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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key reverse mutation test in bacteria (Ames test) according to OECD guideline 471, the test substance proved to be negative for mutagenicity with and without metabolic activation (Wisher, 2020). This was confirmed in one supporting, reliable Ames test.

In a reliable, key mouse lymphoma test performed according to OECD guideline 476 (Litton Bionetics Inc, 1982; Klimisch 2), the test substance tested negative with and without metabolic activation.

in the Balb3T3 in vitro transformation assay, the test substance was considered to be inactive (Litton Bionetics Inc, 1982; Klimisch 2)

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
Study period:
2020-07-22 - 2020-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997 as corrected in 2020
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffamine D-230
- Lot No.: DRW2000305
- Physical state: Pale yellow liquid
- Purity: ca. 100% (w/w). This product is not specified by purity/assay but by titratable total acetylatables and amine content
- Stability under test conditions: not indicated
- Storage condition of test material: Room temperature over silica gel in the dark under nitrogen
- Final preparation of a solid: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction for purity was required. All test item preparation and dosing was performed under yellow safety lighting.
Target gene:
histidine or tryptophan locus
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:
10% rat liver homogenate metabolizing system
Test concentrations with justification for top dose:
The maximum concentration in the first experiment (plate incorporation method) was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully miscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent control
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
In the absence of S9-mix 2 µg/plate for WP2uvrA 3 µg/plate for TA100 5 µg/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In the absence of S9-mix 80 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent control
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
In the absence of S9-mix 0.2 µg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the presence of S9-mix 1 µg/plate for TA100 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent control
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
In the presence of S9-mix 5 µg/plate for TA98
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding::
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Test substance added: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Experiment 2 - not specified
- Exposure duration/duration of treatment: between 48 and 72 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Method: background growth inhibition; dicrease in numbe of revertants, microcolonies
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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:
Statistical significance was confirmed by using Dunnett’s 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.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no 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).

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), 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 were shown to be sterile. The test item formulation was also shown to be sterile.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within or close to the normal range. A single count for TA1535 ( solvent control dosed in the presence of S9-mix after the second mutation test) was just below the minimum level of the in-house historical untreated/vehicle control minima for the tester strain. This count was considered acceptable as the other vehicle and untreated control counts were within the expected range and the tester strain responded very well to 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test, the test substance was considered to be non-mutagenic.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982-03-03 to 1982-07-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Limited information was provided on the test substance (e.g., purity was missing).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
limited information was provided on the test substance (e.g., purity was missing).
Principles of method if other than guideline:
N/A
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy); 4236-44-15 (J-129)
- Substance type: active
- Physical state: clear, colorless liquid
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were maintained in Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10% by volume). Cloning medium consisted of the preceding growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state. Selection medium was cloning medium containing 100 ug/ml of BrdU or 3 ug/ml of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; Laboratory cultures were periodically checked by culturing methods.
- Periodically "cleansed" against high spontaneous background: yes; To reduce the negative control frequency (spontaneous frequency) of TK-/- mutants to as low of a level as possible, cell cultures were exposed to conditions which selected against the TK-/- phenotype (exposure to methotrexate) and were then returned to normal growth medium for three or more days before use.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Under nonactivation conditions, the test material was lethal at 8000 nl/ml, but the data were not shown. Six treatments, from 500, 1000, 2000, 3000, 4000 and 6000 nl/ml were used. These were the concentrations at which the mutation frequencies were analyzed. Additional concentrations were used for treatment, but the study did not provide complete information on all of the concentrations used at the beginning of the test. Complete information was only provided on the analyzed concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test substance was chosen based on solubility.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
water (test substance vehicle)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation: 0.5 µl/ml
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
water (test substance vehicle)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine-0.3 µl/ml
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 or 3 days
- Selection time (if incubation with a selection agent): 10 days


SELECTION AGENT (mutation assays): 5-bromo-2'-deoxyuridine


NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: Relative growth (%) (relative suspension growth x relative cloning efficiency/100)


OTHER: Cell counts were determined daily and appropriate dilutions were made to allow optimal growth rates.
Evaluation criteria:
See below for criteria.
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Although the test substance appeared soluble in the assay medium up to the testing limit of 10 ul/ml (10000 nl/ml), a marked change in pH was noted. The pH indicator in the medium turned pink indicating a basic pH. Since this could cause cellular toxicity, the pH was adjusted to 7.3 with HCL prior to dosing the final trial. Three trials of the mutation assay were initiation but only trial 3 was performed with the pH adjusted to 7.3. Since it was obvious from the results of Trial 3, that the toxicity observed in trials 1 and 2 was caused by the basic pH of the test substance, the first two trials were not used in the evaluation.

ADDITIONAL INFORMATION ON CYTOTOXICITY AND MUTAGENICITY: Under non activation conditions, the test substance was lethal at 8000 nl/ml (data not included in the report). Six treatments from 500-6000 nl/ml were therefore chosen for analysis of mutant induction and moderate to high toxicities were induced (% relative growth, 20.1-7.0 %). All of the assayed treatments induced mutation frequencies that were similar to the backgrounds (solvent and untreated control mutant frequencies). The test substance was therefore considered non mutagenic without metabolic activation.
In the presence of metabolic activation, the test substance was also assayed from 500-6000 nl/ml and a wide range of toxicities were induced (% relative growth, 64.6 to 10.6%). The minimum criterion for mutagenesis in the assay was a mutant frequency exceeding 65.4 x 10^-6 to 44.0 x 10^-6. The test substance was therefore considered nonmutagenic in the assay with S9 activation.
In the assays considered in this evaluation, the average cloning efficiencies for the solvent and untreated negative controls varied from 92.6 % without activation to 95 % with activation which demonstrated excellent cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control substances yielded normal mutant frequencies that were greatly in excess of the background.
Conclusions:
The test substance was evaluated for mutagenic potential in Mouse Lymphoma Forward Mutation Assay using the L5178Y cell line. The test substance failed to induce mutations at the thymidine kinase locus up to a dose level of 6000 nl/ml in the absence or presence of metabolic activation.
Endpoint:
in vitro transformation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982-01-12 to 1982-04-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
To measure ability of test substance to induce cell transformation and loss of contact inhibition in BALB/3T3 cells in vitro.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4236-44-15, #J128
- Physical state: clear, colorless liquid
- Stability under test conditions: The test substance was completely soluble in culture medium at the maximum tested concentration of 100 micorgrams/ml. At this concentration a marked alkalinity was noted; and therefore, the pH of the primary dosing-stock solution was adjusted to pH 7.1 with 1 N HCl.
- Date of receipt: January 6, 1982.
Target gene:
Loss of contact inhibition; formation of foci
Species / strain / cell type:
mammalian cell line, other: Clone 1-13 of Balb/3T3 Cells obtained from Dr. Takeo Kakunaga
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's Minimum Essential Medium supplemented with fetal bovine serum, L-glutamine, penicillin and streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information provided
- Periodically "cleansed" against high spontaneous background: yes, subclones were selected for low spontaneous frequencies of foci formation were used for assays
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not applicable
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.061, 0.122, 0.244, 0.488, 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 nl/ml
Transformation assay: 75.0, 150.0, 225.0, 300.0, 450.0 nl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: no information provided
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
At least 20 dishes
True negative controls:
not specified
Positive controls:
yes
Remarks:
At least 20 dishes were exposed to 2.5 to 10 micrograms/ml for each assay
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 24 hours
- Expression time (cells in growth medium): four weeks

- Fixation time (start of exposure up to fixation or harvest of cells): four weeks

NUMBER OF REPLICATIONS: three culture dished seeded with 200 cells per dish per dose

NUMBER OF CELLS EVALUATED: three culture dished seeded with 200 cells per dish per dose

DETERMINATION OF CYTOTOXICITY
- Method: Transformed cells form a dense mass (focus or colony) that stains deeply (usually blue) and is superimposed on the surrounding monolayer of normal cells. The foci are variable in size.

Scored foci have several variations in morphological features. Most scored foci consist of a dense piling-up of cells and exhibit a random, criss-cross orientation of fibroblastic cells at the periphery of the focus and extensive invasiveness in the contiguous monolayer. Other scored foci are composed of 1) more rounded cells with little criss-crossing at the periphery but with necrosis at the center caused by dense piling-up of a large number of cells, or 2) foci without the necrotic center and large number of cells but which exhibit the criss-cross pattern of overlapping cells throughout most of the colony. Foci that have these characteristics and exceed 2 mm in diameter are scored +++ and those <2mm in diameter are designated ++.

Some densely stained areas are not scored as transformed foci. These include focal areas where some piling-up of rounded cells has occurred but the random orientation of fibroblastic cells is not observed (+). Microscopic examination is employed for scored foci and in the final judgment of transformed character of any marginal foci.




Evaluation criteria:
The assay will be considered acceptable for evaluation for the test results if the following criteria are met:
1. The negative control dishes consist of a contiguous monolayer of cells which may or may not contain transformed foci. The lack of contiguous sheet of cells indicates growth conditions too poor to allow the reliable detection of weak transforming agents.
2. The negative control transformation frequency does not exceed an average of about 2-3 foci/dish after log10 analysis. Attempts are made to isolate and maintain cell stocks (subclones of Balb/3T3 1-13) with a very low spontaneous frequency of transformation.
3. The positive control yields an average number of foci/dish that is significantly different from the negative control at 95 or 99% confidence level.
4. A minimum of 10 flasks per test condition are available for analysis. At least 3 dose levels of test substance are assayed.
5. The dose range of test substance assayed falls within the 10-100% survival range as determined by the preliminary toxicity test, which measures relative cloning efficiencies.

Statistics:
Bailey's modification of Student's t-test (Statistical Methods in Biology, Wiley and Sons, Inc., NY, page 50, 1059) was used to determine whether the results for each treatment condition was significantly different from the experimental negative control.
Key result
Species / strain:
mammalian cell line, other: Clone 1-13 of Balb/3T3 Cells obtained from Dr. Takeo Kakunaga
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was conducted with a series of dilutions ranging from 1.0 micrograms/ml to 0.061 nl/ml in two-fold dilution steps. The treatments resulted in survivals ranging from 0.6% at 500 nl/ml to approximately 102% at 0.061 nl/ml. No survivors were observed for the treatment with 1000 nl/ml. The transformation assay is normally applied to concentrations that cause survivals in the 10% to 100% range and is considered to be most sensitive near 10-20% survival, since maximum transformation frequencies for a series of model carcinogens were obtained for treatments that resulted in survivals over this range. In the present assay, test substance doses were chosen over a wide range of survivals including one dose (450 nl/ml) that resulted in an apparent survival of ~5% as estimated from the results of the preliminary cytotoxicity test. The additional concentrations of 300.0 to 75.0 nl/ml, corresponding to relative survivals of approximately 20 to 75% (estimated graphically from the reported data) were also chosen for the assay.



Summary of Data from Transformation Assay

Test

Total number of foci

Number of Foci/Dish Absolute/Log10

Negative Control

(Culture medium)

1

0.06/0.04

Positive Control

(MCA) 2.5 microgram/ml

91

5.35/4.98

Test material

 

 

450.0 nl/ml

1

0.06/0.04

300.0 nl/ml

2

0.11/0.08

225.0 nl/ml

4

0.21/0.16

150.0 nl/ml

3

0.15/0.11

75.0 nl/ml

2

0.10/0.07

 

 

 

 

Conclusions:
The test substance did not induce the appearance of a significant number of transformed foci over the concentration range fo 450 nl/ml to 75 nl/ml. This concentration range corresponded to approximately 5% to near 10% survival in the preliminary cytotoxicity test. Therefore, the test substance is considered to be inactive in the Balb.3T3 In Vitro Transformation Assay.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1982-01-26 to 1982-02-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
However, no E. coli strain or S. typhimurium TA102 was included. Limited information was provided on the test substance.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No E. coli strain or S. typhimurium TA102 were included in the study. Limited information was provided on the test substance.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)
- Substance type: active
- Physical state: clear, colorless liquid
- Stability under test conditions: There was no apparent change in the physical state of the test substance during the assay.
- Test article 4236-44-15; Order #J-127.
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
other: See table below.
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
other: See table below.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 was used as the metabolic activation. There were 0.6 ml of S-9 supernatant (47 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.
Test concentrations with justification for top dose:
100, 333, 1000, 3333 and 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The test substance was miscible in the solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water (test substance vehicle)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, 1 ug/plate for TA1535 and TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water (test substance vehicle)
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation, 5 ug/plate for TA1538 and TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water (test substance vehicle)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation, 150 ug/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water (test substance vehicle)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine-5 ug/plate for all strains
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation): Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine-0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45 degrees C until used. Using sterile technique, the following were added to tubes in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test substance. S-9 was added in addition when appropriate. The tubes were vortexed and poured onto minimal glucose plates. The samples was evenly distributed on the plate, and the top agar overlay was allowed to harden.

DURATION
- Exposure duration: 48-72 hours
- Selection time (if incubation with a selection agent): 48-72 hours (simultaneous with exposure)


SELECTION AGENT (mutation assays): histidine


NUMBER OF REPLICATIONS: All solvent and positive controls were plated in triplicate, while all test substance groups were plated in duplicate.


DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn

Evaluation criteria:
A positive result was defined as a reproducible, dose related increase in the number of histidine-independent colonies. If the solvent control was within one standard deviation of the historical mean for control values and the test substance produced the highest increase equal to or greater than three times the solvent control value, the test substance was considered mutagenic. A negative result was defined as the absence of a reproducible increase in the number of histidine-independent colonies.
Statistics:
N/A
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 specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: a preliminary toxicity test was performed with S. typhimurium TA1538 and TA100 using dose levels of 100-10,000 ug/plate in the absence of metabolic activation using the plate incorporation assay.. All solvent and test substance groups were performed in duplicate. Plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. The concentration giving moderate inhibition of bacterial lawn growth served as the high dose in the plate incorporation assay. If not toxicity was produced, the high dose for the test substance was prepared at 100 mg/ml. Lower doses would be at log dilutions.
No inhibition at the dose levels tested was observed and therefore, 10,000 ug/plate was chosen as the high dose for the main assay.

COMPARISON WITH HISTORICAL CONTROL DATA: All solvent and positive controls were within the acceptable range of mean historical data. The results for the test substance were negative.

Substance

ug/plate

TA1535

TA1537

TA1538

TA98

TA100

 

-

+

-

+

-

+

-

+

-

+

Solvent

 

12

14

19

21

23

29

31

40

123

103

Sodium Azide

1

601

 

 

 

 

 

 

 

736

 

2-Nitrofluorene

5

 

 

 

 

1136

 

931

 

 

 

9-aminoacridine

150

 

 

2295

 

 

 

 

 

 

 

2-anthramine

5

 

182

 

465

 

2088

 

2832

 

1001

Test Substance

10000

13

10

22

23

18

35

42

29

78

106

3333

15

11

19

23

21

18

46

43

113

110

1000

19

14

21

28

20

22

44

43

117

102

333

15

18

17

24

16

27

40

35

119

117

100

17

16

18

24

24

27

39

29

111

119

 

Conclusions:
The test substance was evaluated for mutagenic activity in the Ames Salmonella typhimurium Assay with and without metabolic activation. Based on the results, the test substance was determined to be non-mutagenic.
Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A reliable, key in vivo micronucleus test on mouse bone marrow erythrocytes was performed according to OECD guideline 474 (Bioreliance, 2010). The test substance demonstrated to be negative up to dose levels of 500 mg/kg.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)-
- Analytical purity: The test substance was a complex mixture, and the purity was considered to be 100%.
- Storage condition of test material: ambient (15-30 degrees C), protected from light
Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD or other alternate Harlan location
- Age at study initiation: ~6-8 weeks
- Weight at study initiation: 25.5-37.0 grams (males), 19.5-31.0 grams (females)
- Assigned to test groups randomly: yes; Animals were assigned to groups using a randomization procedure based on equalization of group mean body weights (MiniTab based program). At the time of randomization, the weight of variation of animals did not exceed +/- 20% of the mean weight.
- Fasting period before study:
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of the system was to supply uninterrupted positive air to each individual rodent Micro-barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage. If needed, alternated housing system was implemented.
- Diet (e.g. ad libitum): ad libitum (Harlan 2018C Certified Global Rodent Diet)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- degrees F
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: As per the Sponsor information, purified water was used as the vehicle based on good workability/solubility of the test substance in the vehicle and compatibility of the vehicle with the test system.
- Concentration of test material in vehicle:
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was prepared prior to dose administration. Each concentration was prepared by mixing an appropriate amount of the test substance with the appropriate volume of the vehicle. The mixtures were vortexed, if needed, homogenized and stirred in order to achieve workable or soluble formulations.
The samples of the dosing formulations were not collected, and analysis of accuracy of preparation, homogeneity or stability of the formulations was not performed. However, by following the standard operating procedures of the laboratory it was to believe that the formulations were within commonly acceptable range of +/-20 % of target.
Animals received 20ml/kg via oral gavage.


Duration of treatment / exposure:
Animals received a single oral gavage administration.
Frequency of treatment:
Animals were treated once.
Post exposure period:
24 and 48 hours
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals/sex/dose/sacrifice time point
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): CP is a known clastogen and is commonly used as the positive control in genetic toxicology assays.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
femoral bone marrow (erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A dose range finding study was used to determine the doses to be used in the definitive study. In the dose range-finding study, a highest guideline recommended dose of 2000 mg/kg was administered to mice five male and five female mice. If mortality occurred at this dose level, additional dose levels were tested. Mice were observed after dose administration and each day thereafter for 3 days for clinical signs of toxicity. Body weights were recorded prior to dose administration (Study Day 0) and on Study Days 1 and 3 (prior to euthanasia). Following observation period, all surviving animals were euthanized by carbon dioxide inhalation followed by incision of the diaphragm, and discarded without further examination. If 2000 mg/kg produced no treatment related morality, the high dose for the definitive micronucleus test would be 2000 mg/kg. Otherwise, the high dose would be the maximum tolerated dose. To additional doses, one half and one-fourth of the high dose, would be tested.


DETAILS OF SLIDE PREPARATION:
In the definitive study, approximately 24 and 48 hours after dose administration, animals were euthanized by carbon dioxide inhalation followed by incision of the diaphragm. Animals in the positive control group were euthanized 24 hours after treatment. Immediately following euthanasia, the femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labelled centrifuge tube containing approximately 1 mL fetal bovine serum.
The bone marrow cells were pelleted by centrifugation and the supernatant was drawn off, leaving a small amount of fetal bovine serum with the remaining cell pellet. The cells were resuspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. Each slide as identified by the experiment and animals number. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with May-Gruenwald-Giemsa, permanently mounted and use d for microscopic evaluation. The other set of slides (not stained) was kept as a backup set.


METHOD OF ANALYSIS:
Slides were coded using a random number table by an individual not involved with the scoring process. Using a light microscope and a medium magnification (400X), an area of acceptable quality was selected such that the cells were well spread and stained. The following cell populations and cell components were recorded using oil immersion (1000X):
1. 2000 polychromatic erythrocytes were scored per animal for the presence of micronuclei resulting in evaluation of 10000 PCEs per group.
2. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes were also enumerated, but were not used to evaluate the response of the test substance.
3. The proportion of polychromatic erythrocytes to total erythrocytes were determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.
4. In the event of high reduction and suppression in PCEs/ECs ration, it may not have been possible to evaluate 2000 PCEs per animal for the presence of micronuclei.



Evaluation criteria:
See below.
Statistics:
Statistical analysis of data was performed using the Kastenbaum-Bowman tables which are based on the binomial distribution.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 750, 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals:
2000 mg/kg bw: mortality observed in all mice
1000 mg/kg bw: mortality observed in 1/5 males and 3/5 females + piloerection in suriving mice
750 mg/kg bw: mortality observed in 1/5 males and 2/5 females + piloerection in suriving mice
500 mg/kg bw: no mortality observed, piloerection in all mice following administration on Day 1
- Evidence of cytotoxicity in tissue analysed: not reported
- Rationale for exposure: 2000 mg/kg bw is the maximum regulatory recommended dose for bone marrow micronucleus assays; and the LD50 oral dose was 2886 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective control groups was observed in male or female mice at 24 or 48 hours after dose administration (p>0.05, binomial distribution, Kastenbaum-Bowman Tables)

- Ratio of PCE/NCE (for Micronucleus assay): No appreciable reduction in the ratio of polychromatic eruthrocytes to total erythrocytes in the test substance groups relative to the concurrent vehicle control groups were observed at 24 or 48 hours post-dose.

Summary of Bone Marrow Micronucleus Analysis Following a Single Oral Administration of Jeffamine D-230 in ICR Mice

Treatment (20 mL/kg)

Sex

Time (hr)

Numbe of animals

**PCE/Total Erythrocytes

(Mean +/- SD

Change

from

Control (%)

Number of MPCE/1000 PCE (Mean +/- SD)

Number of MPCE/1000 PCE (scored)

Purified Water

M

24

5

0.508±0.01

---

0.0±0.00

0 / 10000

F

24

5

0.523±0.02

---

0.0±0.00

0 / 10000

 Jeffamine D-230

125 mg/kg bw

M

24

5

0.519±0.01

2

0.0±0.00

0 / 10000

F

24

5

0.513±0.02

-2

0.0±0.00

0 / 10000

Jeffamine D-230

250 mg/kg bw

M

24

5

0.518±0.03

2

0.0±0.00

0 / 10000

F

24

5

0.522±0.02

0

0.0±0.00

0 / 10000

Jeffamine D-230

500 mg/kg bw

M

24

5

0.526±0.01

4

0.0±0.00

0 / 10000

F

24

5

0.519±0.01

-1

0.0±0.00

0 / 10000

Cyclophosphamide

50 mg/kg

M

24

5

0.522±0.01

3

13.3±4.78

*133 / 10000

F

24

5

0.531±0.02

2

13.9±3.36

*139 / 10000

Purified Water

M

48

5

0.515±0.03

---

0.0±0.00

0 / 10000

F

48

5

0.557±0.02

---

0.1±0.22

1 / 10000

Jeffamine D-230

500 mg/kg bw

M

48

5

0.496±0.02

-4

0.3±0.45

3 / 10000

F

48

5

0.524±0.01

-6

0.1±0.22

1 / 10000

* Statistically significant increase compared to vehicle control, p < 0.05 (Kastenbaum-Bowman Tables)

**PCE: Polychromatic Erythrocytes

Conclusions:
In an in vivo micronucleus test on mouse bone marrow erythrocytes, performed according to OECD 474, the animals were exposed orally (via gavage) with 125, 250 and 500 mg/kg. This did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. No toxicity was observed. Vehicle and positive controls were valid.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro

- Bacterial reverse mutation assay: In a key bacterial reverse gene mutation assay (Wisher, 2020; Klimisch 1; according to OECD 471) with multiple strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia Coli (WP2 uvrA), the test item was negative both with and without metabolic activation.

In a supporting bacterial reverse gene mutation assay (Pharmakon Research International Inc., 1982; Klimisch 2; similar to OECD 471) with multiple strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538), the test substance was negative both with and without metabolic activation.

 - Gene mutation in mammalian cells: In a mammalian cell gene mutation assay with L5178Y mouse lymphoma cells, exposure to the test substance (Litton Bionetics, Inc., 1982; Klimisch 2; similar to OECD 476) in the presence and absence of metabolic activation did not induce mutations at the thymidine kinase locus up to a dose level of 6000 nl/ml.

 - In vitro transformation assay in mammalian cells : In an in vitro BALB/3T3 cell transformation assay (Litton Bionetics, Inc., 1982, Klimisch 2), the test substance did not induce a significant increase in the number of transformed foci over the concentration range of 450 nl/ml to 75 nl/ml. Therefore, the test substance is considered to be inactive in the BALB/3T3 in vitro transformation assay.

 - an in vitro cytogenicity study in mammalian cells or an in vitro micronucleus tests does not need to be performed as an adequate in vivo study is available

In vivo

- micronucleus test: In an in vivo micronucleus test on mouse bone marrow erythrocytes, performed according to OECD 474, the animals were exposed orally (via gavage) with 125, 250 and 500 mg/kg. This did not induce a statistically significant increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. No toxicity was observed. Vehicle and positive controls were valid.

Justification for classification or non-classification

Based on the results and according to the criteria laid down in the CLP regulation (EC)1272/2008, the test substance is considered to be non genotoxic in vitro and in vivo. Therefore, the substance does not warrant classification.