N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

From December 17, 2001 to March 25, 2002

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)

Version / remarks:

Cited as Directive 2000/32/EC, B.13/14

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA100, TA98, and TA102.

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 100, 500, 1,000, 2,500 and 5,000 µg/plate, +/- S9.

Main study, with S9:
1st experiment: 6.25, 12.5, 25, 50 and 100 µg/plate (TA 98 and TA 1537), 12.5, 25, 50, 100 and 200 µg/plate (other strains).
2nd experiment: 3.125, 6.25, 12.5, 25 and 50 µg/plate (TA 98, TA 1537 and TA 1535); 12.5, 25, 50, 100 and 200 µg/plate (TA 100 and TA 102)

Main study, without S9:
1st experiment: 12.5, 25, 50, 100 and 200 µg/plate, for all tester strains
2nd experiment: 3.125, 6.25, 12.5, 25 and 50 µg/plate (TA 98, TA 1537 and TA 1535), 12.5, 25, 50, 100 and 200 µg/plate (TA 100 and TA 102).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (TA 1535 and TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), mitomycin C (TA 102). +S9: 2-anthramine (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation and preincubation (experiment 2,+ S9)
The test substance was dissolved in distilled water the vehicle previously heated at approximately 50°C. The preparations were made immediately before use.
- The direct plate incorporation method was performed as follows: test substance solution (0.1 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
- The preincubation method was performed as follows: test substance solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

DURATION
- Preincubation period: 60 min (experiment 2, + S9)
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: 3 plates/dose level in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Decrease in the number of revertant colonies and/or a thinning of the bacterial lawn

Evaluation criteria:

The study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with laboratory historical data;
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with laboratory historical data.
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA100, TA98, and TA102.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Since the test substance was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main study in the experiments without S9 mix, a moderate to marked toxicity was induced generally at dose-levels ≥50 μg/plate. In the experiments with S9 mix, a moderate to marked toxicity was induced generally at dose-levels ≥100 μg/plate in the first experiment and ≥50 μg/plate in the second experiment.
In the preliminary cytotoxicity test, with S9, toxicity was induced at dose levels ≥100 µg/plate in TA 98 strain and ≥500 µg/plate in other strains.

Conclusions:

Under the study conditions, the test substance was not considered to be mutagenic in the bacterial reverse mutation assay in the presence and absence of S9 mix.

Executive summary:

An in vitro bacterial reverse mutation study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU method B.13/14, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA100, TA98, and TA102 were exposed to the substance in the presence and absence of S9. The assay was performed in two phases, using the plate incorporation and/or pre-incubation method. The first phase (preliminary toxicity) was to establish the dose-range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. The second phase (confirmatory assay) was to evaluate and confirm the mutagenic potential of the test substance. The main study was conducted in two independent experiments with at least five dose levels of the test substance using three plates/dose-level with and without S9 mix:

Without S9-mix:

First experiment (plate incorporation method): 6.25, 12.5, 25, 50 and 100 µg/plate, for the TA 98 and TA 1537 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the remaining strains.

Second experiment (plate incorporation method): 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains.

With S9 mix:

First experiment (plate incorporation method): 12.5, 25, 50, 100 and 200 µg/plate for all tester strains.

Second experiment (pre-incubation method): 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains). Concurrent solvent (water) and positive controls (without S9: sodium azide (TA 1535 and TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), mitomycin C (TA 102); with S9: 2-anthramine (all strains), were also included.

In the preliminary toxicity test, no precipitate was observed in the Petri plates when the revertants were scored at all dose levels. Both with and without S9 mix, the test substance was strongly toxic at dose-levels ≥500 µg/plate. In the TA 98 strain, the test substance was also toxic at 100 µg/plate. In the mutagenicity assay, moderate to marked toxicity was induced generally at dose-levels ≥50 µg/plate without S9-mix and at ≥100 µg/plate with S9 mix. No increase in the number of revertants was noted in all tester strains in both the experiments. All the validity criteria were met.

Under the study conditions, the test substance was not considered to be mutagenic in the bacterial reverse mutation assay in the presence and absence of S9 mix (Haddouk, 2002).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

see, principles of method if other than guideline

Principles of method if other than guideline:

Range finder study concentration were prepared on the assumption of the test substance being a 30% w/v solution. The actual test substance was a 30% w/w solution, hence range-finder concentrations were 3 to 4% lower than intended.

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium, other: S. typhimurium: TA 1535, TA 1537, TA 98, TA 100, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

Range-finding experiment :
+S9 and –S9 : 8, 40, 200, 1000 and 5000µg a.i./plate
Expt. 1:
- S9: 0.24, 1.2, 6, 30, 150 µg a.i./plate
+S9: 0.96, 4.8, 24, 120, 600 µg a.i./plate
Expt. 2:
-S9: 9.4, 18.8, 37.5, 75, 150 µg a.i./plate
+S9: 37.5, 75, 150, 300, 600 µg a.i./plate

Positive controls:

yes

Positive control substance:

other: +S9: 2-aminoanthracene (TA98, TA100, TA1535) -S9: 2-nitrofluene (TA98, TA1538) sodium azide (TA100, TA1535) 9-aminoacridine (TA1537)

Key result

Species / strain:

S. typhimurium, other: S. typhimurium: TA 1535, TA 1537, TA 98, TA 100, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the range-finder the test substance was toxic to TA100 at concentrations of 200 µg/plate in the absence of S9 and 1000 µg/plate in the presence of S9. Expt. 1: -S9: TA 98 > 30 µg a.i./plate TA 1535, TA1537 >= 150 µg a.i./plate +S9: All strains >= 600

Positive controls validity:

valid

Additional information on results:

Interpretation of results (migrated information): negative

Conclusions:

Under the study conditions, no significant increase in the number of revertant colonies could be observed with and without metabolic activation. The test substance was not mutagenic in the Salmonella typhimurium strains.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to EU Method B.13/14, in compliance with GLP. The test substance was examined using five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 1538). The test was performed in three experiments in the presence and absence of metabolic activation (S9 -mix). The substance concentrations ranged from 0.24 to 5000 µg a.i./plate. In the range finding test, the actual test substance was a 30% w/w solution, hence range-finding concentrations were 3 to 4% lower than intended. In the range finding test, the test substance was positive for cytotoxicity. The other tests (Experiment 1 and 2) were negative for genotoxicity. Under the study conditions, no significant increase in the number of revertant colonies could be observed with and without metabolic activation. The test substance was not mutagenic in the Salmonella typhimurium strains (Kennelly, 1988).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Expt. 1:
+/-S9: 10.0, 5.0, 2.5,1.0, 0.5, 0.1 µg/mL
Expt. 2:
-S9: 2.5, 1.0, 0.25 µg/mL
+S9: 10.0, 5.0, 1.0 µg/mL

Positive controls:

yes

Positive control substance:

other: -S9: Methyl-methane-sulfonate (MMS) +S9: Cyclophosphamide (CPA)

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9: 10.0, 5.0 µg/mL (3.0, 1.5 µg a.i./mL), +S9: 10.0 µg/mL (3.0 µg a.i./mL)

Positive controls validity:

valid

Additional information on results:

Interpretation of results (migrated information): negative

Conclusions:

Under the study conditions, the test substance did not induce gene mutations in V79 cells in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to determine the potential of the test substance to induce mutations in Chinese Hamster V79 lung fibroblast cells according to OECD 473, in compliance with GLP. The assay was performed in two independent experiments both with and without metabolic activation using an Aroclor-induced rat liver S9 system. In Experiment I, 6 concentrations of the test substance were used within the range of 0.1 to 10 μg/mL (nominal) and in Experiment II, only 3 concentrations were used within the same range. The test substance caused cytotoxicity at the highest concentration with metabolic activation and at the two highest concentrations without metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration of the test substance. Under the study conditions, the test substance did not induce gene mutations in V79 cells in the absence and presence of metabolic activation (Bosse, 1991).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 29, 2002 to July 29, 2002

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)

Version / remarks:

Cited as Directive 2000/32/EC, B.10

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

1st experiment: +/- S9 mix: 0.137, 0.41, 1.23, 3.69, 11.07, 33.22, 99.67 and 299 µg/mL ( corresponding to dose-levels ranging from 0.46 x 10-3 mM to 1 mM)
2nd experiment: +/- S9 mix: 0.31, 0.63, 1.25, 2.5, 5 and 7.5 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: mytomycin C; -S9: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Incubation period: 48 h
- Exposure duration: 1st experiment: 3 h. 2nd experiment: 3 h or continuous until harvest
- Fixation time (start of exposure up to fixation or harvest of cells): 1st experiment: 20 h. 2nd experiment: 20 and 44 h.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

NUMBER OF REPLICATIONS: Duplicate cultures in two independent experiments

NUMBER OF CELLS EVALUATED: 200 metaphases were examined/dose level, with 100 metaphases/culture when possible. When at least 10% cells with structural chromosome aberrations were observed only 50 metaphases/culture were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

Evaluation criteria:

Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

This study was considered valid when the following criteria were met:
- the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with the historical data of the test lab.
- the frequency of cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and consistent with the historical data of the test lab.
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the X2 test, in which p = 0.05 was used as the lowest level of significance. A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p = 0.05 was used as the lowest level of significance.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix: A slight to strong decrease in the mitotic index was induced at dose-levels ≥7.5 μg/mL. With S9 mix: A slight to strong toxicity was generally induced at dose-levels ≥5 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: For the choice of the highest dose for treatment in the first experiment of the present study, significant variations in pH and strong toxicity induced in CIT Study No. 23283 MLY (In vitro mammalian cell gene mutation test performed on the same compound) were taken into account.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without S9 mix: A slight to strong decrease in the mitotic index was induced at dose-levels ≥7.5 μg/mL. With S9 mix: A slight to strong toxicity was generally induced at dose-levels ≥5 μg/mL.

PRECIPITATION CONCENTRATION: No precipitation observed
MITOTIC INDEX: 
First experiment
-S9 mix:  (3 hour treatment, 20 hour harvest)
Doses	0      0.137  0.41  1.23  3.69  11.07 	
MI	7.70   5.90   6.35  7.30  6.95  0.90	   
OC 	100    77     82    95    90    12        

Doses   33.22  99.67  299   *Pos. control
MI      0.00   0.00   0.00   230 
OC      0      0      0      30 	

+ S9 mix: (3 hour treatment, 20 hour harvest)
Doses	0      0.137  0.41  1.23  3.69	11.07	
MI      6.55   5.45   5.9   6.25  5.65	0.00		
OC      100    83     90    95	  86	0		

Doses   33.22  99.67   299  *Pos. control
MI      0.00   0.00    0.00  315/1.75    
OC      0      0       0     48/27

MI = Mitotic index in % (mean)
OC = % of control
* -S9 mix =MCC  3 ug/mL, +S9-mix = CPA 25 ug/mL/ CPA 50
ug/mL

Second experiment
1 -S9 mix: 
Doses	0     0.31  0.63  1.25 2.5  5    7.5 *Pos. control
MI           4.10  4.75  4.60  5.25 5.20 4.50 4.05 3.65 
OC	100   116   112  128  127  110  99   98
2 - S9 mix:  
MI	5.00  4.95  5.65  4.75 4.25 5.10 2.90	-
OC	100   99    113	  95   85   102	 58	-
3 + S9 mix: 	
MI      4.50  3.75  3.95  3.65 3.30 2.70 0.00 1.50/0.75
OC      100   83    88 81   73   60 0    33/17
4 + S9 mix: 
MI      2.05  3.25  1.75  3.00  2.80 2.80 0.00	-
OC      100   159   85    146	137  137  0     -

MI = Mitotic index in % (mean)
OC = % of control
*-S9 mix =MCC  0.2 ug/mL, +S9-mix = CPA 25 ug/mL/ CPA 50 ug/mL 
1: 3 h treatment, 20 h harvest
2: 44 h treatment, 44 h harvest
3: 3 h treatment, 20 h harvest
4: 3 h treatment, 44 h harvest

CYTOTOXIC CONCENTRATION: 
- With metabolic activation: First and second experiment >= 5 µg/mL: 40 to 100% decrease in mitotic index.- Without metabolic activation: First experiment: After 3 h treatment >= 11.07 µg/mL: 88 to 100% decrease in mitotic index; Second experiment: After 44 h treatment at 7.5 µg/mL:42% decrease in mitotic index.


STATISTICAL RESULTS: 
Chromosomal aberration analysis:  
Experiments without S9 mix: The dose-levels selected for metaphase analysis were as follows:   0.41, 1.23 and 3.69 µg/mL, for the 3-h treatment (first experiment), higher dose-levels being strongly toxic,   2.5, 5 and 7.5 µg/mL, for the 20-h treatment (second experiment), 7.5 µg/mL, for the 44-h treatment (second experiment), 7.5 µg/mL being the highest dose used for treatment in this second experiment.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44 h treatments

Experiments with S9 mix: The dose-levels selected for metaphase analysis were as follows: 0.41, 1.23 and 3.69 µg/mL, for the 20-h harvest time in the first experiment, 1.25, 2.5 and 5 µg/mL, for the 20-h harvest time in the second experiment, 5 µg/mL, for the 44-h harvest time.
The highest doses selected for metaphase analysis were the highest readable doses (higher dose-levels being strongly toxic).
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

Conclusions:

Under the study conditions, the test substance did not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

An in vitro chromosomal aberration assay was conducted to determine the clastogenic potential of the test substance in human lymphocytes according to OECD Guideline 473 and EU method B.10, in compliance with GLP. The cells were tested at the following dose levels of the test substance using duplicate cultures, with and without S9 mix:

First experiment both with and without S9 mix (for 3 h): 0.137, 0.41, 1.23, 3.69, 11.07, 33.22, 99.67 and 299 µg/mL

Second experiment both with S9 mix (for 3 h) and without S9 mix (continuous until harvest i.e. 44 h): 0.31, 0.63, 1.25, 2.5, 5 and 7.5 µg/mL.

Concurrent solvent (culture medium) and positive controls (cyclophosphamide (without S9) and mitomycin C (with S9) were also included. Harvest times were 20 h and 44 h from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 h later.

The cytotoxicity of the test substance which was evaluated using the mitotic index (number of cells in mitosis/1,000 cells examined) indicated a slight to strong decrease in the mitotic index at dose levels ≥7.5 μg/mL without S9 mix and at dose levels ≥5 μg/mL with S9 mix.

Hence, the dose-levels selected for metaphase analysis were as follows:

Without S9 mix: 0.41, 1.23 and 3.69 µg/mL, for the 3 h treatment (first experiment), higher dose-levels being strongly toxic, 2.5, 5 and 7.5 µg/mL, for the 20 h treatment (second experiment), 7.5 µg/mL for the 44 h harvest time (second experiment); With S9 mix: 0.41, 1.23 and 3.69 µg/mL, for the 20 h harvest time in the first experiment, 1.25, 2.5 and 5 µg/mL, for the 20 h harvest time in the second experiment, 5 µg/mL, for the 44 h harvest time. One and a half hour before the harvest, each culture was treated with a colcemid solution to block cells at the metaphase-stage of mitosis followed by fixing in a methanol/acetic acid mixture (3/1; v/v), staining with Giemsa and blind scoring. Two hundred metaphases were examined/dose level, with 100 metaphases/culture when possible. When at least 10 % cells with structural chromosome aberrations were observed only 50 metaphases/culture were analysed.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at all harvest times. Further, the frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Under the study conditions, the test substance did not induce chromosome aberrations in cultured human lymphocytes (Haddouk, 2002).

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 29, 2002 to July 29, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Cited as Directive 2000/32/EC, B.17

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Preliminary toxicity test, +/- S9: 0.02, 0.2, 1, 2, 5 and 10 mM

Main study, -S9:
1st experiment (3-h treatment): 0.31, 0.63, 1.25, 2.5, 5 and 10 µM
2nd experiment (24-h treatment): 0.063, 0.125, 0.25, 0.5, 0.75 and 1 µM

Main study, +S9:
1st experiment 0.63, 1.25, 2.5, 5, 10 and 20 µM
2nd experiment 0.63, 1.25, 2.5, 5, 10 and 15 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: methylmethane sulfonate; + S9: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

MEDIUM: Cells were grown in RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL), supplemented by heat inactivated horse serum at 5% (RPMI 5), 10% v/v (RPMI 10) or 20% v/v (RPMI 20)

DURATION
- Exposure duration: 3 or 24 h
- Expression time (cells in growth medium): 11-12 d

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: One 96-well plate/culture = two plates/dose-level, except for the vehicle control where two 96-well plates/culture were used = total of four plates. Two independent experiments.

NUMBER OF CELLS EVALUATED: 2000 sells/well

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

CONTROLS:
The following controls were included using duplicate cultures:
- Vehicle controls: cultures treated with the vehicle,
- Positive controls: cultures treated with:
- Methylmethane sulfonate, in the absence of S9 mix,
- Cyclophosphamide, in the presence of S9 mix

Evaluation criteria:

Evaluation criteria:
A reproducible two-fold increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered.

The study was considered valid when the following criteria were fulfilled:
- the cloning efficiency of the vehicle controls are between 0.6-1.4 for cloning efficiency CE0 and between 0.7-1.3 for cloning efficiency CE2
- the mutation frequency of the vehicle controls are between 60-250* 10E-6
- the mutation frequency of the positive controls are higher than that of the vehicle controls (more than two fold) and consistent with our historical data.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without S9 mix, 3 h treatment: >= 10 µM without S9 mix, 24 h treatment: >= 0.75 µM

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: To assess the cytotoxicity of the test substance, at least six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
A treatment of 3 h was performed using a final concentration of 10E6 cells/mL. At the end of treatment, cells were washed and then cell concentration was adjusted in order to seed 1.6 cells per well in the 96-well microtiter plates. After at least 7 d of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- S9: A marked toxicity was noted mainly at 10 μM, after 3 h of treatment. After 24 h treatment, a marked to strong toxicity was noted at 0.75 μM. +S9: a marked to strong toxicity was induced at dose-levels ≥10 μM.

Conclusions:

Under the study conditions, cytotoxicity was observed with and without S9 mix, 3 h treatment, from 10 µM and without S9 mix, 24 h treatment, from 0.75 µM. Further, no noteworthy increase in the mutation frequency was induced, both after 3 and 24 h treatments. Hence, the test substance did not show mutagenic activity in the in vitro mouse lymphoma assay.

Executive summary:

An in vitro mouse lymphoma study was conducted to determine the mutagenic potential of the test substance in L5178Y TK+/-mouse lymphoma cells according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. The mouse lymphoma L5178Y TK+/-(3.7.2C) cells were exposed at concentrations of 0.02, 0.2, 1, 2, 5 and 10 mM (one culture/dose-level) both with and without metabolic activation to assess the cytotoxicity of the test substance. Further, two independent mutagenicity experiments were performed using 3 and 24 h exposure durations:

Without S9 mix: First experiment (3 h treatment), 0.31, 0.63, 1.25, 2.5, 5 and 10 µM; Second experiment (24 h treatment, because first experiment was negative), 0.063, 0.125, 0.25, 0.5, 0.75 and 1 µM.

With S9 mix: First experiment 0.63, 1.25, 2.5, 5, 10 and 20 µM; Second experiment 0.63, 1.25, 2.5, 5, 10 and 15 µM.

Concurrent solvent (culture medium) and positive controls (cyclophosphamide (with S9) and methylmethane sulfonate (without S9) were also included. Under the study conditions, cytotoxicity was observed with and without S9 mix, 3 h treatment, from 10 µM and without S9 mix, 24 h treatment, from 0.75 µM. Further, no noteworthy increase in the mutation frequency was induced, both after 3 and 24 h treatments. Hence, the test substance did not show mutagenic activity in the in vitro mouse lymphoma assay (Haddouk, 2002).

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991

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)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Range-finding test
+/- S9: 5.0, 1.0, 1.0, 0.2, 0.04, 0.008, 0.0016 mg/mlL
Expt. 1:
+/- S9: 5.0, 2.5, 1.0, 0.5, 0.1, 0.05 µg/mL
Expt. 2:
-S9: 5.0, 2.5, 1.0, 0.5, 0.1, 0.05 µg/mL
+S9: 5.0, 2.5, 1.0, 0.5, 0.1, 0.05 µg /mL

Positive controls:

yes

Positive control substance:

other: -S9: Ethyl-methane sulfonate (EMS) +S9: 7,12-dimethylbenzanthracene (DMBA)

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Range-finding test: +/- S9: 5.0 – 0.0016 mg/mL no survival; Main study: + S9 > 5 µg/mL; - S9 = 5 µg/mL

Positive controls validity:

valid

Additional information on results:

Interpretation of results: negative

Conclusions:

Under the study conditions, the test substance did not induce gene mutations in V79 cells in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce mutations in Chinese Hamster V79 lung fibroblast cells according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments with and without metabolic activation using an Aroclor-induced rat liver S9 system. The range finding test concentrations were, with and without metabolic activation, 5.0 to 0.0016 mg/mL. The range of concentrations selected for the two main exposures was 5 to 0.5 µg/mL. The test substance caused cytotoxicity in the range finding test at all concentrations tested with and without S9 and in the main test with metabolic activation at >5 µg/mL. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration. Under the study conditions, the test substance did not induce gene mutations in V79 cells in the absence and presence of metabolic activation (Bosse, 1991).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genototoxicity by polyamines such as the test substance is not expected. In physiological circumstances, the polyamines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil.

Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

Ames test

An in vitro bacterial reverse mutation study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU method B.13/14, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA100, TA98, and TA102 were exposed to the substance in the presence and absence of S9. The assay was performed in two phases, using the plate incorporation and/or pre-incubation method. The first phase (preliminary toxicity) was to establish the dose-range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. The second phase (confirmatory assay) was to evaluate and confirm the mutagenic potential of the test substance. The main study was conducted in two independent experiments with at least five dose levels of the test substance using three plates/dose-level with and without S9 mix:

Without S9-mix:

First experiment (plate incorporation method): 6.25, 12.5, 25, 50 and 100 µg/plate, for the TA 98 and TA 1537 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the remaining strains.

Second experiment (plate incorporation method): 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains.

With S9 mix:

First experiment (plate incorporation method): 12.5, 25, 50, 100 and 200 µg/plate for all tester strains.

Second experiment (pre-incubation method): 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains). Concurrent solvent (water) and positive controls (without S9: sodium azide (TA 1535 and TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), mitomycin C (TA 102); with S9: 2-anthramine (all strains), were also included.

In the preliminary toxicity test, no precipitate was observed in the Petri plates when the revertants were scored at all dose levels. Both with and without S9 mix, the test substance was strongly toxic at dose-levels ≥500 µg/plate. In the TA 98 strain, the test substance was also toxic at 100 µg/plate. In the mutagenicity assay, moderate to marked toxicity was induced generally at dose-levels ≥50 µg/plate without S9-mix and at ≥100 µg/plate with S9 mix. No increase in the number of revertants was noted in all tester strains in both the experiments. All the validity criteria were met.

Under the study conditions, the test substance was not considered to be mutagenic in the bacterial reverse mutation assay in the presence and absence of S9 mix (Haddouk, 2002).

A study was conducted to determine the mutagenic potential of the test substance according to EU Method B.13/14, in compliance with GLP. The test substance was examined using five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 1538). The test was performed in three experiments in the presence and absence of metabolic activation (S9 -mix). The substance concentrations ranged from 0.24 to 5000 µg a.i./plate. In the range finding test, the actual test substance was a 30% w/w solution, hence range-finding concentrations were 3 to 4% lower than intended. In the range finding test, the test substance was positive for cytotoxicity. The other tests (Experiment 1 and 2) were negative for genotoxicity. Under the study conditions, no significant increase in the number of revertant colonies could be observed with and without metabolic activation. The test substance was not mutagenic in the Salmonella typhimurium strains (Kennelly, 1988).

Chromosome aberration assay

An in vitro chromosomal aberration assay was conducted to determine the clastogenic potential of the test substance in human lymphocytes according to OECD Guideline 473 and EU method B.10, in compliance with GLP. The cells were tested at the following dose levels of the test substance using duplicate cultures, with and without S9 mix:

First experiment both with and without S9 mix (for 3 h): 0.137, 0.41, 1.23, 3.69, 11.07, 33.22, 99.67 and 299 µg/mL

Second experiment both with S9 mix (for 3 h) and without S9 mix (continuous until harvest i.e. 44 h): 0.31, 0.63, 1.25, 2.5, 5 and 7.5 µg/mL.

Concurrent solvent (culture medium) and positive controls (cyclophosphamide (without S9) and mitomycin C (with S9) were also included. Harvest times were 20 h and 44 h from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 h later.

The cytotoxicity of the test substance which was evaluated using the mitotic index (number of cells in mitosis/1,000 cells examined) indicated a slight to strong decrease in the mitotic index at dose levels ≥7.5 μg/mL without S9 mix and at dose levels ≥5 μg/mL with S9 mix.

Hence, the dose-levels selected for metaphase analysis were as follows:

Without S9 mix: 0.41, 1.23 and 3.69 µg/mL, for the 3 h treatment (first experiment), higher dose-levels being strongly toxic, 2.5, 5 and 7.5 µg/mL, for the 20 h treatment (second experiment), 7.5 µg/mL for the 44 h harvest time (second experiment); With S9 mix: 0.41, 1.23 and 3.69 µg/mL, for the 20 h harvest time in the first experiment, 1.25, 2.5 and 5 µg/mL, for the 20 h harvest time in the second experiment, 5 µg/mL, for the 44 h harvest time. One and a half hour before the harvest, each culture was treated with a colcemid solution to block cells at the metaphase-stage of mitosis followed by fixing in a methanol/acetic acid mixture (3/1; v/v), staining with Giemsa and blind scoring. Two hundred metaphases were examined/dose level, with 100 metaphases/culture when possible. When at least 10 % cells with structural chromosome aberrations were observed only 50 metaphases/culture were analysed.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at all harvest times. Further, the frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Under the study conditions, the test substance did not induce chromosome aberrations in cultured human lymphocytes (Haddouk, 2002).

A study was conducted to determine the potential of the test substance to induce mutations in Chinese Hamster V79 lung fibroblast cells according to OECD 473, in compliance with GLP. The assay was performed in two independent experiments both with and without metabolic activation using an Aroclor-induced rat liver S9 system. In Experiment I, 6 concentrations of the test substance were used within the range of 0.1 to 10 μg/mL (nominal) and in Experiment II, only 3 concentrations were used within the same range. The test substance caused cytotoxicity at the highest concentration with metabolic activation and at the two highest concentrations without metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration of the test substance. Under the study conditions, the test substance did not induce gene mutations in V79 cells in the absence and presence of metabolic activation (Bosse, 1991).

Mammalian gene mutation assay

An in vitro mouse lymphoma study was conducted to determine the mutagenic potential of the test substance in L5178Y TK+/-mouse lymphoma cells according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. The mouse lymphoma L5178Y TK+/-(3.7.2C) cells were exposed at concentrations of 0.02, 0.2, 1, 2, 5 and 10 mM (one culture/dose-level) both with and without metabolic activation to assess the cytotoxicity of the test substance. Further, two independent mutagenicity experiments were performed using 3 and 24 h exposure durations:

Without S9 mix: First experiment (3 h treatment), 0.31, 0.63, 1.25, 2.5, 5 and 10 µM; Second experiment (24 h treatment, because first experiment was negative), 0.063, 0.125, 0.25, 0.5, 0.75 and 1 µM.

With S9 mix: First experiment 0.63, 1.25, 2.5, 5, 10 and 20 µM; Second experiment 0.63, 1.25, 2.5, 5, 10 and 15 µM.

Concurrent solvent (culture medium) and positive controls (cyclophosphamide (with S9) and methylmethane sulfonate (without S9) were also included. Under the study conditions, cytotoxicity was observed with and without S9 mix, 3 h treatment, from 10 µM and without S9 mix, 24 h treatment, from 0.75 µM. Further, no noteworthy increase in the mutation frequency was induced, both after 3 and 24 h treatments. Hence, the test substance did not show mutagenic activity in the in vitro mouse lymphoma assay (Haddouk, 2002).

A study was conducted to investigate the potential of the test substance to induce mutations in Chinese Hamster V79 lung fibroblast cells according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments with and without metabolic activation using an Aroclor-induced rat liver S9 system. The range finding test concentrations were, with and without metabolic activation, 5.0 to 0.0016 mg/mL. The range of concentrations selected for the two main exposures was 5 to 0.5 µg/mL. The test substance caused cytotoxicity in the range finding test at all concentrations tested with and without S9 and in the main test with metabolic activation at >5 µg/mL. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration. Under the study conditions, the test substance did not induce gene mutations in V79 cells in the absence and presence of metabolic activation (Bosse, 1991).

Justification for classification or non-classification

Based on the results of in vitro genotoxicity studies, the substance does not warrant classification for this endpoint according to CLP (EC 1272/2008) criteria.