N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +/- S9, negative, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, similar to OECD TG 471, Deparade 1988.

- In vitro chromosome aberration study, +/- S9 negative, Chinese Hamster Ovary (CHO) cells, according to OECD TG 473, Strasser 1988.

- In vitro gene mutation assay, +/- S9 negative, Chinese Hamster lung cells (V79), according to OECD TG 476, Dollenmeier 1988.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Feb 1988 to 14 Apr 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Aroclor 1254 induced rat liver S9
- Method of preparation of S9 mix: The S9 is prepared from rats (Tif: RAIf(SPF)) induced with Aroclor 1254 and 0.7 mL of a solution of co-factors.

Test concentrations with justification for top dose:

Pre-Experiment: 0.08 - 5000 μg/0.1 mL
Main experiment: 20, 78, 313, 1250 and 5000 μg/0.1 mL

Vehicle / solvent:

- Solvent used: acetone.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cyclophosphamide

other: Daunorubicin-HCl

Details on test system and experimental conditions:

PRELIMINARY TEST FOR TOXICITY
A preliminary toxicity test was carried out with strain TA 100 without activation with 9 concentrations ranging from 0.08 to 5000 μg/0.1 mL. The protocol used is the same as in the mutagenicity test. Accordingly, the concentration of 5000 µg/0.1 mL was used as the highest in the mutagenicity test and the tests were performed with the following concentrations of the trial substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg /0.1 mL.

EXPERIMENTAL PERFORMANCE
The substance is dissolved in acetone. Acetone alone is used for the negative controls (the substances and vehicles used for the positive controls are indicated below). Each Petridish contains:
- approx. 20 mL of minimum agar (agar bacterial grade, plus salts and glucose).
- 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, 2.5 %) in 2.0 mL of soft agar.
- The soft agar is composed of: 100 mL of 0.6 % agar solution with 0.6 % NaCl and 10 mL of a solution of L-histidine, 0.5 mM and + biotin, 0.5 mM.
- In the experiments in which the substance is metabolically activated, 0.5 mL of an activation mixture is added also.
- 1 mL activation mixture contains: 0.3 mL S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 mL of a solution of co-factors.
- In the experiments without and with the addition of microsomal activation mixture three petri dishes are prepared per strain and per group (i.e. per concentration or per control group). The plates are incubated for about 48 hours at 37 ±1.5 °C in darkness.

DATA PRESENTATION
The data reported include individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration. Historical values of negative controls obtained with each strain without and with microsomal activation are listed in a separate table, which contains mean values of controls covering an experimental period of 1 year as well as standard deviations and acceptable minimal and maximal values of spontaneous revertants for each bacterial strain.

ANALYTICAL CONTROL
To prove that the indicator organisms are really exposed to the intended concentrations of the test substance, determination of the substance in solution is performed by the analytical unit. This determination is performed with the lowest concentration of the solutions used in the mutagenicity tests.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable.

ACCEPTABILITY OF THE ASSAY
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision has to be based on scientific judgement.

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100

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:

TOXICITY TEST
Nine concentrations of the test substance ranging from 0.08 to 5000 µg/0.1 mL were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 µg/0.1 mL

MUTAGENICITY TEST
In two separate experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar. The sensitivity of the test system, and the metabolic activity of the S9-mix, was clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

Conclusions:

No induction of point mutation by the test substance or by its metabolites was detectable in the tested strains. It was concluded that the test substance showed no evidence of genotoxic activity in this assay system.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test and the pre-incubation test. For this purpose, the Salmonella typhimurium histidine-auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100 and Aroclor 1254 pre-treated liver microsomes of rats (Tif: RAIf(SPF)) were used according to GLP principles and OECD TG 471.

A preliminary toxicity test was carried out with strain TA 100 without activation with 9 concentrations ranging from 0.08 to 5000 μg/0.1 mL. The protocol used is the same as in the mutagenicity test. The investigations were performed on Salmonella strains with 20, 78, 313, 1250 and 5000 μg/0.1 mL trial substance without and with microsomal activation (S9-mix) using liver microsomes of rats (Tif: RAIf(SPF)) pre-treated with Aroclor 1254. The substance was dissolved in acetone. Acetone alone was used for the negative controls. In the experiments without and with the addition of microsomal activation mixture 3 Petri dishes are prepared per strain and per group (i.e. per concentration or per control group). The plates are incubated for about 48 hours at 37 ± 1.5 °C in darkness.

The test material in solution was analysed to confirm the intended concentrations to be used in the mutagenicity tests. The values of the lowest concentrations to be analysed were found to be somewhat above the calculated concentrations, mainly due to the fast evaporation of the vehicle acetone. This, however, did not affect the test results.

From the results obtained in the toxicity test, the highest concentration suitable for the mutagenicity test was found to be 5000 μg/0.1 mL. In two separate experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar. The sensitivity of the test system, and the metabolic activity of the S9-mix, was clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

To conclude, no induction of point mutation by the test substance or by its metabolites was detectable in the tested strains and this assay system did not show no evidence of genotoxic activity.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct 1988 to 16 Mar 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

May 1983

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

May 1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Sep 1984

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

ATCC - CCL 61

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
The cell line CHO CCL 61 has been used for cytogenetic studies for several years. The stability of the genome of these cells is assessed regularly on the basis of the cytogenetic analysis of control cultures in the course of the cytogenetic studies. It is judged to be adequate for the particular purpose of cytogenetic studies.

MEDIA USED
- Type and composition of media: The cell line CCL 61 (Chinese hamster ovary cells) was maintained in Nutrient Mixture F-12 supplemented with 10 % fetal calf serum + Penicillin/Streptomycin 100 units/mL/100 µg/mL.

Cytokinesis block (if used):

Two hours before harvesting, the cultures were treated with Colcemide (0.4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: rat liver microsomal fraction S9 was prepared from Aroclor 1254 (induced livers of male RAI rats.
- Concentration or volume of S9 mix and S9 in the final culture medium: The co-factors used were NADP and Iso-citric acid. 1.0 mL activation mixture contained: 0.15 mL S9 fraction and 0.2 mL of the solution with the co-factors and 0.65 mL medium. In the experiments in which the substance was metabolically activated, 1.0 mL of the activation mixture was added to 9.0 mL of culture medium.

Test concentrations with justification for top dose:

Toxicity test range: 3.13 to 1600 µg/mL
Main (-S9): 50.0, 100.0, 200.0 µg/mL
Main (+S9): 400.0, 800.0, 1600.0 µg/mL

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

CYTOTOXICITY TEST
The preincubation time before treatment was 22 hours. The substance dissolved in DMSO was added (1:100) to the cells in culture medium. The cells were exposed for three hours to ten concentrations ranging from 3.13 to 1600.0 µg/mL of the test substance. After removal of the test substance, the cells were washed and incubated in culture medium for 17 hours. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration. This preliminary toxicity test was performed with and without metabolic activation.  The highest concentration used in the toxicity test or the concentration calculated to produce about a 50 % suppression of mitotic activity in comparison with the control is used as the highest in the mutagenicity experiments together with three further concentrations corresponding to factors of 0.5, 0.25 and 0.125.  

MUTAGENICITY TEST (treatment and chromosome preparation)
The mutagenicity tests were carried out by treating the cultures with the selected concentrations (25.0, 50.0, 100.0, 200.0 µg/mL without microsomal activation and 200.0, 400.0, 800.0, 1600.0 µg/mL with microsomal activation). About 22 hours before exposure to the test substance, a series of Falcon flasks was seeded with Chinese hamster ovary cells. Subsequently, the cells were treated for three hours, both in the presence and in the absence of rat S9 liver activation system, with the preselected concentrations of the test substance, with the positive control, or with the vehicle as negative control. In the experiments in which the substance was metabolically activated, 1.0 mL of an activation mixture was added to 9.0 mL of culture medium. 1.0 mL activation mixture contained: 0.15 ml 89 fraction of liver from rats induced with Aroclor 1254 and 0.2 mL of a solution of co-factors and 0.65 mL medium. Mitomycin-C 1.0 µg/mL, a mutagen not requiring S 9 activation, and cyclophosphamide 40.0 µg/mL, which requires activation, were used as positive controls.  
After treatment, the cells were washed with 10 mL Hanks BSS to remove the test substance, after that new culture medium was added and the cells were allowed to grow for 4 hours and for 21 hours. Two hours prior to harvesting, the cultures were treated with Colcemide 0.4 µg/mL. The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation methanol:acetic acid, 3:1). Drop preparations were made by the air-drying technique.  

ANALYTICAL CONTROL
To prove that the cells were really exposed to the intended concentrations of the test substance, determination of the substance in solution was performed by the analytical unit. This determination was performed with the lowest concentration of the stock solution used in the mutagenicity test.

SCORING OF THE SLIDES
Prior analysis the slides were coded. The cultures treated with the upper three concentrations of the test material showing enough scorable metaphases were selected for the evaluation, likewise the cultures treated with the vehicle alone as well as the positive control. 100 metaphase figures with 19 to 21 chromosomes from cultures of two falcon flasks in the vehicle control, in the treated groups and in the positive control (exception: a total of 50 metaphases in the positive controls of the 21 hours experiment) were examined for the following aberrations:
a) specific aberrations: breaks, exchanges, deletions, fragments and minutes,
b) marker aberrations: specific aberrations, occurring spontaneously at particular chromosomes: iso-chromatid breaks, isochromatid fragments, deletions, dicentric chromosomes
c) unspecific aberrations: gaps, premature chromosome condensation and chromosome decay
d) numerical aberrations (metaphases with >21 chromosomes).

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
- A test substance is considered to be active in this test system if in comparison to the negative control a marked increase in the number of specific chromosomal aberrations appear or if an increased number of exchange figures appear together with a high number of other specific chromosomal aberrations such as breaks and fragments.
- A concentration-related response in the number of aberrations should be demonstrable.
 
ASSAY ACCEPTANCE CRITERIA
The quality of the slides should allow, at least to a large extent, the chromosomes to be easily identifiable.

SCORING OF THE SLIDES
Prior analysis the slides were coded. The cultures treated with the upper three concentrations of the test material showing enough scorable metaphases were selected for the evaluation, likewise the cultures treated with the vehicle alone as well as the positive control. 100 metaphase figures with 19 to 21 chromosomes from cultures of two falcon flasks in the vehicle control, in the treated groups and in the positive control (exception: a total of 50 metaphases in the positive controls of the 21 hours experiment) were examined for the following aberrations:
a)   specific aberrations: breaks, exchanges, deletions, fragments and minutes,
b)   marker aberrations: specific aberrations, occurring spontaneously at particular chromosomes: iso-chromatid breaks, isochromatid fragments, deletions, dicentric chromosomes
c)   unspecific aberrations: gaps, premature chromosome condensation and chromosome decay
d)   numerical aberrations (metaphases with >21 chromosomes).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY TEST
The results obtained from the preliminary toxicity test were based on the mitotic index. The highest concentration for the mutagenicity assay without microsomal activation selected was 200.0 µg/mL (46.6% mitotic index frequency of control) and 1600.0 µg/mL (59.4 % mitotic index frequency of control) for that with activation.
 
MUTAGENICITY TEST
In the experiment with a harvesting time of 4 hours and in that with a harvesting time of 21 hours performed with and without microsomal activation, the number of cells with chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. The incidence of changes observed was within the range of spontaneous aberrations inherent in this particular cell line used (historical control data). The treatment of the cultures with mitomycin-C and cyclophosphamide (positive controls), was followed by a high incidence of specific chromosomal aberrations (38 % and 26 %) in the experiment with the harvesting time of 21 hours, but not in the experiment with the short harvesting time of 4 hours, it was presumed by the study author to be an effect of the reference compound on the cell cycle.
 
AMENDMENT NO. 1 TO THE REPORT
Results of the re-evaluation showed in the negative control groups, 0 to 1.5 % of metaphases with specific aberrations. These values were consistent with current historical negative control data. Therefore, it could be demonstrated, that the high level of specific aberrations found in the negative control groups in the original scoring, were due to the scoring protocol used at that time. This becomes also obvious when the historical negative control range shown in the original report (1987/88) is compared with the one from 1992. With respect to effects induced by the test substance, the re-evaluation confirmed the findings of the original scoring. Based on current assay evaluation criteria and on statistical analysis, no evidence of an induction of chromosome aberrations by the test substance was found.

Legend to Tables 2 - 5 (see below)

ct del = Chromatid deletions (including deletions, breaks, fragments)

ct exc = Chromatid exchanges (including triradials, quadriradials, endfusions and acentric rings)

cs del = Chromosome deletions (including deletions, breaks, fragments)

cs exc Chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings)

mab = Multiple aberrations: metaphases containing more than 10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps)

gaps = Chromatid and chromosome type gaps

pol = Polyploid metaphases (>30 centromers)

end = Endoreduplications

CPA = Cyclophosphamide

Mito-C = Mitomycin-C

*) = Statistical significance: 0.05 P > 0.01

**) = Statistical significance: 0.01 P > 0.01

***) = Statistical significance: P ≤ 0.001

#) = % cells with aberrations excluding gaps and numerical alterations (pol, end)

Table 2 The effect of test substance on Chinese hamster ovarv cells in vitro without metabolic activation

Treatment 3 h / Harvest time treatment after 4 h
Treatment	Tot no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exe	cs del	cs exe	mab	pol	end
negative control	200	1.5	3	1		1	1		7
50 μg/mL test substance	200	1.0	3			2	1		2
100 μg/mL test substance	200	0.5	2				1		4
200 μg/mL test substance	200	2.0	3		1	3			4
positive control (Mito-C, 1.0 μg/mL)	200	5.5*	6	8	1	2			5

Table 3 The effect of test substance on Chinese hamster ovarv cells in vitro with metabolic activation

Treatment 3 h / Harvest time treatment after 4 h
Treatment	Tot no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exe	cs del	cs exe	mab	pol	end
negative control	200	1.5	5	1		1	1		1
400 μg/mL test substance	200	2.5	14	2		1	2
800 μg/mL test substance	200	3.5	11	4		2	1		3
1600 μg/mL test substance	200	3.5	13			3	5		4
positive control (CPA, 40 μg/mL)	200	5.5*	9	6		5			4

 

Table 4 The effect of test substance on Chinese hamster ovarv cells in vitro without metabolic activation

Treatment 3 h / Harvest time treatment after 21 h
Treatment	Tot no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exe	cs del	cs exe	mab	pol	end
negative control	200	1.0				2			2
50 μg/mL test substance	200	0.5	1			1			8
100 μg/mL test substance	200	1.5	4	1		2			9
200 μg/mL test substance	200	1.0	1			2			10
positive control (Mito-C, 1.0 μg/mL)	200	52.0***	8	20	7	1		2

 

Table 5 The effect of test substance on Chinese hamster ovarv cells in vitro with metabolic activation

Treatment 3 h / Harvest time treatment after 21 h
Treatment	Tot no of cells examined	% cells with specific aberrations #	total number of cells with aberrations
			gaps	ct del	ct exe	cs del	cs exe	mab	pol	end
negative control	200	0	2						6
400 μg/mL test substance	200	2.0	2	3		1			7	2
800 μg/mL test substance	200	1.5		1	1	1			11	4
1600 μg/mL test substance	200	1.5	2	1	1	1			5	3
positive control (CPA, 40 μg/mL)	200	22.0 ***	3	4	5	3				1

Conclusions:

It is concluded that under the given experimental conditions no evidence of mutagenic effects was obtained in Chinese hamster ovary cells in vitro treated with test substance.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation, according to GLP principles and OECD TG 473. The substance dissolved in DMSO was added (1:100) to the cells line CCL 61 in culture medium. The cells were exposed for 3 hours to 10 concentrations ranging from 3.13 to 1600.0 µg/mL of the test substance. After removal of the test substance, the cells were washed and incubated in culture medium for 17 hours. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration. This preliminary toxicity test was performed with and without metabolic activation.

In the mutagenicity test, cultures were treated for a period of 3 hours in both the presence and absence of S9 mix with the pre-selected test substance concentrations with the positive control, or with the vehicle as negative control. The test substance dissolved in DMSO was used as the negative control. Positive controls were mitomycin C, 1.0 µg/mL (in the absence of S9 mix) and cyclophosphamide, 40.0 µg/mL (in the presence of S9 mix). After treatment, the cells were washed to remove the test substance, after that new culture medium was added and the cells were allowed to grow for 4 hours and for 21 hours. Two hours prior to harvesting, the cultures were treated with Colcemide (0.4 µg/mL). The lowest concentration of test material in solution was analysed to confirm the intended concentration to be used in the mutagenicity tests. The value found was in agreement with the calculated concentration.

In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using a chi-square test. The non-significant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly a chi square test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell was taken as the experimental unit.

As a result in the cytotoxicity test, the highest concentration for the mutagenicity assay without microsomal activation was 200.0 µg/mL (46.6 % mitotic index frequency of control) and 1600.0 µg/mL (59.4 % mitotic index frequency of control) for that with activation.

From the mutagenicity test, it could be demonstrated that the high level of specific aberrations found in the negative control groups in the original scoring, were due to the scoring protocol used at that time. Based on current assay evaluation criteria and on statistical analysis, no evidence of an induction of chromosome aberrations by the test substance was found.

Hence, it was concluded that under the given experimental conditions no evidence of mutagenic effects was obtained in CHO cells in vitro treated with test substance.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Mar 1988 to 14 Jun 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Apr 1984

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

May 1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 1988

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: cells were cultured in 25 mL growth medium (Ham's F10 plus 10 % pre-tested foetal calf serum plus 100 U penicillin and 100 μg streptomycin /mL) in 75 cm2 tissue-culture (plastic) flasks. The humidity in the incubator is adjusted to 92 (± 3) %RH, the air is enriched to 5 ± 2) % CO2 and the temperature is 37 (± 1) °C.
s

MEDIA USED
- Type and composition of media: HAM F10 medium and 3 % foetal calf serum and 1 % DMSO (plus activation mixture).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: S9 liver of male RAI rats induced with Aroclor 1254.
- Method of preparation of S9 mix: The co-factors used were essentially NADP, glucose-6-phosphate, Ca2+ and Mg2+. The activation mixture was added to the medium at a concentration of 10 % in both the cytotoxicity test and the mutagenicity test and the final concentration of S9 fraction was 2 % in the treatment medium.

Test concentrations with justification for top dose:

Cytotoxicity test:
- Without metabolic activation: 183 – 6000 µg/mL
- With metabolic activation: 183 – 6000 µg/mL

Mutagenicity test (original exp.):
- Without metabolic activation: 25.0, 50.0, 100, 200, 300, 400 and 500 µg/mL
- With metabolic activation: 37.5, 75.0, 150, 300, 450, 600 and 750 µg/mL

Confirmatory experiment:
- Without metabolic activation: 25.0, 50.0, 100, 200, 300, 400 and 500 µg/mL
- With metabolic activation: 45.0, 90.0, 180, 360, 540, 720 and 900 µg/mL

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF TREATMENT/EXPOSURE:
- Cell density at seeding: 1xE06 cells plated in culture flask and incubated.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 18 hour
- Exposure duration/duration of treatment: The exposure period was 5 hours (with metabolic activation) or 21 hours (without metabolic activation).

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Following the end of the treatment period the cells were resuspended in fresh growth medium and incubated at 37 °C for five days to express the mutant phenotype. The cytotoxicity was estimated from the cloning efficiency after treatment.
- Selection time (if incubation with a selective agent): At the end of the expression period the cultures were harvested and from each culture 18xE05 cells were plated on 18 dishes and incubated in presence of 8 µg/mL 6-TG for mutant selection. Only cells mutated at the HGPRT locus can survive the 6-TG treatment.
- Fixation time (start of exposure up to fixation or harvest of cells): After seven to eight days incubation at 37 °C, the cultures were fixed with methanol and stained with Giemsa stain. The colonies of the viability control were enumerated with a colony counter. The sensitivity of the counter was adjusted to detect clones of about twenty or more cells. The mutant clones were counted with the naked eye. In parallel the viability at the end of the expression period was estimated by culturing six dishes per concentration at 100 cells per dish for 7 days.

OTHER:
- The test substance in solution was analysed from both experiments to confirm the intended concentrations to be used in the mutagenicity test. The values found are in agreement with the calculated concentrations.

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100 % at the end of the expression period. If this cloning efficiency is lower than 15 %, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mutant factor, which is defined as the ratio of the mutant frequencies of the treated and the negative control cultures, will be calculated. The sensitivity of the test is restricted to a lower limit of the mutant frequency of 4 x E-06. If the mutant frequency measured is below this limit, it will be reported to be smaller than 4 x E-06. and the mutant factor will be calculated assuming a mutant frequency of 4 x E-06

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

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

CYTOTOXICITY TEST:

In the preliminary toxicity test, a 90 % reduction in the viability of the cells treated with test substance was obtained at the concentration of 750 μg/mL in the experiment with microsomal activation and between the concentrations of 375 and 750 μg/mL in the experiment without microsomal activation, a 100 % cytotoxicity was found at 1500 μg/mL and above..

ORIGINAL MUTAGENICITY AND CONFIRMATORY MUTAGENICITY TESTS

The original mutagenicity test without microsomal activation was performed with concentrations of test substance ranging from 25.0 to 500 μg/mL and the original mutagenicity test with microscmal activation was performed with concentrations of test substance tech. ranging from 37.5 to 750 μg/mL.

In the original mutagenicity test without activation, after screening with 6-thioguanine the mutant frequencies in the solvent controls were 11.4x E-06 and 10.3x E-06 (mean used for calculation: 10.85x E-06.

At the highest concentration, the calculated mutant frequency value Was 6.0 x E-06. Comparison of this value with the mean of the two solvent controls revealed a mutant factor of 1.0. At the six lower concentrations the mutant-frequency values calculated were <4.0x E-06 (factor 1.0), 9.4 x E-06 (factor 1.0), 7.1x E-06 (factor 1.0), 14.6 x E-06 (factor 1.3), 13.5 x E-06 (factor 1.2) and at the lowest concentration, the mutant frequency was <4.0x E-06 (factor 1.0).

The positive control treated with 300 nL ethylmethanesulfonate / mL medium gave a mutant frequency of 1517.3x E-06 and a corresponding mutant factor of 139.5

The mutant frequency values in the solvent controls of the microsomal-activated cultures were 5.2x E-06 and <4x E-06 (the lower limit of the sensitivity of the test system) (mean: 4.6x E-06. The concentrations of the microsomal activated cultures treated. with test substance yielded a mutant frequency of 17.7 x E-06 (factor 3.8) at the highest concentrations and mutant frequencies of 14.4x E-06 (factor 3.1), 13.5x E-06 (factor 2.9), 9.7x E-06 (factor 2.1), 7.8x E-06 (factor 1.7), (factor 3.8) and 8.2x E-06 (factor 1.8) down to the lowest.

The positive control treated with 1 μL DMN/mL medium revealed a mutant frequency of 55.9 x E-06 giving a mutant factor of 12.2.

 

CONFIRMATORY MUTAGENICITY TEST

The confirmatory mutagenicity test without microsomal activation was performed. with concentrations of test substance ranging from 25.0 to 500μg/mL and the original mutagenicity test with microsomal activation was performed with concentrations of test substance ranging from 45.0 to 900 μg/mL.

 

In the confirmatory mutagenicity test without activation after screening with 6-thioguanine the mutant frequency values in the solvent controls were <4x E-06 and 5.3x E-06 (mean: 4.65 x E-06 The mutant-frequency values calculated were <4 x E-06 (factor 1.0). at all concentrations tested with the exception at 300μL/mL, where a mutant-frequency value of 9.1 x E-06 and a corresponding mutant factor of 1.9 were calculated. The positive control treated with 300 nL ethylmethanesulfonate /mL medium gave a mutant frequency of 1480.8 x E-06 and a corresponding mutant factor of 317.8.

 

Both mutant-frequency values in the microsomal-activated cultures were <4 x E-06 (mean used for calculation 4.0 x E-06) in the negative (solvent) controls The highest concentration of the microsomal activated cultures in the negative treated with test substance. gave a mutant-frequency value of each <4 x E-06 and a corresponding mutant factor of 1.0. The six lower concentrations yielded mutant frequencies of <4 x E-06 (factor 1.0), 13.9x E-06 (factor 3.5), <4x E-06 (factor 1.0), 7.4x E-06 (factor 1.8), 5.5 x E-06 (factor 1.4) and <4 x E-06 (factor 1.0) down to the lowest. The positive control treated with 1μL DMN/mL medium revealed a mutant frequency of 142.9x E-06 giving a mutant factor of 35.7.

Conclusions:

It is concluded that, under the given experimental conditions, the test substance and its metabolites induced no mutagenic effects in this forward mutation system.

Executive summary:

The test substance was evaluated for induction of point mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V79, derived from Chinese hamster lung cells according to OECD TG 476 and GLP principles. A cytotoxicity test was performed on the cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. The highest concentration to be selected for the mutagenicity assay was that causing about 90 % reduction of viable cells in the cytotoxicity test in comparison with the solvent controls. In the subsequent mutagenicity test, the potential of the test substance to cause gene mutation in mammalian cells (V79) treated in vitro. The auxiliary metabolic system (S9-mix) used was derived from the liver of rats and treated with Aroclor 1254. Mutant frequencies were assessed by cell growth in the presence of 6-thioguanine (6-TG). The cells were treated with 37.5, 75.0, 150, 300, 450, 600 and μg/mL with and 25.0, 50.0, 100, 200, 300, 400 and 500 μg/mL without metabolic activation. The exposure duration was 5 hours (with metabolic activation) or 21 hours (without metabolic activation).

In the preliminary toxicity test, a 90 % reduction in the viability of the cells treated with test substance was obtained at the concentration of 750 μg/mL in the experiment with microsomal activation and between the concentrations of 375 and 750 μg/mL in the experiment without microsomal activation, a 100 % cytotoxicity was found at 1500 μg/mL and above.

In both investigations, each performed without and with microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 1x E06 cells plated was not detected at any concentration of the test substance, and there was also no indication of any concentration-mutant frequency relation in either experiment.

It was therefore concluded that, under the given experimental conditions, the test substance and its metabolites induced no mutagenic effects in this forward mutation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test in mice, negative, according to OECD TG 474, Meyer 1989.

- in vivo mammalian cell study: DNA damage and/or repair, negative, according to OECD TG 486, Ogorek 2000.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Apr 1988 to 07 Jul 1989

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:

May 1983

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Sep 1984

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

Sep 1987

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Tif:MAGf

Remarks:

(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: females 24-27 g, males 28-32 g (tolerability test); females 22-29 g, males 29-37 g (mutagenicity test, first part): females 20-26g, males 24-29 g (mutagenicity test, second part)
- Housing: The animals were individual caged.
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 -23
- Humidity (%): 46 - 52.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 Apr 1988 To: 07 Jul 1989

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % Carboxymethyl Cellulose (CMC)
- Amount of vehicle: 20 mL/kg body weight

Duration of treatment / exposure:

Part 1: Negative control + high dose: 16, 24 and 48 hours
Part 2: Intermediate dose + low dose + high dose+ positive control: 24 hours

Frequency of treatment:

Once

Post exposure period:

Part 1: 16, 24 or 48 hours
Part 2: 24 hours

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Intermediate dose

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral 20 mL/kg body weight.
- Doses: 64 mg/kg in CMC 0.5 %

Tissues and cell types examined:

Bone marrow, Polychromatic erythrocytes (PCEs) and the number of mature erythrocytes (RBCs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary (tolerability) test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. Three groups of 4 mice (2/sex) were treated with 3 single doses, one receiving the maximum dose of 5000 mg/kg bw, or the highest applicable dose, and the other two doses of 1/5 and 1/25 of that amount respectively. The observation period corresponded to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. The highest dose causing no death was used as the highest in the mutagenicity test. In this experiment all animals survived and the dose of 5000 mg/kg bw was determined as the highest to be applied in the mutagenicity assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Part 1: The preparation was administered orally to groups of 24 mice/sex in the treatment group (5000 mg/kg bw test substance in 20 mL/kg CMC 0.5 %) and in the negative control group (20 ml/kg bw CMC 0.5%). 16, 24 and 48 hours after treatment, 8 mice/sex/sampling time were sacrificed. Positive control was conducted on 8 mice/sex with 64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5 % which were sacrificed at 24 h.
- Part 2: The preparation was administered orally to groups of 8 mice/sex in the treatment groups (1250, 2500 and 5000 mg/kg bw, each in 20 mL/kg bw CMC 0.5%), in the negative control group (20 ml/kg bw CMC 0.5 %) and in the positive control group (64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5 %). 24 hours after treatment all animals were sacrificed.
Animals were treated once by gavage, after each sampling time, bone marrow smears were prepared from the shafts of both femurs. Prior to analysis the slides were coded. Thereafter the quality of staining was evaluated. The slides of 5 animals/sex showing the best differentiation between mature and polychromatic erythrocytes were selected for later scoring from each group and sampling tim

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs with foetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a glass slide and the preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grunwald solution for 3 min then in May-Grünwald solution / water 1/1 for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted. Giemsa solution (16.6 %) for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

METHOD OF ANALYSIS:
Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later scoring. In the first experiment the slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. In the second experiment the slides of five female and five male animals each of the negative control group, the positive control group and of the three dosage groups sacrificed at 24 hours post treatment. were examined.
1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

OTHER:
Analytical control: to prove that the animals were really exposed to the intended doses of the test substance, determination of the substance in suspension was performed by the analytical unit. This determination was performed with the highest dose of the suspension used in part 1 and in part 2 of the mutagenicity test.

Evaluation criteria:

INTERPRETATION OF RESULTS
A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any dose and sampling time respectively.

ASSAY ACCEPTANCE CRITERIA
- The quality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes.
- The result obtained with the positive control has to fulfil the criteria given for a positive response.

Statistics:

The significance of difference was assessed by x2-test. A test substance was considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any dose and sampling time respectively.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

RESULTS

The analysed concentrations used were in agreement with the calculated concentrations. In the first experiment (see Table 1 in 'Any other information on results incl. tables) 2 male animals died within the treatment period of 16 hours. The bone marrow smears from the animals treated with the dose of 5000 mg/kg of lufenuron showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours.

The second experimental part (see Table 2 in 'Any other information on results incl. tables) was performed with dosages of 1250, 2500 and 5000 mg/kg bw test substance at the sampling time of 24 hours. Also in this second experiment the bone marrow smears at each dose levels showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the animals of the negative control group.

The respective positive control experiments with cyclophosphamide yielded an average of 0.98% (first part) and 1.06 % (second part) polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06 % and 0.05 % respectively) treated with the vehicle (CMC 0.5 %) alone (p<0.05). The ratio of polychromatic/normochromatic erythrocytes was similar in all groups.

Table 1 Number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (p/n) erythrocytes, arithmetic mean per sex and group. Part 1

Sacrifice	Dose	sex	PCE	NCE	Ratio of PCE/NCE	n.0 of PCE with micronuclei	% of PCE with micronuclei
16 h	Control 0.5 % CMC	F	426	574	0.7	0.2	0.02
		M	469	531	0.9	0.4	0.04
	Test substance 5000 mg/kg	F	544	456	1.2	0.4	0.04
		M	482	518	0.9	0.4	0.04
24 h	Control 0.5 % CMC	F	470	530	0.9	0.8	0.08
		M	444	556	0.8	0.4	0.04
	Test substance 5000 mg/kg	F	484	516	0.9	0.2	0.02
		M	447	553	0.8	0.2	0.02
48 h	Control 0.5 % CMC	F	433	567	0.8	0.6	0.06
		M	462	538	0.9	0.2	0.02
	Test substance 5000 mg/kg	F	477	523	0.9	0.2	0.02
		M	452	548	0.8	0.2	0.02
Positive control
24 h	Control 0.5 % CMC	F	470	530	0.9	0.8	0.08
		M	444	556	0.8	0.4	0.04
	CP 64 mg/kg	F	399	601	0.7	8.2	0.82
		M	383	617	0.6	11.4	1.14

PCE : polychromatic erythrocytes

NCE : normochromatic erythrocytes

Table 2 Number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (p/n) erythrocytes, arithmetic mean per sex and group. Part 2

Sacrifice	Dose	sex	PCE	NCE	Ratio of PCE/NCE	n.0 of PCE with micronuclei	% of PCE with micronuclei
24 h	Control 0.5 % CMC	F	424	576	0.7	0.6	0.06
		M	415	585	0.7	0.4	0.04
	Test substance 1250 mg/kg	F	460	540	0.9	2	0.2
		M	484	516	0.9	0.2	0.02
	Test substance 2500 mg/kg	F	474	526	0.9	1	0.1
		M	478	522	0.9	0.8	0.08
	Test substance 5000 mg/kg	F	440	560	0.8	1	0.1
		M	454	546	0.8	1.4	0.14
	CP 64 mg/kg	F	467	533	0.9	7.8	0.78
		M	470	530	0.9	13.4	1.34

PCE : polychromatic erythrocytes

NCE : normochromatic erythrocytes

Conclusions:

The results indicate that under the given experimental conditions no evidence of mutagenic effects was obtained in mice treated with test substance.

Executive summary:

The test substance was evaluated for its ability to induce micronuclei in polychromatic erythrocytes (PCE’s) of male and female Tif: MAGF(SPF) mouse bone marrow in a study following GLP principles and performed similar to OECD TG 474. A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. Three groups of 4 mice (2/sex) were treated with 3 single doses, one receiving the maximum dose of 5000 mg/kg bw, or the highest applicable dose, and the other two doses of 1/5 and 1/25 of that amount respectively. The highest dose causing no death was used as the highest in the mutagenicity test. In this experiment all animals survived and the dose of 5000 mg/kg bw was determined as the highest to be applied in the mutagenicity assay. In the subsequent mutagenicity the test substance was administered orally to groups of 24 mice/sex in the treatment group (5000 mg/kg bw test substance in 20 mL/kg CMC 0.5%) and in the negative control group (20 mL/kg bw CMC 0.5%). 16, 24 and 48 hours after treatment, 8 mice/sex/sampling time were sacrificed. Positive control was conducted on 8 mice/sex with 64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5% which were sacrificed at 24 h.

In the first experiment, 2 male animals died within the treatment period of 16 hours. The bone marrow smears from the animals treated with the dose of 5000 mg/kg of test substance showed no statistically significant increase (p>0.05) in the number of micronucleated PCE’s in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours. The second experimental part was performed with dosages of 1250, 2500 and 5000 mg/kg bw test substance at the sampling time of 24 hours. Also in this second experiment the bone marrow smears at each dose levels showed no statistically significant increase (p>0.05) in the number of micronucleated PCE’s in comparison with the animals of the negative control group. The respective positive control experiments with cyclophosphamide yielded an average of 0.98 % (first part) and 1.06 % (second part)PCE’s with micronuclei. This is significantly different from the controls (0.06 % and 0.05 % respectively) treated with the vehicle (CMC 0.5 %) alone (p<0.05). The ratio of polychromatic/normochromatic erythrocytes was similar in all groups. It was concluded that, under the given experimental conditions, no evidence of mutagenic effects was obtained in mice treated with treated.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jul 1999 to 16 Dec 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 1997

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

HanIbM: WIST(SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 -10 weeks
- Weight at study initiation: 217 - 297 g
- Assigned to test groups randomly: Yes
- Diet: Pelleted, certified standard diet ad libitum up to 12 hours before dosing
- Water: Tap water ad libitum (quality according to the specifications of the "Schweizerisches Lebensmittelbuch, Edition 1972)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -21
- Humidity (%): 33 – 80
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Jul 1999 To: 16 Dec 1999

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % w/v carboxymethylcellulose (CMC) in bi-distilled water.
- Concentration of test substance in vehicle: 100 and 200 mg/mL
- Amount of vehicle: 10 mL/kg for test substance and vehicle control; 10 mL/kg for positive control

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The suspension of the test substance and the solutions of the positive control substances were prepared freshly for each application.
A tolerability test was not performed. Since in vivo-data indicated that the substance is well tolerated by laboratory animals at the test limit dose of 2000 mg/kg bw, the doses of 1000 and 2000 mg/kg bw were applied in the DNA repair test. Hence, test substance doses of 1000 and 2000 mg/kg body weight were applied in the DNA repair test.
A negative control (vehicle, 10 mL/kg was included in each part of the study. In the first part, the promutagen dimethylnitrosamine (DMN, 10 mg/kg, 10 mL/kg) dissolved in bi-distilled water was chosen as the positive control. In the second part, the positive control was 2-acetylaminofluorene (2-AAF, 100 mg/kg, 10 mL/kg) dissolved in arachis oil.

Frequency of treatment:

Single application

Post exposure period:

Part 1: 4 hours
Part 2: 16 hours

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

3 males per treatment and vehicle control group, 2 males per positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

In the first part: dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): Known to elicit a positive response in the in vivo UDS
- Route of administration: Oral gavage
- Doses: 10 mg/kg bw

In the second part: 2-acetylaminofluorene (2-AAF)
- Justification for choice of positive control(s): Known to elicit a positive response in the in vivo UDS
- Route of administration: Oral gavage
- Doses: 100 mg/kg bw

Tissues and cell types examined:

Liver tissue; hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Guideline limit dose as determined in Phase I of the study

TREATMENT AND SAMPLING TIMES:
- DETAILS OF SLIDE PREPARATION:
Primary hepatocytes were freshly isolated by in situ-collagenase perfusion according to a method described by Berry and Friend, 1969 and by L.R. Schwarz et al, 1979. The procedure was as follows: the liver was perfused in situ through the portal vein for 8 to 12 minutes with calcium-free Hanks' solution (BSS) supplemented with EGTA (ethylene glycol-bis[β-aminoethyl ether] N, N, N’,N’- acid, 0.5 mmol/L) and NaHCO3 and aerated with carbogen (95 % O2, 5 % CO2) to adjust the pH to about 7.3 to 7.4. Its temperature was kept at about 37 °C. Thereafter, the perfusion was continued for about a further 8 to 12 minutes with BSS, which was supplemented with 0.05 % collagenase, 2 mmol/L CaCl2 and NaHCaO3 and aerated with carbogen to adjust the pH to about 7.3 to 7.4. The liver was then carefully excised and placed into a dish containing the following wash solution: BSS supplemented with 2 mmol/L CaCl2, 0.4 mMol/L MgSO4, 0.5 % BSA (bovine serum albumin) and NaHCO3 and aerated with carbogen to adjust the pH to about 7.3 to 7.4 (4 °C). After opening the Glisson's capsule, the cells were dispersed by gently shaking of the liver in the solution. The cells were then filtered (mesh width of 61 µm) and washed twice with the same wash solution (sedimentation rate of 50 g for 3 minutes at about 2 to 4 °C). Finally, the cells were suspended in Williams's medium E and analysed for viability by Trypan blue exclusion. The viability of hepatocytes prepared in this way, was tabulated.
Freshly isolated male rat hepatocytes were cultured in Williams’s medium E containing 10 % foetal bovine serum, 1 % glutamine, 100 U penicillin, 100 µg/mL streptomycin and 2.5 µg/mL amphotericin (culture medium) and incubated in a humidified atmosphere with 5 % CO2 at 37 °C. A series of compartments in multiplates containing gelatinised cover-slips were seeded with 4x E05 cells per compartment (density E06 cells/mL; 4 mL/compartment). The cells were allowed to attach to the cover-slips during an attachment period of 1.5 - 2 hours. Unattached cells were then removed by washing with BSS and the cultures were refed with culture medium (2 mL/compartment).
The UDS assay was initiated by adding 10 µCi/mL 6-3H-thymidine activity, 24 Curies/mmol). After 4 hours incubation the cells were washed twice with BSS and further incubated overnight in renewed Williams's medium. In the morning, the cells were washed twice with BSS. The nuclei were swollen by treatment with 1 % sodium citrate for 10 min. Then the cells were fixed with ethanol/acetic acid, 3/1, v/v. The coverslips were mounted on microscope slides and prepared for autoradiography. Four slides from each animal were autoradiographed; the remainder were kept in reserve. Preparation and development of autoradiographs were performed in a room at 20 °C. Autoradiographic emulsion Ilford K.5 (diluted with two vol. H2O) was used as film material. Preparing the emulsion and coating the slides were performed under red light conditions.
The air-dried slides were exposed in light- and airproof boxes containing desiccants at 4 °C for five days.
Autoradiographs were developed in Kodak Developer D-l9, rinsed in acetic acid (1 %) and fixed in Hypam solution (Ilford: diluted 1:10 in water). The autoradiographs were stained in hematoxylin solution, rinsed in tap water and counter-stained in eosine..

METHOD OF ANALYSIS
Prior to scoring, the slides were coded. Silver grains over the nuclei and of the hepatocytes were counted using an electronic counter (DOMINO) attached to a microscope (ZElSS) at a magnification of 2000x. Two slides (50 cells/slide) from each animal of the treatment groups and of the respective positive and the negative controls were scored. The number of silver grains over the (nuclei grain counts) were counted and the mean values with the standard deviations were calculated.
The incorporation of radioactive material in the cytoplasm was determined by counting the silver grains in three cytoplasm regions adjacent to the nucleus, each with an area equivalent to that of the nucleus (cytoplasmic grain counts). The results were tabulated. The net nuclear grain counts were calculated by subtracting the average grain count over the cytoplasm from the average grain count over the nuclei.
To make sure that not only effect on the majority of the cells within the treated hepatocyte population were recognised but also effects on particularly sensitive subpopulations of the hepatocyte culture, the percentage of cells in repair (percentage of cells with a net nuclear grain count value of 2.0 or more) was calculated for each slide.
Cells which are in the S-phase show >120 silver grains/nucleus. These cells were excluded from the of the determination of the silver grain/nucleus count.

Evaluation criteria:

a) The results of the experiments should not be influenced by a technical error, contamination or a recognised artefact.
b) The nuclear labelling in the vehicle control cultures should not exceed an average of eight total grains/nucleus.
c) The net nuclear labelling in the vehicle control cultures should not exceed an average of two grains per nucleus.
d) The positive control should full the criteria given for a positive response.
e) Grain count data for a given treated animal must be obtained from at least two replicate cultures and at least 50 cells per culture

Statistics:

Not performed

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

RESULTS

QUALITY OF HEPATOCYTE PREPARATIONS

Hepatocyte preparations of good quality were obtained with all animals. Viability after perfusion was determined to be between 84 and 96 %

 

UNSCHEDULED DNA SYNTHESIS (UDS)

In both parts of the study, the test substance was administered to 2 groups of 3 animals each at the doses of 2000 and 1000 mg/kg body weight and the animals were sacrificed 4 hours thereafter (part 1) or 16 hours thereafter (part 2). No symptoms of toxicity were observed with any of the animals. Evaluation of the mean nuclear counts of silver grains in the vehicle controls and after treatment with test substance revealed no marked differences. In consideration of the silver grain number over the cytoplasm, the mean net nuclear grain counts were calculated. At both dose levels (2000 and 1000 mg/kg) there was no marked difference compared with the negative control. The percentage of cells in repair (net nuclear grain count 2.0) in part 1 at both concentrations and in part 2 at the concentration of 2000 mg/kg was slightly higher than the value of the corresponding negative control. Such effects are also occasionally found in other DNA repair studies and are attributed to spontaneous fluctuations in the number of cells with ≥ 2.0 of net nuclear grain counts. Since the three elevated values concerned are clearly within the historical negative control range, and the negative controls are near the lower limit of the historical negative control range, the effects observed in this study are considered to be of no biological relevance.

By contrast, the positive controls dimethylnitrosamine (10 mg/kg) and 2-acetylaminofluorene (100 mg/kg) yielded a marked increase in the mean nuclear grain counts and the mean net nuclear grain counts in comparison with the negative control. The percentage of cells in repair was found also clearly increased.

 

EXTRACELLULAR BACKGROUND

The background in the autoradiographs (outside he cells) determined microscopically cell-free in areas was found to be negligible low (data not shown).

 

ANALYTICAL RESULTS

The test material in suspension was analysed by HPLC with UV detection to confirm the intended concentrations to be used in the DNA repair test and the stability of the test substance in the vehicle used.

Table 2 Summary of results: group mean values: in vivo/in vitro UDS test in rat hepatocytes

Treatment	NG ± SD	CG ± SD	NNG ± SD	NG ± 2	NG ± 2 (%)
16 hours treatment (part 1)
Vehicle control (CMC 0.5 %)*	2.65 ± 0.50	2.98 ± 0.60	- 0.33 ± 0.10	2.4 ± 0.0	5.7 ± 4.2
Positive control (DMN)**	19.13 ± 1.49	3.52 ± 0.30	15.60 ± 1.19	15.6 ± 1.2	100.0 ± 0.0
Test substance, 2000 mg/kg bw*	3.18 ± 0.12	3.32 ± 0.19	- 0.15 ± 0.21	2.8 ± 0.1	10.0 ± 1.7
Test substance, 1000 mg/kg bw*	3.20 ± 0.08	3.12 ± 0.16	0.08 ± 0.21	2.4 ± 0.3	10.7 ± 3.5
4 hours treatment (part 2)
Vehicle control (CMC 0.5 %)*	2.77 ± 0.21	2.70 ± 0.10	0.07 ± 0.12	2.5 ± 0.3	8.3 ± 4.2
Positive control (2-AAF)**	14.06 ± 1.16	3.57 ± 0.36	10.49 ± 0.80	10.6 ± 0.9	99.5 ± 0.7
Test substance, 2000 mg/kg bw*	3.36 ± 1.06	2.93 ± 0.30	0.44 ± 0.82	3.2 ± 0.7	19.0 ± 14.7
Test substance, 1000 mg/kg bw*	2.84 ± 0.24	2.76 ± 0.21	0.08 ± 0.25	3.1 ± 0.3	10.3 ± 5.1

NG: Mean nuclear grain counts

CG: Mean cytoplasmic grain count (mean of 3 per cell)

NNG: Mean net nuclear grain count

NG 2: Net grain count of cells in repair

NG 2 (%): Percentage of cells in repair

SD: Standard deviation

*) mean from 3 animals, 2 slides per animal, for a total of 300 nuclei scored

**) mean from 2 animals, 2 slides per animal, for a total of 200 nuclei scored

Table 3 Historical controls Percent cells in repair (NG ≥ 2) means per animal

Study No.	Vehicle control (2-4h)	Vehicle control (12-16h)	Positive control (2-AAF, 100 mg/kg bw)	Positive control (DMN, 10 mg/kg bw)
933031	31.0^b	--	--	99.0^d
	33.0^b	--	--	100.0^d
	24.0^b	--	--	99.0^d
	25.0^b	--	--	100.0^d
932124	--	17.0^b	--	100.0^d
	--	13.0^b	--	99.0^d
	--	29.0^b	--	100.0^d
	--	16.0^b	--	100.0^d
941062	11.0^a	11.0^a	--	100.0
	14.0^a	5.0^a	--	98.0
	17.0^a	9.0^a	--	100.0
	19.0^a	8.0^a		100.0
	10.0^a	7.0^a		99.0
	12.0^a	7.0^a		100.0
961122	--	12.0^c	--	100.0
	--	11.0^c	--	100.0
	--	4.0^c	--	100.0
	--	8.0^c	--	100.0
972809	7.0^a	28.0^a	81.0	98.0
	16.0^a	31.0^a	84.0	100.0
	13.0^a	43.0^a	--	--
983065	7.0^b	8.0^b	99.0	98.0
	11.0^b	7.0^b	98.0	100.0
	4.0^b	6.0^b	--	100.0
983139	12.0^b	9.0^b	93.0	99.0
	12.0^b	12.0^b	99.0	96.0
	6.0^b	8.0^b	--	--

a) Arachis oil

b) CMC, 0.5 %

c) CMC, 0.5% / Tween 80, 0.1 %

d) DMN: 15 mg/kg

Conclusions:

It is concluded from the results of both experiments, that no evidence of induction of DNA repair activity by the test substance was found in this test system.

Executive summary:

The current study was performed to report on the in vivo/in vitro unscheduled DNA synthesis in rat hepatocytes according to GLP; and was carried out according to OECD TG486 and E.U. method B.39. The assay was designed to measure unscheduled DNA synthesis (UDS, DNA-repair synthesis in isolated rat liver cells as a consequence of DNA-damage induced after in vivo treatment of the animals with the test substance.

Primary hepatocytes were freshly isolated from 9 - 10 weeks old male rat HanIbM: WIST(SPF) rats previously given the test substance. In total 3 rats/test group and vehicle control group and 2 rats/positive control group were treated once orally by a stomach tube at 1000 and 2000 mg/kg bw/day. The high dose was the test limit dose. The vehicle used was carboxymethylcellulose (CMC) at 0.5 % w/v in dissolved in bi-distilled water.

In the first part of the study, hepatocytes from animals treated with the test material, the negative or positive control (dimethylnitrosamine) were isolated 4 hours after application. In the second part of the study, hepatocytes from animals treated with the test material, the negative or positive control (2-acetylaminofluorene) were isolated 16 hours after application. The liver of the rats was perfused with collagenase containing buffer in order to prepare

hepatocytes. Two slides (50 cells/slide) from each animals of each group were scored. DNA-repair was determined by scoring autoradiographs (nuclear grain counts, cytoplasmic grain counts, net nuclear grain counts and percentage of cells in repair were calculated).

No signs of toxicity were observed in the animals treated with the test material. Hepatocyte preparations of good quality were obtained with all animals. Viability after perfusion was determined to be between 84 and 96 %. Evaluation of the mean nuclear counts of silver grains in the vehicle controls and after treatment with test substance revealed no marked differences.

The percentage of cells in repair (net nuclear grain count ≥2.0) in part 1 at both concentrations and in part 2 at the concentration of 2000 mg/kg bw was slightly higher than the value of the corresponding negative control. Such effects were noted to be also occasionally found in other DNA repair studies and were attributed to spontaneous fluctuations in the number of cells with ≥2.0 of net nuclear grain counts. Since the 3 elevated values concerned were clearly within the historical negative control range, and the negative controls were near the lower limit of the historical negative control range, the effects observed in this study were considered to be of no biological relevance.

It is concluded under the given experimental conditions, no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA. A test strain to identify cross-linking mutagens is missing in the AMES test, however, since sufficient in vitro and vivo data is available, this deficiency is not considered to affect the overall conclusion on genotoxicity. All of the assays conducted were negative and it is concluded that the test substance is not genotoxic. A metabolite of the test substance did not induce revertants in the Ames test.

AMES, Deparade 1988

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test and the pre-incubation test. For this purpose, the Salmonella typhimurium histidine-auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100 and Aroclor 1254 pre-treated liver microsomes of rats (Tif: RAIf(SPF)) were used according to GLP principles and OECD TG 471. A preliminary toxicity test was carried out with strain TA 100 without activation with 9 concentrations ranging from 0.08 to 5000 μg/0.1 mL. The protocol used is the same as in the mutagenicity test. The investigations were performed on Salmonella strains with 20, 78, 313, 1250 and 5000 μg/0.1 mL trial substance without and with microsomal activation (S9-mix) using liver microsomes of rats (Tif: RAIf(SPF)) pre-treated with Aroclor 1254. The substance was dissolved in acetone. Acetone alone was used for the negative controls. In the experiments without and with the addition of microsomal activation mixture 3 Petri dishes are prepared per strain and per group (i.e. per concentration or per control group). The plates are incubated for about 48 hours at 37 ± 1.5 °C in darkness.

The test material in solution was analysed to confirm the intended concentrations to be used in the mutagenicity tests. The values of the lowest concentrations to be analysed were found to be somewhat above the calculated concentrations, mainly due to the fast evaporation of the vehicle acetone. This, however, did not affect the test results.

From the results obtained in the toxicity test, the highest concentration suitable for the mutagenicity test was found to be 5000 μg/0.1 mL. In two separate experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar. The sensitivity of the test system, and the metabolic activity of the S9-mix, was clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

To conclude, no induction of point mutation by the test substance or by its metabolites was detectable in the tested strains and this assay system did not show no evidence of genotoxic activity.

 

In vitro chromosome aberration assay, Strasse 1988

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation, according to GLP principles and OECD TG 473. The substance dissolved in DMSO was added (1:100) to the cells line CCL 61 in culture medium. The cells were exposed for 3 hours to 10 concentrations ranging from 3.13 to 1600.0 µg/mL of the test substance. After removal of the test substance, the cells were washed and incubated in culture medium for 17 hours. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration. This preliminary toxicity test was performed with and without metabolic activation.

In the mutagenicity test, cultures were treated for a period of 3 hours in both the presence and absence of S9 mix with the pre-selected test substance concentrations with the positive control, or with the vehicle as negative control. The test substance dissolved in DMSO was used as the negative control. Positive controls were mitomycin C, 1.0 µg/mL (in the absence of S9 mix) and cyclophosphamide, 40.0 µg/mL (in the presence of S9 mix). After treatment, the cells were washed to remove the test substance, after that new culture medium was added and the cells were allowed to grow for 4 hours and for 21 hours. Two hours prior to harvesting, the cultures were treated with Colcemide. The lowest concentration of test material in solution was analysed to confirm the intended concentration to be used in the mutagenicity tests. The value found was in agreement with the calculated concentration.

In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using a chi-square test. The non-significant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly a chi square test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell was taken as the experimental unit.

As a result in the cytotoxicity test, the highest concentration for the mutagenicity assay without microsomal activation was 200.0 µg/mL (46.6 % mitotic index frequency of control) and 1600.0 µg/mL (59.4 % mitotic index frequency of control) for that with activation.

From the mutagenicity test, it could be demonstrated that the high level of specific aberrations found in the negative control groups in the original scoring, were due to the scoring protocol used at that time. Based on current assay evaluation criteria and on statistical analysis, no evidence of an induction of chromosome aberrations by the test substance was found.

Hence, it was concluded that under the given experimental conditions no evidence of mutagenic effects was obtained in CHO cells in vitro treated with test substance.

 

In vitro gene mutation assay, Dollenmeier 1988

The test substance was evaluated for induction of point mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V79, derived from Chinese hamster lung cells according to OECD TG 476 and GLP principles. A cytotoxicity test was performed on the cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. The highest concentration to be selected for the mutagenicity assay was that causing about 90 % reduction of viable cells in the cytotoxicity test in comparison with the solvent controls. In the subsequent mutagenicity test, the potential of the test substance to cause gene mutation in mammalian cells (V79) treated in vitro. The auxiliary metabolic system (S9-mix) used was derived from the liver of rats and treated with Aroclor 1254. Mutant frequencies were assessed by cell growth in the presence of 6-thioguanine (6-TG). The cells were treated with 37.5, 75.0, 150, 300, 450, 600 and μg/mL with and 25.0, 50.0, 100, 200, 300, 400 and 500 μg/mL without metabolic activation. The exposure duration was 5 hours (with metabolic activation) or 21 hours (without metabolic activation).

In the preliminary toxicity test, a 90 % reduction in the viability of the cells treated with test substance was obtained at the concentration of 750 μg/mL in the experiment with microsomal activation and between the concentrations of 375 and 750 μg/mL in the experiment without microsomal activation, a 100 % cytotoxicity was found at 1500 μg/mL and above.

In both investigations, each performed without and with microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 1x E06 cells plated was not detected at any concentration of the test substance, and there was also no indication of any concentration-mutant frequency relation in either experiment.

It was therefore concluded that, under the given experimental conditions, test substance and its metabolites induced no mutagenic effects in this forward mutation system.

 

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus, Meyer 1989

The test substance was evaluated for its ability to induce micronuclei in polychromatic erythrocytes (PCE’s) of male and female Tif: MAGF(SPF) mouse bone marrow in a study following GLP principles and performed similar to OECD TG 474.A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. Three groups of 4 mice (2/sex) were treated with 3 single doses, one receiving the maximum dose of 5000 mg/kg bw, or the highest applicable dose, and the other two doses of 1/5 and 1/25 of that amount respectively. The highest dose causing no death was used as the highest in the mutagenicity test. In this experiment all animals survived and the dose of 5000 mg/kg bw was determined as the highest to be applied in the mutagenicity assay. In the subsequent mutagenicity the test substance was administered orally to groups of 24 mice/sex in the treatment group (5000 mg/kg bw test substance in 20 mL/kg CMC 0.5%) and in the negative control group (20 mL/kg bw CMC 0.5%). 16, 24 and 48 hours after treatment, 8 mice/sex/sampling time were sacrificed. Positive control was conducted on 8 mice/sex with 64 mg/kg bw cyclophosphamide in 20 mL/kg bw CMC 0.5% which were sacrificed at 24 h.

In the first experiment, 2 male animals died within the treatment period of 16 hours. The bone marrow smears from the animals treated with the dose of 5000 mg/kg of test substance showed no statistically significant increase (p>0.05) in the number of micronucleated PCE’s in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours. The second experimental part was performed with dosages of 1250, 2500 and 5000 mg/kg bw test substance at the sampling time of 24 hours. Also in this second experiment the bone marrow smears at each dose levels showed no statistically significant increase (p>0.05) in the number of micronucleated PCE’s in comparison with the animals of the negative control group. The respective positive control experiments with cyclophosphamide yielded an average of 0.98 % (first part) and 1.06 % (second part)PCE’s with micronuclei. This is significantly different from the controls (0.06 % and 0.05 % respectively) treated with the vehicle (CMC 0.5 %) alone (p<0.05). The ratio of polychromatic/normochromatic erythrocytes was similar in all groups. It was concluded that, under the given experimental conditions, no evidence of mutagenic effects was obtained in mice treated with treated.

 

In vivo mammalian cell study: DNA damage and/or repair, Ogorek 2000

The test substance has been evaluated, using an autoradiographic technique, for its ability to induce unscheduled DNA synthesis (UDS) in the liver of male HanIbM: WIST(SPF) rats according to OECD TG 486 and GLP principles. The test substance was administered orally by stomach tubes to groups of 3 male rats at a dose level of 2000 mg/kg rats/test group and vehicle control group and 2 rats/positive control group.A tolerability test was not performed. Since in vivo-data indicated that the substance is well tolerated by laboratory animals at the test limit dose of 2000 mg/kg bw, the doses of 1000 and 2000 mg/kg bw were applied in the DNA repair test. The compound was dissolved in CMC (0.5 %) and administered orally at single doses of 1000 and 2000 mg/kg bw. The high dose was the test limit dose. In the first part of the study, hepatocytes from animals treated with the test material, the negative or positive control (dimethylnitrosamine, 10 mg/kg) were isolated 4 hours after application. In the second part of the study, hepatocytes from animals treated with the test material, the negative or positive control (2-acetylaminofluorene, 100 mg/kg) were isolated 16 hours after application.

The liver of the rats was perfused with collagenase containing buffer in order to prepare hepatocytes. Following attachment of the cells to coverslips they were incubated in Williams’s medium containing 10 µCi/mL 3H-thymidine. Autoradiographs were prepared and slides were coded prior to analysis. Silver grains over the nuclei and cytoplasm of the hepatocytes were counted using a microscope with a video camera connected to an image analysis system. Two slides (50 cells/slide) from each animals of each group were scored. DNA-repair was determined by scoring autoradiographs (nuclear grain counts, cytoplasmic grain counts, net nuclear grain counts and percentage of cells in repair were calculated). The test material in suspension was analysed by HPLC with UV detection to confirm the intended concentrations used in the DNA repair tests and the stability of the test substance in the vehicle.

Analytical results of the test material in suspension confirmed the intended concentrations used in the DNA repair test and the stability of the test substance in the vehicle. No signs of toxicity were observed in the animals treated with the test material. Hepatocyte preparations of good quality were obtained with all animals. Viability after perfusion was determined to be between 84 and 96 %. The background in the autoradiographs (outside the cells) determined microscopically in cell-free areas was found to be negligibly low. Evaluation of the mean nuclear counts of silver grains in the vehicle controls and after treatment with test substance revealed no marked differences. In consideration of the silver grain number over the cytoplasm, the mean net nuclear grain counts were calculated. At both dose levels (2000 and 1000 mg/kg bw) there was no marked difference compared with the negative control. The percentage of cells in repair (net nuclear grain count 2.0) in part 1 at both concentrations and in part 2 at the concentration of 2000 mg/kg bw was slightly higher than the value of the corresponding negative control. Such effects were noted to be also occasionally found in other DNA repair studies and were attributed to spontaneous fluctuations in the number of cells with 2.0 of net nuclear grain counts. Since the 3 elevated values concerned were clearly within the historical negative control range, and the negative controls were near the lower limit of the historical negative control range, the effects observed in this study were considered to be of not of biological relevance. By contrast, the positive controls yielded a marked increase in the mean nuclear grain counts, in the mean net nuclear grain counts as well as in the percentage of cells in repair in comparison with the negative control.

Under the given experimental conditions, it was concluded that no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

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.