clone A31-1.

It was observed that even at 0.03 µg/mL with 2 hours exposure there was approximately 33% death. This indicates that BALB/ 3T3 was about 10 times more sensitive to the cytotoxic effects of the test substance than were the V-79 cells.

At 0.03 µg/mL, the test substance was not effective in inducing ouabain resistant mutants without activation using BALB/3T3 as the target cells. BP was used as the positive control. BP, in the presence of exogenous mammalian S-9 enzyme fraction was converted to 11.5 nM of 3-OH BP in 2 hours incubation. This activating system was effective in inducing mutation frequency of 17.1 x 10^-6. The test chemical failed to increase the mutation frequency above the spontaneous level.

It must be pointed out that the presence of the S-9 preparation allowed us to increase the concentration of the test substance that could be evaluated. A concentration of 0.3 µg/mL (10 times higher than could be used without activation or in the presence of heat inactivated S- 9) was evaluated and yet this higher dose still failed to significantly increase the mutation frequency at the ouabain resistant locus.

Under the conditions of this bioassay, the test substance did not induce point mutations at the ouabain resistant locus in both V-79 clone 8 Chinese hamster cells or BALB/3T3 clone A subline 1 cells either with or without the addition of an exogenous source of liver enzyme (S-9) for metabolic activation of the test agent.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Oct 2016 to 10 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

E. coli, other: WP2 pKM101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/ß-naphthoflavone induced rat liver
- method of preparation of S9 mix: The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week.
- quality controls of S9: Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo[a]pyrene

Test concentrations with justification for top dose:

Strains TA1537 and WP2 uvrA pKM101: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Strains TA1535, TA98, TA100, and WP2 pKM101: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Vehicle / solvent:

- Solvent used: DMSO (purity > 99% )
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: Two, plate incorporation and pre-incubation experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).
- Test substance in the plate (experiment I, plate incorporation) or in suspension (experiment II, with preincubation)

TREATMENT SCHEDULE:
- Pre-incubation period: For the pre-incubation method test solution (100 µL) (solvent or reference mutagen solution (positive control)), S9 mix / S9 mix substitution buffer* (500 µL) and bacteria suspension (100 µL) were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Treatment: After preincubation overlay agar (2.0 mL, 45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for 72 hours at 37°C in the dark, plates were then stored at 4°C until counted.

FOR GENE MUTATION:
- Selection time: 72 h
- Method used: agar
- If a selective agent is used: selective agar (without Histidine/Tryptophan)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test). Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
- Pre-experiment: In the pre-experiment the concentration range of the test substance was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since cytotoxic effects were observed in experiment I, at least eight concentrations were tested in experiment II. The concentration range included two logarithmic decades. The following concentrations were tested in experiment II: Strains TA1537 and WP2 uvrA pKM101: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. Strains TA1535, TA98, TA100, and WP2 pKM101: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colonies were counted using a validated computer system, which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test substance the colonies were partly counted manually.

Evaluation criteria:

A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Key result

Species / strain:

other: TA1537, WP2 uvrA pKM101, TA1535, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test substance was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since cytotoxic effects were observed in experiment I, seven concentrations were tested in experiment II. 5000 µg/plate was chosen as the maximal concentration for experiment II. The concentration range included two logarithmic decades. The following concentrations were tested in experiment II: Strains TA1537 and WP2 uvrA pKM101: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate; Strains TA1535, TA98, TA100, and WP2 pKM101: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

STUDY RESULTS
- Positive control data : Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
- Ames test: No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations and all mutation rates were within the range of normal biological variability

Table 1. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	333 – 5000	2500 – 5000	1000 – 2500	1000 – 2500
TA 1537	1000 – 5000	/	5000	1000 – 5000
TA 98	333 – 5000	333 – 5000	100 – 2500	333 – 2500
TA 100	333 – 5000	333 – 5000	100 – 2500	100 – 2500
WP2 pKM101	1000 – 5000	2500 – 5000	1000 – 2500	/
WP2uvrApKM101	/	/	/	/

/ = no cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is
considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA pKM101 and WP2 pKM101. The plates incubated with the test substance showed normal background growth in all strains used. In experiment II reduced background growth was observed in strain TA 98 without metabolic activation from 333 to 2500 µg / plate.

Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used, except of strain WP2 uvrA pKM101. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Dec 1985 to 13 Feb 1986

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 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time or proliferation index: 10-14 hours
- Modal number of chromosomes: 20

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Exponentially growing CHO-K1 cells will be seeded in complete medium (Ham's F-12 or McCoy's 5A mediun containing 10% fetal bovine serun, 2 mM L-glutamine, 100 units penicillin/mL and 100 µg streptomycin/mL) for each treatment condition at approximately 5 x 10^5 cells/25 cm2 flask. 37+1°C in a humidified atmosphere of 5+1% CO2 in air for 16 -24 hours.

Cytokinesis block (if used):

Colcemid (0.1 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The liver homogenate (S-9 fraction) used in the metabolic activation system was obtained from Sprague-Dawley rats treated with 500 mg/kg bw Aroclor 1254 for five days prior to sacrifice.
- method of preparation of S9 mix: Immediately prior to use, the S-9 ismixed with a cofactor pool containing 1.4 mg NADP, 2.7 mg lsocitric acid and and 15 µL S-9 per mL growth mediun with 2.5% serum.

Test concentrations with justification for top dose:

Main test in presence of S9 (14 hours): 0.005, 0.01, 0.03, 0.08, 0.15 and 0.3 µg/mL
Main test in absence of S9 (2 hours): 0.3, 0.6, 1.5, 3.0 and 6.0 µg/mL

Vehicle / solvent:

- Solven used: Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10^5 cells/25 cm2
- Test substance added in medium

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colcemid, 0.1 µg/mL, two hours incubation
- Methods of slide preparation and staining technique used including the stain used: The cells will be collected by centrifugation, swollen with 0.075 M KCl, washed with two consecutive changes of Carnoy's fixative, capped and stored overnight or longer at approximately 4 °C. To prepare slides, the cells will be collected by centrifugation, resuspended In fresh Carnoy's fixative. One to two drops of fixed cells will be dropped onto a wet slide and air -dried. The slide will be identified by the experiment number, treatment condition and date. One to 2 slides will be prepared from each treatment flask. The slides will be stained with Giemsa and permanently mounted.
- Number of cells spread and analysed per concentration: Whenever possible, a minimum of 100 metaphase spreads from each dose level (50 per duplicate flask) will be examined and scored for chromatid and chromosome gaps and breaks, chromatid and acentric fragments, dicentrics, rings, triradials, quadriradials, complex rearrangements, pulverized chromosome(s), pulverized cells and severely damaged cells (>10 aberrations). The XY coordinates for each cell with chromosomal aberrations will be recorded using a calibrated microscope stage.
- Determination of polyploidy and endoreplication: The frequency of cells with numerical aberrations (endoreduplication and polyploidy) will be recorded as the number per 100 metaphase cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method; relative total growth (RTG)

Evaluation criteria:

The test article will be considered to induce a positive response when the number of structural aberrations per cell Is significantly increased (p<0.05) in a dose-responsive manner. A significant increase at only one dose level with no dose-response will be considered equivocal.

Statistics:

Statistical analysis of the frequency of structural aberrations per cell will be performed using analysis of variance and Dunnett's multiple comparison tables to judge significance of differences. Chi-square analysis using a 2 x 2 contingency table will be used to ascertain significant differences between the percentage of cells with numerical aberrations in each treatment group relative to the vehicle control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test based upon cell proliferation after treatment relative to the solvent control. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 1000-0.1 µg/mL in the presence and absence of an S-9 reaction mixture. The test article was partially insoluble at final concentrations of 1000-30 µg/mL in the treatment flasks. Based upon these findings, dose levels of 0.3, 0.15, 0.08, 0.03, 0.01 and 0.005 µg/mL in the absence of exogenous metabolic activation and 6, 3, 1.5, 0.6 and 0.3 µg/mL in the presence of an S-9 activation system were selected for further study. The test substance induced substantial cell cycle delay in the nonactivated system, therefore, cell harvest was delayed to 16 hours in order to assure analysis of first division metaphase cells after treatment. Cell cycle delay was minimal in the S-9 activated system, therefore, harvest was conducted at the standard 10 hours.

All dose levels tested were soluble in the treatment medium in absence of S-9 mix. At the time of harvest, 0.3 µg/mL was observed to be slightly toxic upon microscopic examination of the cell monolayer. The four highest dose levels with scorable metaphase cells were evaluated for aberrations. No significant effects on numerical aberrations were observed. The frequency of with structural aberrations was significantly increased above that of the solvent control at 0.3 µg/mL (p<0.05, Dunnett's t test). TEM induced 1.22 aberrations per cell which was statistically increased above the untreated control (p<0.05, Dunnett's t test).

All dose levels were soluble in the treatment medium in presence of S-9 mix. At the time of harvest, 6 µg/mL was observed to be slightly toxic upon microscopic examination of the cell monolayer. No significant effects an numerical aberrations were observed. The frequency of cells with structural aberrations in the test article-treated groups was not statistically increased above that of the solvent control (p>0.05, Dunnett's t test). CP induced 0.42 aberrations per cell which was statistically increased above the untreated control (p<0.05, Dunnett's t test)

Conclusions:

In this study, the test substance was shown to be mutagenic in CHO cells in absence of a metabolic activation system.

Executive summary:

The test article was tested in the chromosome aberration assay using Chinese hamster ovary cells (similar to OECD 473, GLP). The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.3, 0.15, 0.08, 0.03. 0.01 and 0.005 µg/mL in the absence of exogenous metabolic activation and at 6, 3, 1.5, 0.6 µg/mL and 0.3 µg/mL in the presence of S-9 activation. Dose levels of 0.3-0.03 µg/mL in the absence of S-9 and 6-0.6 µg/mL in the presence of S-9 were selected far metaphase analysis. A delayed harvest was carried cut in the nonactivated study because of cell cycle delay. A statistically significant and dose-responsive increase in chromosome aberrations was observed in the nonactivated test system only.

In conclusion, the test substance was concluded to be positive in the CHO chromosome aberration assay..

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No clastogenic effects were found in the following study:

- rat, mouse and hamster bone marrow micronucleus assay, Siou 1983

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: rats of approximately 150 g (males)
- Diet: ad libitum
- Water: ad libitum
- Acclimation: All animals will be held for a minimal period of time to assure that all the animals used for the preliminary or main test are in good health.
- Housing: Currently acceptable practices of good animal husbandry was followed at all times

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % (w/w) Methocel E15 Premium (hydroxy-propyl methylcellulose) in ion-exchanged water.
- Amount of vehicle: 2 mL

Details on exposure:

All the suspensions or solutions were prepared extemporeanously in a solution at 0.5 % methocel. Test substance was crushed in a mortar in small quantities of aqueous solution of methacel. The volume was then adjusted with the same vehicle. The animals were not be fasted prior to the administration of the test material, vehicle or the positive control.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Two doses with a 24 hours interval in between

Post exposure period:

6 hours

Dose / conc.:

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

Remarks:

Group II, suspension at 0.4 mg/mL

Dose / conc.:

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

Remarks:

Group III, suspension at 2 mg/mL

Dose / conc.:

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

Remarks:

Group IV, suspension at 10 mg/mL

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group V, suspension at 50 mg/mL

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Group VI, suspension at 250 mg/mL

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: Methy methasulfonate (MMS)
Route of administration: oral gavage
Doses: 65 mg/kg, suspended at 2.5 μL/mL (Group VII)

Tissues and cell types examined:

Polychromatic erythrocytes (PCEs) in bone morrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study was conducted to determine the maximum dose of test substance which could be administered by incubation.

METHOD OF ANALYSIS: The principle of the method consists to search the presence of a rest of chromosome in the young erythrocits of the marrow. Clastogenic agents can cause breakdown of a chromosome at the mitosis and the fragment which is not, at the telophase, included in the cell, appears in the cytoplasm as a small ball (from where the name of micronucleus). Micronuclei have been observed on animals treated with agents considered as mutagenic, at the level of different cells of the marrow, namely the myelocyts and the erythroblasts. However, the most are observed in the polychromatophilic erythrocits, cells particularly interesting for this study, because a few hours after the end of their last mitosis, these cells expel their nucleus. This chromosomal rest is easily recognisable. The young erythrocits, less than 24 hours old, have besides, the advantage to possess tinctorial properties different from the adult erythrocits.
The product is administered in two shots at 24 hours interval and the animals are sacrificed 6 hours after the second administration. The femur is taken and cut at its two ends in order to loosen the medular tubular. The marrow is recovered in serum of veal’s embryon. After centrifugation, the bottom is homogenized and one drop is laid out on a lamina. The smear is dried and coloured by the May Grünwald-Giemsa. The counting of the cells carrying micronuclei is realized on 2000 polychromatophilic erythrocits. The readings are done by two observers which read each on a different lamina 1000 polychromatophilic erythrocits. The average of these two reading is then calculated.

Statistics:

The statistical analysis consisted by a comparison of two measures done with the Student’s method, valid for small samples. The value of t calculated has been compared to the value of t theoric given in a distribution table of t.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One animal died approximately 4 hours after the second administration.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the preliminary study four rats were orally dosed with 5000 mg/kg test substance. The rats were dosed twice with a 25 hour interval between doses. One rat died before the second dosing, the other three animals survived at least six hours after the second dose. The maximum dose selected for the micronucleus test was set at 5000 mg/kg.

MICRONUCLEUS TEST
- Dose levels for the micronucleus test in the rat were 0, 8, 40, 200, 1000 and 5000 mg/kg. Ten males rats per group were dosed twice with a 24 hour interval between doses. One high dose rat died four hours after the second administration of test material and was not included in the micronuclei evaluation. The mean per cent polychromatic erythrocytes with micronuclei did not differ statistically from the vehicle control. The mean per cent polychromatic erythrocytes with micronuclei from rats treated with MMS was statistically higher than controls.

Table 1: Polychomatophilic erythrocits average (P.E.) carying micrnuclei in rats

Products	Doses mg/kg (PO)	Number of animals	% of P.E. with micronuclei m -/+ 2 Sm
Control methocel		10	0.16 ± 0.08
Test substance	2 x 8	10	0.21 ± 0.06
	2 x 40	10	0.22 ± 0.04
	2 x 200	10	0.17 ± 0.07
	2 x 1000	10	0.23 ± 0.04
	2 x 5000	9	0.23 ± 0.06
MMS	2 x 65	10	3.22 ± 1.10

Conclusions:

Orally administered test substance to male rats at 2 x 8, 2 x 40, 2 x 200, 2x 1000 and 2 x 5000 mg/kg has not determined at the level of the marrow any increasing of the average of polychromatophilic erythrocits carrying micronuclei (Schmid’s micronuclei technic). By the same way methylmethanesulfonate utilised as positive control at the dose of 2 x 65 mg/kg appeared as a clastogenic agent. The absence of clastogenic property of the test substance indicates then, according to the interpretation admitted by the technic applied, an absence of mutagenic effect.

Executive summary:

An OECD 474 -like micronucleus test in compliance with GLP was conducted in rats to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to rats did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible Mutagenic potentiality has been searched for by the technic of Micronucleus in Rat. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 males rats, 8, 40, 200, 1000 or 5000 mg/kg/day. The animals were sacrificed by cervical dislocation 6 hours after the second shot. Hematogenic marrow smears taken from the femur, were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methyl-methanesulfonate, utilised as positive control and orally administered at 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of Mutagenic effect.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

other: Mice

Strain:

Swiss

Remarks:

Swiss C.F.L.P

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: mice of 25 - 30 g (males)
- Diet: ad libitum
- Water: ad libitum
- Acclimation: All animals will be held for a minimal period of time to assure that all the animals used for the preliminary or main test are in good health.
- Housing: Currently acceptable practices of good animal husbandry will be followed at all times

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % (w/w) Methocel E15 Premium (hydroxy-propyl methylcellulose) in ion-exchanged water.
- Amount of vehicle: 0.25 mL

Details on exposure:

All the suspensions or solutions were prepared extemporeanously in a solution at 0.5 % methocel. Test substance was crushed in a mortar in small quantities of aqueous solution of methacel. The volume was then adjusted with the same vehicle. The animals were not be fasted prior to the administration of the test material, vehicle or the positive control.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Two doses with a 24 hours interval in between

Post exposure period:

6 hours

Dose / conc.:

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

Remarks:

Group II, suspension at 0.16 mg/mL

Dose / conc.:

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

Remarks:

Group III, suspension at 0.8 mg/mL

Dose / conc.:

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

Remarks:

Group IV, suspension at 4 mg/mL

Dose / conc.:

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

Remarks:

Group V, suspension at 20 mg/mL

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Group VI, suspension at 100 mg/mL

No. of animals per sex per dose:

10 (additional animals were treated in case of death)

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: Methy methasulfonate (MMS)
Route of administration: oral gavage
Doses: 65 mg/kg, suspended at 2.0 μL/mL (Group VII)

Tissues and cell types examined:

Polychromatic erythrocytes (PCEs) in bone morrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study was conducted to determine the maximum dose of test substance which could be administered by incubation.

METHOD OF ANALYSIS: The principle of the method consists to search the presence of a rest of chromosome in the young erythrocits of the marrow. Clastogenic agents can cause breakdown of a chromosome at the mitosis and the fragment which is not, at the telophase, included in the cell, appears in the cytoplasm as a small ball (from where the name of micronucleus). Micronuclei have been observed on animals treated with agents considered as mutagenic, at the level of different cells of the marrow, namely the myelocyts and the erythroblasts. However, the most are observed in the polychromatophilic erythrocits, cells particularly interesting for this study, because a few hours after the end of their last mitosis, these cells expel their nucleus. This chromosomal rest is easily recognisable. The young erythrocits, less than 24 hours old, have besides, the advantage to possess tinctorial properties different from the adult erythrocits.
The product is administered in two shots at 24 hours interval and the animals are sacrificed 6 hours after the second administration. The femur is taken and cut at its two ends in order to loosen the medular tubular. The marrow is recovered in serum of veal’s embryon. After centrifugation, the bottom is homogenized and one drop is laid out on a lamina. The smear is dried and coloured by the May Grünwald-Giemssa. The counting of the cells carrying micronuclei is realized on 2.000 polychromatophilic erythrocits. The readings are done by two observers which read each on a different lamina 1.000 polychromatophilic erythrocits. The average of these two reading is then calculated.

Statistics:

The statistical analysis consisted by a comparison of two measures done with the Student’s method, valid for small samples. The value of t calculated has been compared to the value of t theoric given in a distribution table of t.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Two groups of mice (5 mice/group) were dosed with 5000 or 10000 mg/kg test substance. In the high dose group all animals died within 24 hours. One death occurred in the low dose group. All remaining animals in this group were reported to be moribund 24 hours after administration of the test material. The maximum dose selected for the micronucleus test was 2500 mg/kg.

MICRONUCLEUS TEST
- Dose levels were 4, 20, 100, 500 and 2500 mg/kg test substance. Animals were dosed twice with a 24 hour interval between doses. Ten males mice were evaluated in the 4, 20 and 100 mg/kg groups. Due to deaths, only nine mice were evaluated in the 500 and 2500 mg/kg groups. In the substance-treated groups, the mean per cent polychromatic erythrocytes with micronuclei did not differ statistically from controls. Micronuclei in the MMS treated group were statistically increased when compared with the vehicle control group.

Table 1: Polychomatophilic erythrocits average (P.E.) carying micrnuclei in mice

Products	Doses mg/kg (PO)	Number of animals	% of P.E. with micronuclei  m -/+ 2 Sm
Control methocel		10	0.24 ± 0.09
Test substance	2 x 4	10	0.25 ± 0.06
	2 x 20	10	0.18 ± 0.06
	2 x 100	10	0.12 ± 0.05
	2 x 500	9	0.21 ± 0.05
	2 x 2500	9	0.35 ± 0.10
MMS	2 x 65	10	1.36 ± 0.24

Conclusions:

Orally administered test substance to male rats at 2 x 4, 2 x 20, 2 x 100, 2 x 500 and 2 x 2500 mg/kg has not determined at the level of the marrow any increasing of the average of polychromatophilic erythrocits carrying micronuclei (Schmid’s micronuclei technic). By the same way methylmethanesulfonate utilised as positive control at the dose of 2 x 65 mg/kg appeared as a clastogenic agent. The absence of clastogenic property of the test substance indicates then, according to the interpretation admitted by the technic applied, an absence of mutagenic effect.

Executive summary:

An OECD 474-like micronucleus test in compliance with GLP was conducted in mice to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to mice did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible mutagenic potentiality has been searched for by the technic of micronucleus in Mouse. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 males mice at 4, 20, 100, 500 or 2500 mg/kg/day. The animals were sacrificed by cervical dislocation 6 hours after the second shot. Hematogenic marrow smears taken from the femur, were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methyl-methanesulfonate, utilised as positive control and orally administered at 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of mutagenic effect.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: hamsters of appoximatively 30 - 35 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation: All animals will be held for a minimal period of time to assure that all the animals used for the preliminary or main test are in good health.
- Housing: Currently acceptable practices of good animal husbandry will be followed at all times

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % (w/w) Methocel E15 Premium (hydroxy-propyl methylcellulose) in ion-exchanged water.

Details on exposure:

All the suspensions or solutions were prepared extemporeanously in a solution at 0.5 % methocel. Test substance was crushed in a mortar in small quantities of aqueous solution of methacel. The volume was then adjusted with the same vehicle. The animals were not be fasted prior to the administration of the test material, vehicle or the positive control.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Two doses with a 24 hours interval in between

Post exposure period:

6 hours

Dose / conc.:

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

Remarks:

Group II, suspension at 0.16 mg/mL

Dose / conc.:

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

Remarks:

Group III, suspension at 0.8 mg/mL

Dose / conc.:

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

Remarks:

Group IV, suspension at 4 mg/mL

Dose / conc.:

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

Remarks:

Group V, suspension at 20 mg/mL

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Group VI, suspension at 100 mg/mL

No. of animals per sex per dose:

10 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: Methy methasulfonate (MMS)
Route of administration: oral gavage
Doses: 65 mg/kg, suspended at 2.0 μL/mL in mice and hamsters (Group VII)

Tissues and cell types examined:

Polychromatic erythrocytes (PCEs) in bone morrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study was conducted to determine the maximum dose of test substance which could be administered by incubation.

METHOD OF ANALYSIS: The principle of the method consists to search the presence of a rest of chromosome in the young erythrocits of the marrow. Clastogenic agents can cause breakdown of a chromosome at the mitosis and the fragment which is not, at the telophase, included in the cell, appears in the cytoplasm as a small ball (from where the name of micronucleus). Micronuclei have been observed on animals treated with agents considered as mutagenic, at the level of different cells of the marrow, namely the myelocyts and the erythroblasts. However, the most are observed in the polychromatophilic erythrocits, cells particularly interesting for this study, because a few hours after the end of their last mitosis, these cells expel their nucleus. This chromosomal rest is easily recognisable. The young erythrocits, less than 24 hours old, have besides, the advantage to possess tinctorial properties different from the adult erythrocits.
The product is administered in two shots at 24 hours interval and the animals are sacrificed 6 hours after the second administration. The femur is taken and cut at its two ends in order to loosen the medular tubular. The marrow is recovered in serum of veal’s embryon. After centrifugation, the bottom is homogenized and one drop is laid out on a lamina. The smear is dried and coloured by the May Grünwald-Giemsa. The counting of the cells carrying micronuclei is realized on 2000 polychromatophilic erythrocits. The readings are done by two observers which read each on a different lamina 1000 polychromatophilic erythrocits. The average of these two reading is then calculated.

Statistics:

The statistical analysis consisted by a comparison of two measures done with the Student's method, valid for small samples. The value of t calculated has been compared to the value of t theoric given in a distribution table of t.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the preliminary study 10 hamsters were dosed with 5000 mg/kg test substance. The hamsters were dosed twice with a 24 hour interval between doses. Two hamsters died after the first dose, four after the second administration. The maximum dose selected for the micronucleus test 250 mg/kg.

MICRONUCLEUS TEST
- Dose levels for the micronucleus test were 0, 4, 20, 100, 500 and 500 mg/kg. Animals were dosed twice with a 24 hour interval between doses. Ten male hamsters per group were dosed. Two hamsters died during the dosing period; one in the 100 mg/kg group and one in the 500 mg/kg group. These animals were not included in the evaluation. In the substance-treated groups the mean per cent polychromatic erythrocytes with micronuclei did not differ statistically frαn the vehicle controls. Mean values for the MMS treated animals were statistically higher than controls.

Table 1: Polychomatophilic erythrocits average (P.E.) carying micrnuclei in hamsters

Products	Doses mg/kg (PO)	Number of animals	% of P.E. with micronuclei  m -/+ 2 Sm
Control methocel		10	0.19 ± 0.05
Test substance	2 x 4	10	0.09 ± 0.05
	2 x 20	10	0.14 ± 0.03
	2 x 100	9	0.13 ± 0.05
	2 x 500	9	0.12 ± 0.05
	2 x 2500	10	0.12 ± 0.06
MMS	2 x 65	10	4.08 ± 0.80

Conclusions:

Orally administered test substance to male Chinese hamsters at 2 x 4, 2 x 20, 2 x 100, 2 x 500 and 2 x 2500 mg/kg has not determined at the level of the marrow any increasing of the average of polychromatophilic erythrocits carrying micronuclei (Schmid’s micronuclei technic). By the same way methylmethanesulfonate utilised as positive control at the dose of 2 x 65 mg/kg appeared as a clastogenic agent. The absence of clastogenic property of the test substance indicates then, according to the interpretation admitted by the technic applied, an absence of mutagenic effect.

Executive summary:

An OECD 474-like micronucleus test in compliance with GLP was conducted in Chinese hamsters to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to Chinese hamsters did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible mutagenic potentiality has been searched for by the technic of micronucleus in Chinese Hamster. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 Chinese Hamsters 2 x 4, 2 x 20, 2 x 100, 2x 500 or 2 x 2500 mg/kg/day. The dose of 2 x 2500 mg/kg has been taken as the high dose for the experiment after a preliminary test of toxicity. The animals were sacrificed by diethylether 6 hours after the second shot. Hematogenic marrow smears taken from the femur were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methylmethanesulfonate, utilised as positive control and orally administered at 2 x 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of mutagenic effect.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Summary

Bacterial gene mutation (Ames Test) - The test conducted according to guideine and reliable gives a negative result.
Gene mutation in vitro in mammalian cells - The test conducted according to guideine and reliable gives a negative result.
As both the test for bacterial gene mutation and the test on gene mutation in vitro in mammalian cells are negative it can be concluded that the test substance is negative for this endpoint and accordingly no additional in vivo tests are necessary for this endpoint. 

Chromosomal aberration in vitro - the test was conducted using Chinese hamster ovary cells (similar to OECD 473, GLP) and is reliable. The result was positive.
Chromosomal aberration in vivo - three micronucleus tests in compliance with GLP were conducted in Chinese hamsters, rats and mice respectively, generally compliant to OECD 474 and deemed fully reliable. All three tests gave a negative result for the test substance. 
These negative results from the in vivo studies superseed the positive result from the in vitro study for chromocomal aberration. Therefore the test substance is deemed negative for choromosomal abberation.

As the test substance is negative for both endpoints, gene mutation and chromosome abberation, it does not need to be classified for mutagenicity and no further testing is required. 

Ames test

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA pKM101 and WP2 pKM101.The plates incubated with the test substance showed normal background growth in all strains used. In experiment II reduced background growth was observed in strain TA 98 without metabolic activation from 333 to 2500 µg / plate.

Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used, except of strain WP2 uvrA pKM101. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Gene mutation test in mammalian cells

Mammalian cell mutagenesis tests with Chinese hamster cells and/or mouse fibroblasts are performed according to the method of Schechtman et al. Ouabain is used as the selective agent to detect point mutations in treated cells. This mutant cell is resistant to ouabain and hence forms mutant colonies in the presence of ouabain. Plates, with treated cells are incubated in ouabain containing medium for 14 days for Chinese hamster cells and 25 days for mouse fibroblasts. Ouabain resistant mutant colonies are counted and compared with controls. Positive controls and negative controls are included in each experiment. N-methyl-N-nitro-N-nitrosoguanidine (MNNG) serves as the positive control without activation and Benzo (a) pyrene (BP) serves as the positive control with activation. Acetone is the negative control without activation and S-9 alone, heat-inactivated S-9 + BP, are the negative controls without activation.

The cytotoxicity in the preliminary test in V79 cells was determined at concentrations of 30, 10, 3, 1 and 0.3 µg/mL. The test substance is toxic, even at 0.3 µg/mL. Mutation frequency in the presence of test chemical is not significant even though the spontaneous mutation frequency is ten times higher than it should be. Addition of an exogenous source of liver enzyme (i.e., S-9) did not affect the ability of the test agent to increase the frequency of ouabain resistant mutation. As mentioned in an earlier report, the spontaneous mutation frequency is much too high in this specific clone of V-79, the studies were repeated using a different cell line, namely BALB/ 3T3 clone A31-1.

It was observed that even at 0.03 µg/mL with 2 hours exposure there was approximately 33% death. This indicates that BALB/ 3T3 was about 10 times more sensitive to the cytotoxic effects of the test substance than were the V-79 cells.

At 0.03 µg/mL, the test substance was not effective in inducing ouabain resistant mutants without activation using BALB/3T3 as the target cells. BP was used as the positive control. BP, in the presence of exogenous mammalian S-9 enzyme fraction was converted to 11.5 nM of 3-OH BP in 2 hours incubation. This activating system was effective in inducing mutation frequency of 17.1 x 10^-6. The test chemical failed to increase the mutation frequency above the spontaneous level.

It must be pointed out that the presence of the S-9 preparation allowed us to increase the concentration of the test substance that could be evaluated. A concentration of 0.3 µg/mL (10 times higher than could be used without activation or in the presence of heat inactivated S- 9) was evaluated and yet this higher dose still failed to significantly increase the mutation frequency at the ouabain resistant locus.

Under the conditions of this bioassay, the test substance did not induce point mutations at the ouabain resistant locus in both V-79 clone 8 Chinese hamster cells or BALB/3T3 clone A subline 1 cells either with or without the addition of an exogenous source of liver enzyme (S-9) for metabolic activation of the test agent.

 

In vitro chromosome aberration test

The test article was tested in the chromosome aberration assay using Chinese hamster ovary cells (similar to OECD 473, GLP). The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.3, 0.15, 0.08, 0.03. 0.01 and 0.005 µg/mL in the absence of exogenous metabolic activation and at 6, 3, 1.5, 0.6 µg/mL and 0.3 µg/mL in the presence of S-9 activation. Dose levels of 0.3-0.03 µg/mL in the absence of S-9 and 6-0.6 µg/mL in the presence of S-9 were selected far metaphase analysis. A delayed harvest was carried cut in the nonactivated study because of cell cycle delay. A statistically significant and dose-responsive increase in chromosome aberrations was observed in the nonactivated test system only. In conclusion, the test substance was concluded to be positive in the CHO chromosome aberration assay.

 

In vivo micronucleus tests, rat

An OECD 474 -like micronucleus test in compliance with GLP was conducted in rats to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to rats did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible Mutagenic potentiality has been searched for by the technic of Micronucleus in Rat. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 males rats, 8, 40, 200, 1000 or 5000 mg/kg/day. The animals were sacrificed by cervical dislocation 6 hours after the second shot. Hematogenic marrow smears taken from the femur, were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methyl-methanesulfonate, utilised as positive control and orally administered at 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of Mutagenic effect.

 

In vivo micronucleus tests, mice

An OECD 474-like micronucleus test in compliance with GLP was conducted in mice to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to mice did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible mutagenic potentiality has been searched for by the technic of micronucleus in mice. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 males mice at 4, 20, 100, 500 or 2500 mg/kg/day. The animals were sacrificed by cervical dislocation 6 hours after the second shot. Hematogenic marrow smears taken from the femur, were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methyl-methanesulfonate, utilised as positive control and orally administered at 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of mutagenic effect.

 

In vivo micronucleus tests, hamster

An OECD 474-like micronucleus test in compliance with GLP was conducted in Chinese hamsters to determine if the substance, orally administered, caused formation of micronuclei in polychromatic erythrocytes. Only male animals were used for the tests. Animals were given two doses of substance by gavage with a 24 hour interval between doses. Polychromatic erythrocytes in the bone marrow were evaluated for the presence of micronuclei. Oral administration of test substance to Chinese hamsters did not cause an increased incidence of micronuclei in polychromatophilic erythrocytes in the bone marrow of these animals.

A possible mutagenic potentiality has been searched for by the technic of micronucleus in Chinese Hamster. Suspension of test substance in an aqueous solution at 0.5 % of methocel has been orally administered in two shots at 24 hours interval to groups of 9 or 10 Chinese Hamsters 2 x 4, 2 x 20, 2 x 100, 2x 500 or 2 x 2500 mg/kg/day. The dose of 2 x 2500 mg/kg has been taken as the high dose for the experiment after a preliminary test of toxicity. The animals were sacrificed by diethylether 6 hours after the second shot. Hematogenic marrow smears taken from the femur were prepared. The polychromatophilic erythrocits count which carry micronuclei was done on 2000 elements per animal, by two observers which read each 1000 elements.

The administration of test substance has not induced any significant increasing of the polychromatophilic erythrocits average carrying micronuclei; an increasing should have indicated a clastogenic effect of the product. On the other hand, methylmethanesulfonate, utilised as positive control and orally administered at 2 x 65 mg/kg/day, increases the polychromatophilic erythrocits average with micronuclei. In conclusion, the absence of the clastogenic effect of test substance expresses, according to the interpretation of the technic utilized, the absence of mutagenic effect.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.