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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Benzylated Polyamine was considered to be non-mutagenic under the conditions of this test (Envigo, 2017).

Benzylated Polyamine did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro (Envigo, 2018).

The test item, Benzylated Polyamine did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay (Envigo, 2018).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test item was tested using the following method. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were initially assayed
in triplicate against each tester strain, using the direct plate incorporation method. However, bacterial tester strains TA100 and TA1535 dosed in the absence of
S9-mix showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and, therefore these particular strains had
to be repeated employing an amended test item dose range as follows:
Salmonella strains TA100 and TA1535 (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Vehicle / solvent:
Dimethyl sulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1712138 07/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No
correction was made for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement
of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

Salmonella strains TA100, TA1535 and TA1537 (presence of S9) and TA98 (absence and presence of S9) and E.coli strain WP2uvrA (absence of S9):
0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA (presence of S9):
0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

Eight test item dose levels per bacterial tester strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item
following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer. 3.3.2.4 Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were
required, predominantly due to revertant colonies spreading slightly, thus distorting the actual
plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
S. typhimurium TA 1535
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:
S. typhimurium TA 1537
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:
S. typhimurium TA 98
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:
S. typhimurium 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:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and
the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Experiment 2.
In the initial first experiment the maximum dose level of the test item was selected as the maximum recommended dose level of 5000 μg/plate. However, two of the Salmonella
strains showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and had to be repeated employing the toxic limit of test item as
the maximum dose. In the first mutation test (plate incorporation method) the test item induced toxicity evident as visible reductions in the growth of the bacterial background lawns
of all of the tester strains, initially from 50 μg/plate in the absence of metabolic activation (S9-mix) and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit of the test
item was employed as the maximum dose level in the second mutation test. The test item induced a similar toxic response after employing the pre-incubation method in the second
mutation test with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA1535, TA98 and TA1537) and 150 μg/plate (WP2uvrA). In the
presence of S9-mix, weakened bacterial lawns were noted from 150 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without
metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases
in revertant colony frequency were observed in the first mutation test at 50 μg/plate (TA100) and 15 μg/plate (TA1535) in the presence of S9-mix only. These increases were considered
to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant
dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle controls.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. A single count for WP2uvrA (vehicle control dosed in the absence
of S9-mix after the second mutation test) was just below the minimum level. This count was still considered acceptable as the other vehicle and untreated control counts were within
expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the
test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were
validated.
Conclusions:
Benzylated Polyamine was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Benzylated Polyamine

using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Benzylated Polyamine was considered to be non-mutagenic under the conditions of this test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Mammalian Chromosome Aberration Test
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/NF S9 20/08/17 was used in this study.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
Test concentrations with justification for top dose:
The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL and the purity of the test item was 100% which was not accounted for in the test item formulations.
Dose level
µg/mL 0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000

There was a significant change in pH when the test item was dosed into media at the maximum concentrations but the osmolality did not increase by more than 50 mOsm (Scott et al., 1991). Therefore, the maximum dose level selected for the Preliminary Toxicity Test was limited to 2500 µg/mL and an intermediate dose level of 1875 µg/mL was selected in an effort to ensure the pH was not too high during testing. Therefore, the maximum practical dose level of the test item was 2500 µg/mL.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 26 years
Main Experiment: female, aged 26 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Key result
Species / strain:
lymphocytes: Primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 19.53 to 2500 µg/mL. The maximum dose was the maximum practical dose level due to concerns over an increase in pH.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the exposure groups.
Hemolysis was observed following exposure to the test item at 2500 µg/mL in the 4(20)-hour exposure groups and at and above 1250 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 625 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24 hour continuous exposure was 312.5 µg/mL. The test item induced marked evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 1280 µg/mL for the 4(20)-hour exposure groups and was 240 µg/mL for the continuous exposure group.

Chromosome Aberration Test – Main Experiment
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of Benzylated Polyamine (µg/mL)
4(20)-hour without S9 0*, 120, 240*, 320*, 560*, 640, 960, 1280, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 120, 240, 320*, 560*, 640*, 960, 1280, CP 3*
24-hour without S9 0*, 10, 20*, 40*, 80*, 120, 160, 240, MMC 0.2*
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 560, 640 and 160 µg/mL in the 4(20)-hour in the absence of S9, the 4(20)-hour in the presence of S9 and 24-hour exposure groups, respectively.
No precipitate of the test item was observed in the blood cultures at the end of the exposure in any of the exposure groups.

Hemolysis was observed following exposure to the test item at and above 640 µg/mL in the 4(20)-hour in the absence of S9 and at and above 960 µg/mL the 4(20)-hour in the presence of S9, respectively. No hemolysis was observed in the 24-hour continuous exposure group.
The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in all three exposure groups.
In the 4(20)-hour exposure group in the absence of S9, 22% and 73% mitotic inhibition was achieved at 320 and 560 µg/mL, respectively. Above this dose level, there were no metaphases available for scoring. Therefore, the maximum dose level selected for metaphase analysis was 560 µg/mL because this was the highest dose level with scorable metaphases even though this concentration exceeded the maximum range of optimum toxicity.
In the presence of S9, an inhibition of mitotic index of 28%, 32% and 53% was noted at 320, 560 and 640 µg/mL, respectively. Above this dose level there were no metaphases available to score. Therefore, the maximum dose level selected for metaphase analysis was 640 µg/mL because this concentration achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%).
In the 24-hour continuous exposure group, a plateau of toxicity was noted across the selected dose range. An inhibition of mitotic index of 50%, 26%, 52%, 83% and 70% was noted at 20, 40, 80, 120 and 160 µg/mL, respectively. Above this dose level there were very few scorable metaphases available to for analysis. Therefore, the maximum dose level selected for metaphase analysis was 80 µg/mL because this was the highest concentration which achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%).
The assay was considered valid as it met all of the following criteria:
• The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range
• All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

Discussion

In thePreliminary Toxicity Test, the human lymphocytes exhibited a steep response to the toxicity of the test item which varied with the exposure time. Therefore, a greatly expanded dose range was selected for the main test with intermediate concentrations added in an effort to achieve optimum toxicity. 

This strategy worked for the4(20)-hour in the presence of S9 and the 24-hour continuous exposure groups but fell short for the 4(20)-hour in the absence of S9. The Mitotic Index analysis indicated that many cells were in metaphase on the slides as evidenced by the high numbers recorded for the solvent control. In this exposure group, 22% and 73% mitotic inhibition was achieved at 320 and 560 µg/mL, respectively; above this dose level, there were no metaphases available for scoring. The high numbers of metaphases on the slides indicated that, although the maximum dose level selected for metaphase analysis (560 µg/mL) exceeded optimum toxicity, there were many good quality metaphases available for assessment which did not reflect the Mitotic Index score. Consequently, the maximum dose level selected for metaphase analysis was 560 µg/mL because this was the highest dose level with scorable metaphases even though this concentration exceeded the maximum range of optimum toxicity. It was considered that the scoring of this dose level that exceeded optimum toxicity had no impact on the validity or integrity of the study. No increase in the frequency of cells with aberrations present were recorded in any of the exposure groups tested.

Therefore, the test item is considered to have been adequately tested under the conditions of this study in the 4(20)-hour in the absence of S9 and because optimum toxicity was achieved in both the 4(20)-hour in the presence of S9 and the 24-hour exposure groups.

Conclusions:
Benzylated Polyamine did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Benzylated Polyaminedid not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytesin vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Metabolic activation:
with and without
Metabolic activation system:
Lot No. PB/βNF S9 20/08/17 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
Test concentrations with justification for top dose:
In the Preliminary Toxicity Test were used these concentrations

0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000 μg/mL

In the test were used these concentrations

Group Benzylated Polyamine (μg/mL) Concentrations
4-hour without S9 25, 50, 100, 200, 240, 280, 320, 360
4-hour with S9 (2%) 25, 50, 100, 200, 300, 400, 500, 600
24-hour without S9 2.5, 5, 10, 20, 40, 60, 80, 100

The maximum dose level used was limited by test item induced toxicity.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range was set at 19.53 to 3750 μg/mL in all three exposure groups. The dose levels were selected to avoid the excessive pH issues observed during the solubility check. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.
The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM, whichever is the lowest concentration.
ii) The presence of precipitate regardless of where test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at 8 dose levels of the test item, vehicle and positive controls. 2 mL of S9-mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group) were added to each universal. The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
Evaluation criteria:
See below
Statistics:
See below
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A summary of the results from the test is presented in Table 1.
There was evidence of marked toxicity following exposure to the test item in all three exposure groups as indicated by the %RSG and RTG values. There was evidence of moderate reductions in viability (%V) in the 24-hour exposure group only; therefore indicating that residual toxicity had occurred with the extended exposure time. Optimum levels of toxicity were considered to have been achieved in all three exposure groups. The upper dose levels of 360 μg/mL in the 4-hour –S9, 500 and 600 μg/mL in the 4-hour +S9 and 80 and 100 μg/mL in the 24-hour –S9 exposure groups were not plated out for 5-TFT resistance and viability due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups. A precipitate of the test item was observed at and above 500 in the 4+ S9 exposure only.
Conclusions:
The test item, Benzylated Polyamine did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

The test item, Benzylated Polyamine did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether was tested in three in vitro assays- Ames test, chromosome aberrartion assay and micronucleus test. The results were negative in all three tests indicating that the test material has not to be classified for genotoxicity according to CLP regulation 1727/2008.