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Genetic toxicity in vitro

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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:
From September 2009 to October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
A maximum dose-level of 5000 ug/plate and four lower concentrations of 1580, 500, 158 and 50.0 ug/plate
Vehicle / solvent:
Solvent used: Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strains TA100 and TA1535; -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain TA1537; -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Strain TA98; -S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
All strains; +S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Strain WP2uvrA; -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) - Assay 1; preincubation - Assay 2

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): Histidine (S. typhimurium); tryptophan (E. coli)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Reduced number of spontaneous revertants, microcolony formation, thinning of background lawn

Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose-levels or at the highest practicable dose-level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the initial toxicity test no toxicity was observed at any dose-level with any tester strain in the absence or presence of S9 metabolism at concentrations up to a maximum of 5000 ug/plate.
No precipitation was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In Assay 1, using the plate incorporation method, the substance was tested at the maximum concentration of 5000 μg/plate and at four lower concentrations. No relevant toxicity was observed with any tester strain at any dose-level. No relevant increase in revertant numbers was observed at any concentration tested. A pre-incubation step was included for all treatments of Assay 2 in which the same concentrations were employed. No toxicity was observed at any dose-level with any tester strain.

 

No precipitation of the test item was observed at the end of the incubation period at any concentration in the plate incorporation experiment or in the pre-incubation experiment.

 

The substance did not induce increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.

 

The sterility of the S9 mix and the test solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

 

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.
Executive summary:

A bacterial reverse mutation assay (Ames test) has been undertaken following OECD/EU test methods.

The substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli.

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:
From October 25,2007 to January 16,2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Osmolarity only determined for cultures from the short treatment in the absence of S9. This was considered not to have affected the integrity of the study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Osmolarity only determined for cultures from the short treatment in the absence of S9. This was considered not to have affected the integrity of the study
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Whole blood was collected from a healthy male, non-smoking, volunteer donor not receiving any medication prior to the time of sampling.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
A maximum dose-level of 5000 micrograms/plate and four lower concentrations of 2500, 1250, 625 and 313 micrograms/plate
Vehicle / solvent:
- Solvent used: Ethanol
Untreated negative controls:
yes
Remarks:
untreated cultures
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
yes
Remarks:
untreated cultures
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent assays for chromosomal damage were performed.
For the first experiment, both in the absence and presence of S9 metabolism, the treatment time was 3 hours after which the cells were allowed to recover prior to harvesting. The harvest time of 24 hours, corresponding to approximately 1.5 cell cycle, was used.
As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using a continuous treatment in the absence of S9 metabolism until harvest at 24 hours.

S9 tissue homogenate was prepared from the livers of five young male Sprague-Dawley rats which had received prior treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic metabolising enzymes.

For both assays, dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 and 39.1 µg/ml were used both in the absence and presence of S9 metabolism. Solutions were prepared immediately before use in ethanol.

Appropriate negative and positive control cultures were included in each experiment. Positive control treated cultures received Mitomycin-C in the absence of S9 metabolism at dose levels of 0.75 and 0.50 µg/ml in the first main assay. For the second assay, cultures received Mitomycin-C at dose levels of 0.45 and 0.30 µg/ml. In the presence of S9 metabolism cultures received Cyclophosphamide at dose levels of 18.0 and 23.0 µg/ml.

Two cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained with 3% Giemsa.

Following treatment, the pH and osmolality of the treatment media at higher dose levels (short treatment in the absence of S9) were determined.
No remarkable variation of pH was observed. Slight dose-related reductions of osmolality were observed at the higher dose levels selected for treatment (5000 and 2500 µg/ml).


Evaluation criteria:
Criterion for outcome

In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled:

(i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose-level over the concurrent control.
(ii) The increases are reproduced in both replicate cultures and must be observed in both experiments.
(iii) The increases must exceed historical controls. Any significant increase over the concurrent negative controls is therefore compared with historical control.

The evaluation is based on the set of results which excludes gaps. A more detailed explanation of the criteria for evaluation of the results is given in the Study Protocol.

Evaluation

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce chromosomal aberrations in human lymphocytes cultured in vitro.
Statistically significant increases in aberrant cells compared with the relevant control values were seen in cultures treated with the positive controlsMitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.
Statistics:
For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps.Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps over the control values, was observed in any treatment series at any sampling time.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One hundred metaphase spreads were scored for chromosomal aberrations from each culture.

In the first experiment, following treatment in the absence of S9 metabolism, no remarkable toxicity was observed at any dose-level selected for treatment. In the presence of S9 metabolism, mild or slight toxicity was observed over the whole dose-range (mitotic indexes between 73-92% of the control) with the exception of the two lower dose levels of 78.1 and 39.1 micrograms/ml where no remarkable toxicity was observed.

In the second experiment, following the continuous treatment in the absence of S9 metabolism, moderate toxicity was observed at the three higher dose levels (5000, 2500 and 1250 microg/ml) where the mitotic indexes were reduced in the range between 64-68% of the control. Mild toxicity was observed at the dose levels of 625, 313, 156 and 78.1 micrograms/ml where the mitotic indexes were reduced in the range between 74-78% of the control. Slight toxicity was observed at the lowest dose of 39.1 micrograms/ml where the mitotic index was 86% of the control.

The highest dose level selected for the scoring of aberrations should be a concentration causing moderate toxicity (ideally the reduction of mitotic index should be approximately 50%) and treatments reducing the mitotic index to below 20% should not be scored. In the absence of toxicity the highest treatment level will be selected as the highest dose for scoring. On the basis of the observed toxicity the treatment-levels selected for the scoring of aberrations were 5000, 2500 and 1250 micrograms/ml

Following treatment with the substance, no relevant increase in the incidence of cells bearing aberrations including or excluding gaps over the control values, was observed at any dose level in the absence or presence of S9 metabolism.

Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substance, Mitomycin-C and Cyclophosphamide indicating the correct functioning of the assay system.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 

 TABLE 1 - Mitotic index results - Without metabolic activation

 MAIN ASSAY: 1

 SOLVENT: ETHANOL

 TREATMENT TIME: 3 hours

 SAMPLING TIME: 24 hours

 _____________________________________________________________________

 Treatment       Dose level Culture Cells Metaphases Mean Relative

                  (µg/ml)     No.  Scored           MI(%)  MI(%)

 _____________________________________________________________________

 Untreated          -          1    1000     40     4.1     95

                               2    1000     42

 _____________________________________________________________________

 Solvent            1%         3    1000     44     4.3    100

                               4    1000     42

 _____________________________________________________________________

 Test Item          39.1      19    1000     45     4.3    100

                              20    1000     41

 _____________________________________________________________________

 Test Item          78.1      17    1000     40     4.2     97

                              18    1000     43

 _____________________________________________________________________

 Test Item         156        15    1000     38     4.2     97

                              16    1000     45

 _____________________________________________________________________

 Test Item         313        13    1000     37     3.9     91

                              14    1000     41

 _____________________________________________________________________

 Test Item         625        11    1000     43     4.2     97

                              12    1000     40

 _____________________________________________________________________

 Test Item        1250         9    1000     41     4.0     92

                              10    1000     38

 _____________________________________________________________________

 Test Item        2500         7    1000     40     4.3     99

                               8    1000     45

 _____________________________________________________________________

 Test Item        5000         5    1000     43     4.1     95

                               6    1000     39

 _____________________________________________________________________

 Mitomycin-C        0.50      21    1000     20     1.9     46

                              22    1000     18

 _____________________________________________________________________

 Mitomycin-C        0.75      23    1000     18     1.8     43

                              24    1000     17

 _____________________________________________________________________


TABLE 2 - Mitotic index results - With metabolic activation

MAIN ASSAY: 1

SOLVENT: DMSO

TREATMENT TIME: 3 hours

SAMPLING TIME: 24 hours

 _____________________________________________________________________

 Treatment       Dose level Culture Cells Metaphases Mean Relative

                  (µg/ml)     No.  Scored           MI(%)  MI(%)

 _____________________________________________________________________

 Untreated          -          73   1000     56     5.6    114

                               74   1000     55

 _____________________________________________________________________

 Solvent            1%         75   1000     49     4.9    100

                               76   1000     48

 _____________________________________________________________________

 Test Item          39.1       91   1000     49     5.1    104

                               92   1000     52

 _____________________________________________________________________

 Test Item          78.1       89   1000     46     4.7     97

                               90   1000     48

 _____________________________________________________________________

 Test Item         156         87   1000     35     3.6     74

                               88   1000     37

 _____________________________________________________________________

 Test Item         313         85   1000     42     4.3     88

                               86   1000     43

 _____________________________________________________________________

 Test Item         625         83   1000     44     4.4     90

                               84   1000     43

 _____________________________________________________________________

 Test Item        1250         81   1000     35     3.6     73

                               82   1000     36

 _____________________________________________________________________

 Test Item        2500         79   1000     42     4.5     92

                               80   1000     47

 _____________________________________________________________________

 Test Item        5000         77   1000     37     3.8     78

                               78   1000     39

 _____________________________________________________________________

 Cyclophosphamide  18.0        93   1000     28     2.9     52

                               94   1000     30

 _____________________________________________________________________

 Cyclophosphamide  23.0        95   1000     22     2.3     41

                               96   1000     24

 _____________________________________________________________________


TABLE 3 - Mitotic index results - Without metabolic activation

MAIN ASSAY: 2

SOLVENT: DMSO

TREATMENT TIME: 24 hours

SAMPLING TIME: 24 hours

 _____________________________________________________________________

 Treatment       Dose level Culture Cells Metaphases Mean Relative

                  (µg/ml)     No.  Scored           MI(%)  MI(%)

 _____________________________________________________________________

 Untreated          -          49   1000     54     5.3     98

                               50   1000     51

 _____________________________________________________________________

 Solvent            1%         51   1000     50     5.4    100

                               52   1000     57

 _____________________________________________________________________

 Test Item         39.1        67   1000     45     4.6     86

                               68   1000     47

 _____________________________________________________________________

 Test Item         78.1        65   1000     42     4.2     78

                               66   1000     41

 _____________________________________________________________________

 Test Item        156          63   1000     41     4.0     75

                               64   1000     39

 _____________________________________________________________________

 Test Item        313          61   1000     39     4.0     74

                               62   1000     40

 _____________________________________________________________________

 Test Item        625          59   1000     38     4.0     75

                               60   1000     42

 _____________________________________________________________________

 Test Item       1250          57   1000     38     3.7     68

                               58   1000     35

 _____________________________________________________________________

 Test Item       2500          55   1000     36     3.5     64

                               56   1000     33

 _____________________________________________________________________

 Test Item       5000          53   1000     35     3.6     67

                               54   1000     37

 _____________________________________________________________________

 Mitomycin-C        0.30       69   1000     27     2.9     54

                               70   1000     30

 _____________________________________________________________________

 Mitomycin-C        0.45       71   1000     22     2.3     44

                               72   1000     24

 _____________________________________________________________________

 

 

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that DIPLAST TM/MG does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.
Executive summary:

The test item, DIPLAST TM/MG, was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in-vitro treatment in the absence and presence of S9 metabolic activation.

The substance did not induce chromosomal aberrations in human lymphocytes after in-vitro treatment

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 16,2007 to February 15,2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518).
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
78.1, 156, 313, 625, 1250 and 2500 µg/mL used in the cytogenetic assay.
Vehicle / solvent:
- Solvent used: Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
-S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+S9
Details on test system and experimental conditions:
S9 metabolising system
S9 tissue homogenate was prepared from the livers of five young male Sprague-Dawley rats which had received treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic metabolising enzymes.

Preparation of test cell cultures
A cell suspension (1E6 cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures. The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and in the presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in PBS, cells were resuspended in 20 ml RPMI minimal medium. Cell concentrations were adjusted to 8 cells/ml using complete medium (20%) and, for each dose level, 0.2 ml was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
Treatment of cell cultures
Experiments were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in this experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a long treatment time (24 hours).
After washing in PBS, cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2E5 cells/ml. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Determination of survival
Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/ml using complete medium (20%). A 0.2 ml aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. After incubation, wells containing viable clones were identified by eye using background illumination and then counted.

Expression period
During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2E5 cells/ml.

Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/ml) and an estimated 2E3 cells were plated in each well of four 96-well plates.
Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified . In addition, the number of wells containing large colonies and the number containing small colonies were scored.

Plating for viability
After dilution, in complete medium (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
The assay was considered valid if the following criteria were met:

(i) The cloning efficiencies at Day 2 in the untreated control cultures in the absence of S9 metabolic activation fell within the range of 65-120%.

(ii) The untreated control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8 – 32.

(iii) The mutant frequencies in the untreated control cultures fell within the range of 50 200 x 106 viable cells.

(iv) The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

See "any other information" for details
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cytotoxicity test
Both in the absence and presence of S9 metabolic activation, the test substance was assayed at a maximum dose level of 5000 µg/ml and at lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml.
Slight decreases in relative survival were observed at intermediate dose levels, both in the absence and presence of S9 metabolic activation, using a 3 hour treatment time. No relevant toxicity was observed using a long treatment time in the absence of S9 metabolic activation.

Mutation assays
Two independent assays for mutation to trifluorothymidine resistance were performed.
The mutant frequencies in the solvent control cultures fell within the normal range (5E7 2E8 viable cells). The positive control chemicals induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65-120% and the control growth factor over 2 days fell within the range of 8 – 32 in both experiments.
In the absence of S9 metabolic activation, using the 3-hour treatment time, slight toxicity was observed at the highest dose level (2500 µg/ml) reducing survival to 72% of the concurrent negative control value. The relative total growth was reduced to 78% at the same concentration. Using a long treatment time, no relevant toxicity was observed at any dose level.
In the presence of S9 metabolic activation, no relevant toxicity was observed in the first experiment, while in the second experiment, a slight decrease in relative total growth (60%) was observed only at the intermediate dose level of 313mg/ml.

In Experiment 1, in the presence of S9 metabolic activation, the heterogeneity between replicate cultures for survival at the end of treatment, at the concentration of 625 µg/ml, was higher than usual and thus excluded from the determination of overall heterogeneity. In addition, in the second experiment, in the absence of S9, a slight but statistically significant difference between replicate plates was observed for culture A treated at 625 µg/ml.These results have not affected the validity of the study.

No statistically significant increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test substance at any concentration level.

Osmolality and pH
The test substance did not have any obvious effect on the osmolality or pH of the treatment medium.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that DIPLAST TM/MG does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The substance has been examined for mutagenic activity by assaying for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 

The substance does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assays (Ames test) have shown the substance not to induce reverse mutation in Salmonella typhimurium or Escherichia coli.

 

The ability to cause chromosomal damage in cultured human lymphocytes, following in-vitro treatment has been investigated and the substance found not to induce chromosomal aberrations in human lymphocytes after in-vitro treatment.

 

Potential mutagenic activity has been examined by assaying for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment. The substance does not induce mutation in mouse lymphoma L5178Y cells.

 

The substance was not mutagenic in a dominant lethal assay conducted in the mouse and does not induce unscheduled DNA synthesis in primary rat hepatocytes

 

REACH Regulation 1907/2006 (Annex VIII, 8.4 Column 2) states that appropriate in-vivo mutagenicity studies should be considered in those cases of a positive result in any of the in vitro genotoxicity studies. In vitro investigations were negative and in vivo studies are therefore regarded as inappropriate and not in line with current concerns regarding animal welfare and the use of animals in scientific experiments.


Short description of key information:
Genetic toxicity in-vitro: Negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Non-classification is justified on the basis of negative findings in 3 separate in-vitro tests for gene mutation / mutagenicity. Supporting information from additional in-vitro assays and an in-vivo assay.