4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutation assay (Ames test / Salm. typh.) - key study:  

The study was performed 1985 as GLP-test following EC-test method B.14 on Salmonella typhimurium strains TA 98, 100, 1535, 1537 and 1538. P5367 was dissolved in DMSO and tested at concentrations of 8 – 5000 µg/plate, with and without metabolic activation (Aroclor 1254 induced liver S9-mix). Cytoxicity was observed at concentrations above 5000 µg/plate, however, there was no increase in revertants in any strain following exposure to test substance. In conclusion, the substance is not mutagenic with and without metabolic activation.  

  

Bacterial mutation assay (Ames test / E. coli) - supporting study:  

The study was performed 1996 as GLP-test following EC-test method B.13 on E. coli WP2 uvrA tpr strains. P5367 was dissolved in DMSO and tested at concentrations of 312.5 - 5000 µg/plate, with and without metabolic activation (Aroclor 1254 induced liver S9-mix). Cytoxicity was not observed up to the highest concentration of 5000 µg/plate. No evidence of mutagenic activity was seen at any dose level of P5367 in either mutation test. It is concluded that, when tested in dimethyl sulphoxide, P5367 shows no evidence of mutagenic activity in this bacterial system.  

  

Chromosomal aberration in vitro (human lymphocytes) - key study:  

The study was performed 1987 as GLP-test following EC-test method B.10 on cultured human lymphocytes. The test item was dissolved in DMSO and tested at concentrations of 10 - 333 µg/ml, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was observed at concentrations above 333 µg/ml without S9 -mix and above 1000 µg/ml with S9 -mix. In conclusion, the substance is not clastogenic with and without metabolic activation under the conditions of this test.  

  

Chromosomal aberration in vitro (CHL-cells) - supporting study: 

The study was performed 1996 as GLP-test following OECD-test method 473 and JAPAN: Guidelines for Screening Mutagenicity Testing of Chemicals on Chinese hamster lung cells. The test item was dissolved in DMSO and tested at concentrations of 0.08 - 5000 µg/ml, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was observed starting at concentrations of 78.1 µg/ml without S9-mix and 156.3 µg/ml with S9 -mix. It is concluded that P5367 has shown no evidence of clastogenic or polyploidy-inducing activity in this in vitro cytogenetic test system.  

  

UDS-test in vitro - key study:  

The study was performed 1988 as GLP-test following OECD-test method 482 using rat hepatocytes. The test item was dissolved in DMSO. The original experiment was performed with concentrations of 5, 50, 100, 200, 500 and 700 µg/ml, the confirmatory experiment with concentrations of 1, 5, 12.5, 25, 50, 100, 150 and 200 µg/ml and the repeat experiment with concentrations of 0.2, 1, 5, 10, 20, 30, 40, 50, 60 and 80 µg/ml.  Cytoxicity was observed at concentrations above 60 µg/ml. It is concluded that, under the given experimental conditions, evidence of a weak induction of DNA damage by P 5367 or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance. 

 

Waiving of in-vitro gene mutation assay:

A number of mutagenicity studies on in-vitro and in.-vivo systems have been conducted, having all showed negative results (see above) allowing a proper assessment of the mutagenic/clastogenic potential of the test item. A weakly positive response was observed in an UDS-test in-vitro on rat hepatocytes only. Therefore, an additional in-vitro gene mutation test is not justified.

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:

April - June 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Dr. Bruce Ames, University of California, USA
- Cultures of all organisms were prepared by overnight incubation of freshly inoculated nutrient broth (Oxoid No. 2).

TA 1535:
contains a histidine missense mutation but is also deficient in a DNA repair system (uvr B) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to
frameshift mutagens.

TA 1537:
bears a histidine frameshift mutation. Like TA 1535, it is defective in DNA repair and in the lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.

TA 100
is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.

TA 98
is TA 1538 with the addition of the pKM 101 plasmid

Additional strain / cell type characteristics:

other: see above

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Dr. Bruce Ames, University of California, USA
- Cultures of all organisms were prepared by overnight incubation of freshly inoculated nutrient broth (Oxoid No. 2).

TA 1538
contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat. It is reverted by agents causing deletion of two adjacent basepairs (double frameshift mutations).

Additional strain / cell type characteristics:

other: see above

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

was tested in a separate study (see reference LR2718)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- Concentration range in the main test (with metabolic activation): 8 - 5000 µg/plate
- Concentration range in the main test (without metabolic activation): 8 - 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene: with and without S9-mix

Details on test system and experimental conditions:

A preliminary toxicity test was conducted at eight concentrations from 2.5 µg up to 5000 µg.

Rationale for test conditions:

A preliminary toxicity test was conducted at eight concentrations from 2.5 µg up to 5000 µg. In the main study, concentrations of 8 up to 5000 µg were tested.

Evaluation criteria:

no data

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 98

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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 examined

Positive controls validity:

valid

Additional information on results:

Observations:
No increase in revertants in any strain following exposure to test substance. Positive controls gave the expected response.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The test item was found to be non-mutagenic in this Ames test, in presence and absence of S9-mix.

Executive summary:

The study was performed 1985 as GLP-test following EC-test method B.14 on Salmonella typhimurium strains TA 98, 100, 1535, 1537 and 1538. P5367 was dissolved in DMSO and tested at concentrations of 8 - 5000 ug/plate, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was observed at concentrations above 5000 ug/plate, however, there was no increase in revertants in any strain following exposure to test substance.

In conclusion, the substance is not mutagenic with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: National Collection of Industrial Bacteria, Aberdeen, Scotland

Additional strain / cell type characteristics:

other: tryptophan dependent mutant

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- Concentration range in the main test (with metabolic activation): 312.5 - 5000 µg/plate
- Concentration range in the main test (without metabolic activation): 312.5 - 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene: with S9-mix

Rationale for test conditions:

In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625 and 312.5 µg/plate.

Evaluation criteria:

The mutagenic activity of a test substance is assessed by applying the following criteria:

1. If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with the bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

2. If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with the bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

3. If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989) and will usually be analysis of variance followed by Dunnett's test.

Statistics:

Dunnett's test.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No evidence of mutagenic activity was seen at any dose level of P5367 in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Conclusions:

Interpretation of results:
- negative with metabolic activation
- negative without metabolic activation

It is concluded that, when tested in dimethyl sulphoxide, P5367 shows no evidence of mutagenic activity in this bacterial system (in presence and absence of S9-mix).

Executive summary:

The study was performed 1996 as GLP-test following EC-test method B.13 on E. coli WP2 uvrA tpr strains. P5367 was dissolved in DMSO and tested at concentrations of 312.5 - 5000 µg/plate, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was not observed up to the highest concentration of 5000 ug/plate. No evidence of mutagenic activity was seen at any dose level of P5367 in either mutation test. It is concluded that, when tested in dimethyl sulphoxide, P5367 shows no evidence of mutagenic activity in this bacterial system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - March 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cytogenicity (B10)

Species / strain / cell type:

lymphocytes: cultured human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lympgocytes
- Sex, age and number of blood donors: healthy male adult

Blood samples:
Blood samples were taken from a heal~hy male adul~ volunteer by venapuncture using a sterile syringe. The blood was then immediately transferred to a suitable size sterile vessel containing sodium heparin. The specimen was then gently mixed to prevent clotting. Within 4 h af~er withdrawal lymphocyte cultures were set up.

Cell culture conditions:
0.4 ml blood was added to 5 ml or 4.8 ml (when S9-mix was included) culture medium supplemented with 20% inactivated foetal calf serum (Gibco), L-glutamine (2.0 mM), penicillin/streptomycin (50 U and 50 µg/ml, respectively), sodium bicarbonate (2 g/l) and 0.1 ml Phytohaemagglutinin. Cultures were incubated at 37°C for 72 h in a humid
atmosphere with 5% C02 in air. Approximately 48 h after the start of the culture the test substance was added.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- Concentration range in the main test (with metabolic activation): 10 - 333 µg/ml
- Concentration range in the main test (without metabolic activation): 10 - 100 µg/ml

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Details on test system and experimental conditions:

Fixation time: 23 hours

Evaluation criteria:

A test substance is considered positive (clastogenic) in the chromosome aberration test if:
- it induces a dose-related statistically significant (Chi-square test , P < 0.05) increase in the frequencies of chromosome aberrations
- a statistically significant increase in the frequency of aberrations is observed in the absence of a clear dose-response relationship, but the results are reproducible in an independently repeated experiment

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

333 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

333 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observations:
Mitotic index reduced to 35% to 67% of control without S9- and to 52% to 82% with S9-mix. Positive controls gave the expected responses.

Conclusions:

Interpretation of results:
- negative with metabolic activation
- negative without metabolic activation

The test substance is not clastogenic in human lymphocytes either in the presence or absencen of a metabolic activation system.

Executive summary:

The study was performed 1987 as GLP-test following EC-test method B.10 on cultured Human lymphocytes. The test item was dissolved in DMSO and tested at concentrations of 10 - 333 ug/ml, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was observed at concentrations above 333 ug/ml without S9 -mix and above 1000 ug/ml with S9 -mix. In conclusion, the substance is not clastogenic with and without metabolic activation under the conditions of this test.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster lung (CHL) cells, strain JCRB0030, were obtained from JCRB on 22 February 1988 by Safepharm Laboratories Ltd.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentration ranges:
- Set 1: 6 hours treatment, + S9-mix, harvest at 24 hours: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/ml
- Set 2: 6 hours treatment, - S9-mix, harvest at 24 hours: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/ml
- Set 3: 24 hours treatment, - S9-mix, harvest at 24 hours: 0.15, 0.3, 0.6, 1.2, 2.4, 4.9, 9.8, 19.5, 39.1 and 78.1 µg/ml
- Set 4: 48 hours treatment, - S9-mix, harvest at 48 hours: 0.08, 0.15, 0.3, 0.6, 1.2, 2.4, 4.9, 9.8, 19.5 and39.1 µg/ml

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Carbendazim / with 9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

The cells were then treated with the test substance. Four sets of treatments were used which are summarised as follows:
- Set 1: 6 hours treatment: +S-9, harvest at 24 hours
- Set 2: 6 hours treatment: -S-9, harvest at 24 hours
- Set 3: 24 hours treatment: -S-9, harvest at 24 hours
- Set 4: 48 hours treatment: -S-9, harvest at 48 hours

Evaluation criteria:

A test substance is considered positive (clastogenic) in the chromosome aberration test if:
- it induces a dose-related statistically significant (Chi-square test , P < 0.05) increase in the frequencies of chromosome aberrations
- a statistically significant increase in the frequency of aberrations is observed in the absence of a clear dose-response relationship, but the results are reproducible in an independently repeated experiment

Statistics:

Statistical analysis used done using Fisher's test.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

333 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observations:
Mitotic index reduced to 35% to 67% of control without S9- and to 52% to 82% with S9-mix. Positive controls gave the expected responses.

Conclusions:

Interpretation of results:
- negative with metabolic activation
- negative without metabolic activation

It is concluded that P5367 has shown no evidence of clastogenic or polyploidy-inducing activity in this in vitro cytogenetic test system.

Executive summary:

The study was performed 1996 as GLP-test following OECD test method 473 and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals on Chinese hamster lung cells. The test item was dissolved in DMSO and tested at concentrations of 0.08 - 5000 ug/ml, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was observed starting at concentrations of 78.1 ug/ml without S9 -mix and 156.3 ug/ml with S9 -mix. It is concluded that P5367 has shown no evidence of clastogenic or polyploidy-inducing activity in this in vitro cytogenetic test system.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

- Cell type: Primary hepatocytes freshly isolated from male rat.
- No. of cultures per group: 4
- Animals: Species/sex: Rat/male
- Strain: Tif RAIf (SPF)
- Source: Tierfarm Sisseln, Switzerland

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

none

Test concentrations with justification for top dose:

CONCENTRATION RANGES:

Cytotoxicity test:
57.2 ug/ml - 3300 ug/ml

DNA-repair test - original experiment:
5, 50, 100, 200, 500 and 700 ug/ml

Confirmatory experiment:
1, 5, 12.5, 25, 50, 100, 150 and 200 ug/ml

Repeat experiment:
0.2, 1, 5, 10, 20, 30, 40, 50, 60 and 80 ug/ml

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Aminobiphenyl (4-ABP)

Details on test system and experimental conditions:

- Exposure period (without metabolic activation): 16-18 hours
- Fixation time: 18 hours

Evaluation criteria:

Criteria for a positive response:
The test substance is generally considered to be active in the DNArepair test if one of the following conditions are met:
- The mean number of silver grains per nucleus in relation to the vehicle control is more than doubled at any concentration
- The mean value of the net nuclear grain counts at any concentration is 2.0 together with a difference to the value of the vehicle control of 2.0
- The percentage distribution of silver grains on nuclei reveals a significant difference in comparison with the vehicle control.
- The mean number of silver grains per nucleus in relation to the vehicle control shows a concentration dependent increase and at least at one concentration a statistically significant increase in comparison with the vehicle control is demonstrated. Statistical analysis will be carried out as follows: the values of silver grains per nucleus will be compared using Duncan's multiple range test. Statistical significance will be judged to be achieved if the probability is less than 0.01.

Criteria for a negative response:
The test substance is generally considered to be inactive in the DNA-repair test if the following conditions are met:
- The mean value of net grain counts is less than 2.0 at all concentrations
- The mean value of net grain counts differs from the corresponding vehicle control value by less than 2.0
- The mean number of silver grains per nucleus in relation to the vehicle control is less than doubled at all concentrations and no concentration dependence can be seen

Statistics:

Statistical analysis will be carried out as follows: the values of silver grains per nucleus will be compared using Duncan's multiple range test. Statistical significance will be judged to be achieved if the probability is less than 0.01.

Key result

Species / strain:

hepatocytes: primary hepatocytes from adult male rats (Tif.RAIf (SPF))

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 700 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

A mean value for the net grain counts per nucleus of more than 2 together with a difference from the value of the vehicle control of at least 2 was not observed in Test 1. In Test 2, such a results was recorded at 25 micrograms/ml only (mean value 4.13, compared with vehicle control value of 0.80). In Test 3, exposure to 10, 20 and 30 micrograms/ml resulted in mean values of 2.37, 2.06 and 2.28 respectively, compared with a vehicle control value of -1.53. In the original experiment no significant deviations from control values were observed with substance concentrations of 5, 50 and 100 micrograms/ml (higher concentrations proved to be toxic). In the confimatory experiment (1, 5, 12.5, 25, 50, 100, 50 and 200 micrograms/ml) and in a third experiment (0.2, 1, 5, 10, 20, 30, 40, 50, 60 and 80 micrograms/ml) there was, according to the notifer, some evidence of weak induction of DNA damage by the substance, or its metabolites; at concentrations between 10 and 30 micrograms/ml. The values recorded in results were accompanied in most cases by small increases in the percentage of nuclei with a net grain count value of more than 5 (maximum 33%; maximum in oncurrent vehicle controls 12%), but for neither end-point was there a clear response. These results would represent only a "marginal" response according to Butterworth et al (1987) Mutation Research Vol 189, p.123 -133.

Conclusions:

Interpretation of results: weakly positive without metabolic activation
No evidence for DNA damage and subsequent DNA repair was found in the in vitro UDS Test.

Executive summary:

The study was performed 1988 as GLP-test following OECD-test method 482 using rat hepatocytes. The test item was dissolved in DMSO. The original experiment was performed with concentrations of 5, 50, 100, 200, 500 and 700 ug/ml, the confirmatory experiment with concentrations of 1, 5, 12.5, 25, 50, 100, 150 and 200 ug/ml and the repeat experiment with concentrations of 0.2, 1, 5, 10, 20, 30, 40, 50, 60 and 80 ug/ml.

Cytoxicity was observed at concentrations above 60 ug/ml. It is concluded that, under the given experimental conditions, evidence of a weak induction of DNA damage by the test item or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus test in vivo - key study:  

 

The study was performed 1996 as GLP-test following OECD-test method 474 using bone marrow cells of CD-1 mice. The test item was dissolved in 1% CMC and tested at concentrations of 500 - 2000 µg/ml. Toxic signs were only observed in the high dose group after dosing. Treatment with the test substance did not result in any increase in the number of micro-nucleated polychromatic erythrocytes. In conclusion, the substance is not mutagenic under the conditions of this test.  

  

UDS-Test in vivo/in vitro - key study:  

 

The study was performed 1989 as GLP-test as UDS test in vitro/in vivo using rat hepatocytes. The test item was dissolved in 1.5% CMC. Three rats per dose level were treated with 1000, 500, 250 and 100 mg/kg in volumes which did not exceed 5.1 ml/kg. None of the criteria used to indicate UDS was approached by the treatments and no dose-related response was observed. The test material was therefore evaluated as inactive in the in vivo/in vitro rat hepatocyte Unscheduled DNA Synthesis assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - December 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: approximately 35 days
- Weight at study initiation: between 22 and 24 grams
- Assigned to test groups randomly: yes
- Housing: each group was kept, with the sexes separated, in plastic disposable cages
- Diet (e.g. ad libitum): pelleted Biosure LAD 1 rodent diet
- Water (e.g. ad libitum): tap water
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 55%
- Air changes (per hr): 20 changes/h
- Photoperiod (hrs dark / hrs light): artificial light 12 h/day

Route of administration:

intraperitoneal

Vehicle:

1% aqueous methylcellulose; suspensions ofthe test item were prepared in CMC (obtained from Courtaulds, batch number T32397) on the morning of the test at the concentrations shown overleaf

Details on exposure:

All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight. The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, orally by intragastric gavage. The animals in the positive control group were deprived of diet overnight prior to and for two hours after dosing.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Once

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

pretest; 2000 mg/kg = 100 mg/ml

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

main study; vehicle control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

main study; 500 mg/kg = 25 mg/ml

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

main study; 1000 mg/kg = 50 mg/ml

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

main study; 2000 mg/kg = 100 mg/ml

No. of animals per sex per dose:

Males:
- 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
- 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours

Females:
- 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
- 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes

Positive control(s):

A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Tissues and cell types examined:

Bone marrow smears were obtained from 5 male and 5 female animals in the negative control, test substance groups and the positive control group 24 hours after dosing. Bone marrow smears were also taken from 5 male and 5 female animals from the negative control and high dose level groups 48 hours after treatment. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micro nucleated normochromatic erythrocytes was also kept.

Evaluation criteria:

A positive response is normally indicated by a substantial, statistically significant increase (p < 0.01) in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group for at least one of the sampling times~ individual and/or group mean values should exceed the laboratory historical control range.

Statistics:

Non-parametric statistical methods, based on rank are chosen for analysis of results because:
- They are suited to analysis of data consisting of discrete/integer values such as the incidence of micronucleated polychromatic erythrocytes.
- 'Outliers' are frequently found in the polychromatic erythrocyte to normochromatic erythrocyte ratios for both control and
- The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxic signs were only observed in the high dose group after dosing. Thes signs included lethargy, hunched posture, piloerection and altered gait. The ratio of polychromatic to normochromatic erythrocytes was unaltered by treatment.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment with the test substance did not result in any increase in the number of miconucleated polychromatic erythrocytes.

Only minor clinical signs and no mortalities were obtained in the preliminary toxicity test at a dose level of 2000 mg/kg. Dose levels of 500, 1000 and 2000 mg/kg were therefore selected for use in the micronucleus test. The high dose level of 2000 mg/kg is the standard limit dose for the micronucleus test and we therefore consider it to be an appropriate maximum.

Conclusions:

Interpretation of results: negative
The test substance was found to be non-mutagenic in this test system.

Executive summary:

The study was performed 1996 as GLP-test following OECD test method 474 using bone marrow cells of CD-1 mice. The test item was dissolved in 1% CMC and tested at concentrations of 500 - 2000 ug/ml. Toxic signs were only observed in the high dose group after dosing. Treatment with the test substance did not result in any increase in the number of miconucleated polychromatic erythrocytes. In conclusion, the substance is not mutagenic under the conditions of this test.

Reference 2

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - April 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Laboratories, Incorporated
- Age at study initiation: adults
- Weight at study initiation: between 150-300 grams
- Assigned to test groups randomly: yes

Route of administration:

oral: gavage

Vehicle:

1.5% aqueous methylcellulose; the total volume of the test article solution administered did not exceed 5.1 ml/kg (target of 5 ml/kg ± 10%).

Details on exposure:

A preliminary study was performed to determine the maximum dose and the perfusion time. Four rats were each dosed by oral gavage with the test material suspended in 1.5% high viscosity carboxymethylcellulose. The top dose of 1000 mg/kg was selected by the sponsor according to the recommendations of Butterworth B.E. et al. 1987. Two rats were sacrificed about 4 hours after treatment with the test article and two rats were sacrificed about 15-16 hours after treatment. Hepatocytes were collected and prepared for autoradiography. Microscopic examination of the slides after autoradiography and staining indicated that the distribution of labeling between the nucleus and cytoplasm was similar at both time points. The UDS Revised 9 assay was therefore performed approximately 16 hours after the administration of a single dose of the test material at 1000, 500, 250 and 100 mg/kg..

Duration of treatment / exposure:

4 and 16 hours

Frequency of treatment:

Once

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

pretest

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

main study

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

main study

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

main study

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

main study

No. of animals per sex per dose:

Three male F344 rats were treated by oral gavage at each of four dose levels 100, 250, 500 and 1000 mg/kg) with the test material suspended in 1.5% carboxymethylcellulose (CMC).

Control animals:

yes

Positive control(s):

Dimethylnitrosamine (DMN) at approximately 10 mg/kg was used. Three rats were treated by intraperitoneal injection.

Tissues and cell types examined:

The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting.

Details of tissue and slide preparation:

The cells were fixed in acetic acid:ethanol (1:3) and dried for at least 24 hours. The fixed coverslips were mounted on glass slides, dipped in Kodak NTB2 emulsion, and dried. The emulsion coated slides were stored for 7-10 days at 4°C in light-tight boxes containing packets of Drierite. The emulsions were then developed in D19, fixed, and stained with Williams' modified hematoxylin and eosin procedure.

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations that cause:
1. An increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
2. An increase in the percent of nuclei having six or more net grains to at least 10% of the analyzed population after subtraction of the concurrent negative control value.

Statistics:

no data

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material was therefore evaluated as inactive in the in-vivo/in-vitro rat hepatocyte Unscheduled DNA Synthesis Assay.

Conclusions:

Interpretation of results: negative
The test material was inactive in this UDS Assay.

Executive summary:

The study was performed 1989 as GLP-test as UDS test im-vitro/in-vivo using rat hepatocytes. The test item was dissolved in 1.5% CMC. Three rats per dose level were treated with 1000, 500, 250 and 100 mg/kg in volumes which did not exceed 5.1 ml/kg. None of the criteria used to indicate UDS was approached by the treatments and no dose-related response was observed. The test material was therefore evaluated as inactive in the in-vivo/in-vitro Rat Hepatocyte Unscheduled DNA Synthesis Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the data available the substance is not classified or labelled according to Regulation 1272/2008/EC (CLP).