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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Amines, polyethylenepoly-, triethylenetetramine fraction showed positve, ambigous or negative results in several Ames tests, in vitro gene mutation (mammalian cells) tests, Sister Chromatide Exchange tests and UDS test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study followed OECD 471 guideline and the GLP Regulations. No significant deviations can be observed from the study guidelines; however, strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 were not included. No data on purity.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 were not included
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction of rat liver homogenate obtained from Arcolor 1254-treated Sprague Dawley rats
Test concentrations with justification for top dose:
16.7, 50, 167, 500, 1000 and 1670 µg/plate
Vehicle / solvent:
-solvent(s) used: DMSO;
- Justification for choice of solvent: Maron, D.M., J. Katzenellenbogen and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test, Mutation m., 88:343-350.
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100= 10 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
TA1537=150 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
TA1538 and TA98= 5 µg/plate
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-anthramine
Remarks:
in all tester strains=2.5 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 48h
- Exposure duration: 48h



SELECTION AGENT (mutation assays): biotin and histidine



NUMBER OF REPLICATIONS:
test and control articles (positive and negative): triplicates of all five tested strains
test article in the prescreening: in duplicate



DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition is tested at the following concentrations: 50, 167, 500, 1670 and 5000 µg/plate


Evaluation criteria:
revertant colonies are counted (Artek electronic colony counter interfaced with an IBM PC/AT computer for data acquisation)
-positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertans with at least one dose level inducing solvent control value
-negative results is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants
-equivocal result is defined when the test article dose not induce a statistically significant, dose dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value
Statistics:
Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES:
- Growth inhibition was observed in strain TA1538 and TA100 at doses of 1670 and 5000 µg/plate without metabolic activation.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data:
Strain S9 n Average (± 1SD) Range (mean± 2SD)
TA1535 - 254 9.65±2.82 4.01 – 15.3
+ 253 10.1±2.81 4.48 – 15.7
TA1537 - 255 7.94±2.63 2.68 – 13.2
+ 248 9.28±2.77 3.74 – 14. 8
TA1538 - 262 5.24±2.45 0.340 – 10.1
+ 265 12.5±3.82 4.86 – 20.1
TA98 - 265 19.3±5.18 8. 94 – 29.7
+ 277 27.5±6.74 14. 0 – 41.0
TA100 - 270 86.9±18.0 50. 9 – 123
+ 272 99.0±18.2 62.6 - 135


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Growth inhibition was observed in strain TA1535, TA1537, TA1538, TA98, TA100 at doses 500, 1000 and /or 1670 µg/plate with and /or without metabolic activation.


ADDITIIONAL INFORMATIION
No increase (compared to the negtive control cultures) in revertant frequencies was seen in strain TA1538 with metabolic activation. In contrast, a dose-dependent increase in revertant frequencies to approx 1.9- to 13- fold control values, were observed in strain TA1538, TA1537, TA98 and TA100 with metabolic activation, and in all strains without S9 mix. Therefore, the test substance was re-evaluated under identical conditions in all tester strains with and without S9 mix. Revertant frequencies for all doses in strain TA 1535 without S9 mix approximated or were less than control values. In contrast, statistically significant and/or dose-dependent increases in revertant frequencies, to approximately 1.5- to 5.7-fold control values, were observed in all strains with S9. All positive and negative control values in both assays were within acceptable limits.
Conclusions:
Interpretation of results: positive with and without metabolic activation

The results for Amines, polyethylenepoly-, triethylenetetramine fraction were positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to, the criteria, of the test protocol.
Executive summary:

The test substance was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test).

In the RANGE-FINDING/SCREENING STUDIES Growth inhibition was observed in strain TA1538 and TA100 at doses of 1670 and 5000 µg/plate without metabolic activation. Growth inhibition was observed in strain TA1535, TA1537, TA1538, TA98, TA100 at doses 500, 1000 and /or 1670 µg/plate with and /or without metabolic activation.

A dose-dependent increase in revertant frequencies to approx 1.9- to 13- fold control values, were observed in all strains with and without metabolic activation. Thus, Amines, polyethylenepoly-, triethylenetetramine fraction was considered to be mutagenic in this in vitro bacterial assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 17, 1980 - July 10, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
CHO
Principles of method if other than guideline:
- Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of cultures for analysis (e.g. at least 8 analysable concentrations). Duplicate negative
(solvent) control cultures should be used.
GLP compliance:
no
Remarks:
pre-GLP, inhouse QA in place
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells are maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine.
For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 µg/ml streptomycin and 5% (v/v) of dialyzed bovine serum (F12-D5) is used.
For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
For determination of mutant frequencies, F12-D5 medium containing 2.0 µg/ml TG (6-thioguanine) is used as a "selective medium."
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
80.0, 40.0, 20.0, 10.0, 5.0, 2.5 x 10E-2%
Selection of maximum concentration was based on preliminary toxicity testing and were depended upon an estimate of a dose level which would permit survival of at least 10% of the treated cells.
Vehicle / solvent:
water, dsmo
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water, dmso
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix EMS 200 µg/ml, +S9 mix DMN 3700µg/ml
Details on test system and experimental conditions:
CHO cells were exposed for 5 hours to a minimum of five concent rations of TETA both with and without the addition of an S9 metabolic activation system. Dilutions of TETA for testing were prepared by either direct addition of various aliquots of the test agent into the cell culture media or by making sequential one-half dilutions of the stock solution for the maximum concentrations using sterile H20. The surviving fraction was determined at 20 to 24 hours after treatment and the mutant fraction was determined after a 7-day period to allow "expression" of the mutant phenotype.


Statistics:
Data from the CHO test do not follow a normal distribution according to experience with historical controls. Thus, the Student's t-test was used after transformation of the mutation frequencies (MF) according to the method of Irr and Snee (MF + 1)0.15 (Irr, J. D. and R. Snee, Proceedings of the Cold Spring Harbor-Banbury Conference, II (1979), 263-274).
Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 >20 x 10E-2%
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In tests with and without addition of an S9 metabolic activation system, TETA produced statistically significant increases in the frequency of mutants at all but one dose level in both tests. The effect was not definitively dose-related and the absolute number of mutants obtained in the test without metabolic activation was within the historical experience for the spontaneous occurrence of mutants with the solvent and negative controls in other tests. The presence of a marginal indication of a dose-related response in the test with S9 activation for dose levels between 10 x 10-2% and 40 x 10-2% and the production of highly significant mutation frequencies outside the historical control variation for the test indicated that TETA was an active agent in the induction of gene mutations in CHO cells.
Mutation frequencies for the solvent controls for tests both with and without S9 activation were in an acceptable and low range based upon experience with historical control values. statistically significant increases i n the mutation frequencies were obtained for the DMN and EMS positive controls for both experiments and these values were within the expected range of values observed in historical control data. The statistically significant effect obtained with DMSO, the negative control in the test with metabolic activation, was not considered to be biologically significant because DMSO has been observed in other tests to increase the inherent mutagenic action of the liver homogenate.

Cytotoxicity: A concentration of 0.8% was cytotoxic to the cells.

For detailed results see attached tables.
Conclusions:
Interpretation of results: positive

The pattern of positive responses produced by TETA in the 3 -test battery to determine potential mutagenicity indicated that TETA was active in comparison to known mutagenic agents, such-as the positive controls tested concurrently with the test chemical. The consistent observation of a positive effect in each of the three tests employed indicates that TETA (or one of its many components) should be considered to be an active mutagenic agent.
Executive summary:

TETA was evaluated for potential mutagenic activity with a battery of three in vitro tests, which were: the Chinese Hamster Ovary (CHO) Mutation test , the -- Sister Chromatid Exchange (SCE) test and an assay for induction of Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that TETA produced a statistically significant mutagenic effect in all of the three in vitro tests performed. Increases in the mutation index were observed both with and without metabolic activation. These positive results are consistent with literature reports on positive effects of TETA in the Ames (Salmonella) mutation assay.

RESULTS AND INTERPRETATION

Selection of Test Concentrations - Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of a proximately 10% of the treated cells. A maximum concentration of 80x 10E-2% (by volume) was chosen for the highest dose-level and a total of seven concentrations of TETA were tested for mutation induction because a steep dose response was suggested from prescreening data.

CHO Mutation Test - TETA produced a statistically significant increase in the frequency of mutations of CHO cells at several concentrations between 80 x 10E-2% to 2.5 x 10E-2% (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a definite dose-related effect of treatment suggested that the alkaline effect of the test agent may have interfered with the tests. With S9 metabolic activation, the acidic S9 liver homogenate may have somewhat buffered the alkaline effect and a dose related trend in the mutation index was observed for treatments between 10 x 10E-2% and 40 x 10E-2%.

SCE Test - TETA produced a highly statistically significant and dose-related increase in the frequency of SCE in CHO cells in tests without the incorporation of an S9 metabolic activation system. With metabolic activation, the effect on the frequency of SCE was decreased with respect to the results without S9, but the highest dose level produced a highly statistically significant effect . An overall range of concentrations between 40 x 10E-2% to 1.25 x 10E-2% (by volume) was used. The highly positive effects observed in the test without S9 metabolic activation indicated that TETA was an active mutagenic agent in the production of SCE in CHO cells.

UDS Test - TETA produced statistically significant increases in the amount of UDS activity in evaluations of concentrations between 100 x 10E-2% to 0.1 x 10E-2% (by volume). TETA was considered to be active in the present test with the hepatocyte test system and positive effects were observed in tests using both nuclei and DNA to detect increases in UDS.

Comparative Mutagenicity - The pattern of positive responses produced by TETA in the 3 -test battery to determine potential mutagenicity indicated that TETA was active in comparison to known mutagenic agents, such-as the positive controls tested concurrently with the test chemical. The consistent observation of a positive effect in each of the three tests employed indicates that TETA (or one of its many components) should be considered to be an active mutagenic agent.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 28, 1980 - September 11, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Principles of method if other than guideline:
- The test was not performed with duplicate cultures for each concentration.
- 15 cells were evaluated per culture instead of 25.
GLP compliance:
no
Remarks:
pre-GLP, inhouse QA in place
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
A commercial sample of TETA was hydrogenated with Raney Nickel catalyst to determine if treatment would alter the effects of this chemical observed in previous studies.
Target gene:
interchanges between sister chromatids
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells are maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine.
For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 µg/ml streptomycin and 5% (v/v) of dialyzed bovine serum (F12-D5) is used.
For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
For determination of mutant frequencies, F12-D5 medium containing 2.0 µg/ml TG (6-thioguanine) is used as a "selective medium."
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
50.0/40.0, 30.0, 20.0, 10.0, 5.0, 2.5 x 10E-2%
Test concentrations were selected based on preliminary cytotoxicity studies. Selection of suitable range of concentrations for testing was based upon an estimate of the doses which would not kill over 50% of the treated cells.
Vehicle / solvent:
water, dsmo
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water, dmso
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix EMS 100 µg/ml, +S9 mix DMN 300µg/ml
Details on test system and experimental conditions:
Production of SCE's following exposure to various concentrations of TETA was studied in CHO cells both with and without the incorporation of an S9 metabolic activation system. Selection of a maximum dose level which would permit survival of at least 50% of the treated cells was based on the prescreening test for cytotoxicity performed as part of the CHO Mutation test. Dilutions of TETA for testing were prepared either by direct addition of various aliquots into the culture medium or by making sequential one-half dilutions of the stock solution for the maximum dose level using sterile H20. For determination of direct mutagenic action, CHO cells were exposed to TETA and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU) required to differentiate between the individual "sister" chromatids by SCE staining, was present at a concentration of 3 µg/ml in the growth medium during treatment and during the culture period following exposure. A total of 20 cells/dose level and 5 dose levels, tested either with or without metabolic activation, were examined.

Statistics:
Data from the SCE test were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 >40 x 10E-2% without S9 >30 x 10E-2% after 5 hour treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically significant increase in the SCE frequency was produced by any dose level of TETA-RNT tested for direct action in the absence of a metabolic activation system. However, this first experiment was not considered reliable because there was a statistically significant difference between the solvent and medium controls. The SCE values produced by the medium were significantly lower than those obtained with medium.The experiment was repeated to assure that the sensitivity of the test was not affected by what could be considered a significantly higher value for the water control.
In comparison to the solvent control, we observed a statistically significant increase in the frequency of SCE at four of five doses of the test chemical. Although the response w as non-linear, there also appeared to be a general dose-related effect upon the SCE frequency. This second test indicated that TETA-RNT was an active agent for producing DNA damage in this in vitro test sytem. The number of SCE produced by the concurrent EMS positive control in both experiments was highly statistically different than the values for the concurrent solvent control. These data indicated an appropriate s nsitivity of the test system comparable to our historical positive control data. The numbers of SCE obtained with the H2O solvent and medium contro;s were statistically different in experiment 1 as discussed previously. Both negative controls were essentially identical and within the range of experimental variability in experiment 2.

In the presence of an S9 metabolic activation system highly statistically significant increases in the SCE values were produced at all of the tested concentrations of TETA-RNT. The value of SCE for the medium control in this experiment was significantly greater than the value for the H2O control. The variation in the two negative controls was not a typical result for this assay and the reason for this discrepancy is unknown.
However, four of five of the concentrations of the test agent produced SCE levels which were numerically higher than the historical control values for
this test system and the SCE value produced by the (unusually high) medium control. Also, statistical analyses using the medium control for comparison revealed a highly significant difference for the 30 x 10-2% dose level which was consistent with the statistical indications of a positive effect using the H2O control for comparison. These comparisons indicated that the test agent produced a positive effect on SCE which was relatively weak in comparison to the positive control agents, for example. However, the positive results obtained in this test: with metabolic activation were considered to be
consistent with the findings in the test without addition of S9. TETA-RNT was considered to be weakly active as a mutagenic agent in the induction of SCE in vitro.
The SCE values for the negative and solvent controls in the test with S9 activation were moderately outside the range of variability as encountered in previous experiments with this test system. Additional testing was not performed because the results with metabolic activation were in basic agreement with the positive conclusions from the experiment with S9 activation. Highly statistically significant numbers of SCE were produced by the positive control tested which indicated that the metabolic activation system was suitably active.

Conclusions:
Interpretation of results: positive

TETA-RNT was evaluated for potential mutagenic activity using the Sister Chromatid Exchange (SCE) test and an assay to detect the production of Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that TETA-RNT produced a statistically significant effect upon SCE frequency in CHO cells and weakly positive effects in the UDS test. Thus, TETA-RNT was considered to be an active, but weak, mutagenic agent in the in vitro screening tests employed.
Executive summary:

An appropriate range of test concentrations was chosen from data of previous studies of TETA in our laboratory (BRRC Report Numbers 43-127, 44-11 and 44-12). For the SCE test, a range of concentrations was chosen which would not produce excessive cytotoxicity and inhibition of cell division. For the UDS test, a wide range of concentrations spanning cytotoxic to non-cytotoxic doses were tested.

SCE: TETA-RNT produced a statistically significant and dose-related effect upon the frequency of SCE in CHO cells in tests both with and without the incorporation of an S9 metabolic activation system. An overall range of concentrations betwen 2.5 x 10E-2% to 50 x 10E-2% (by volume) was tested and the effects on the SCE frequency were determined with the highest five concentrations which allowed adequate cell division. The results indicated that TETA-RNT was an active agent in this test and should be considered a probable positive mutagenic agent for production of DNA damage in animal cells in culture.

UDS Test: TETA-RNT produced slight increases in the amount of UDS activity in evaluations of concentrations between 100 x 10E-2% to 0.1 x 10E-2% ( by volume) . TETA-RNT was considered to be weakly active in the present test with the hepatocyte test system because a majority of the UDS levels were significantly greater than historical negative control values for this test system.

Comparative Mutagenicity: The pattern of responses produced by TETA-RNT in the screening tests to determine potential mutagenicity indicated that TETA-RNT was weakly active in comparison to known mutagenic agents, such as the positive controls tested concurrently with the test chemical. The observation of positive effects in the SCE test and weak effects in the UDS test indicated that TETA-RNT was a weakly active mutagen in the in vitro- assays employed.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 19, 1987 - February 20, 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
Target gene:
SCE: interchanges between sister chromatids
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells are maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine.
For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 µg/ml streptomycin and 5% (v/v) of dialyzed bovine serum (F12-D5) is used.
For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
For determination of mutant frequencies, F12-D5 medium containing 2.0 µg/ml TG (6-thioguanine) is used as a "selective medium."
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
-S9 mix 0.2, 0.3, 0.4 mg/ml
+S9 mix 0.5, 0.6, 0.8 mg/ml
Selection of a suitable range of doses for testing was based upon cytotoxicity data obtained from preliminary experiments to determine relative cytotoxicity.
Vehicle / solvent:
cell culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix EMS 100 µg/ml, +S9 mix DMN 300µg/ml
Details on test system and experimental conditions:
Production of SCEs following exposure to various concentrations of triethylenetetramine - sample A was studied with duplicate cultures of CHO cells tested both with and without the incorporation of a rat-liver S9 metabolic activation system. Various concentrations of triethylenetetramine - sample A for testing were attained by direct addition of various aliquots of the diluted test agent into the culture medium. Cell-culture medium was used as the diluent. All dilutions were prepared immediately prior to testing. For determination of direct mutagenic action, CHO cells were exposed to triethylenetetramine - sample A and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU), required to differentiate between the individual sister chromatids by SCE staining, was present at a concentration of 3 µg/ml in the growth medium during treatment and during the culture period following exposure. A total of twenty-five. cells/concentration was examined for SCE frequencies using duplicate cultures. At least 5 dose levels were tested both with and without metabolic activation. SCE production was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division. The number of SCEs/cell, mean number of SCEs/chromosome and the level of statistical significance of the increases above the concurrent solvent control values are presented. The percentages of cells at various mitotic stages of cell division were monitored and recorded for indicating comparative cytotoxic effects.
Evaluation criteria:
The criteria for evaluation of a positive or negative response depend on both on statistical analyses and scientific judgement. The key determinant is whether a dose-dependent increase in SCEs is induced by the test agent and if consistent repsonses are seen in duplicate culture/dose. When no clear dose-response relationship is evident and when one or more repsonses of marginal indications of statistical differencs are obtained, a careful examination of the data in comparison to the concurrent controls and the historical data base is necessary to determine the probable biological significance of the statistical indicators. Clearly positive responses will include any of the following:
(i) Doubling in the SCE frequency by one or more concentrations with both of the duplicate cultures/doses,
(ii) Statistically significant responses of p < 0.01 with one or more consecutive concentrations;
(iii) Induction of a statistically significant, dose-related increase in the number of SCE.
Random statistical indications of positive increases which do not meet the criteria for a positive test result will be concluded to be negative indications of DNA damage potential.
Statistics:
Data from the SCE tests were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
SCE production in CHO cells treated with various dose levels of triethylenetetramine - sample A or with positive and solvent control agents without an S9 metabolic activation system. Statistically significant increases in the numbers of SCEs were observed with two of the three doses of the test agent evaluated for SCE induction. The increases followed a dose-related trend and the 0.4 mg/ml high dose produced approximately a 1.4-fold increase above control values. The data showed that triethylenetetrarnine - sample A was a direct-acting, but weak genotoxic agent in this in vitro test system. The number of SCEs produced by the concurrent EMS positive control was highly statistically different from the values for the concurrent solvent controls. These data indicated an appropriate sensitivity of the test system comparable to the historical positive control data. The number of SCEs obtained with the solvent and medium controls were also in an acceptable range of values included in the variability encountered in historical control values for this test.
SCE values obtained following treatments of CHO cells with triethylenetetramine - sample A in the presence of an S9 metabolic activation system are presented in Table 3. Statistically significant increases in SCEs were produced only by the lowest dose of the test agent evaluated. The quantitative increases in SCEs showed an inverse trend in contrast to the test without S9 activation. SCE values attained approximately a 1.3-fold increase over concurrent controls. Triethylenetetramine - Sample A was not considered to be a genotoxic agent in the test with S9 activation because of the lack of a dose-related response.
The SCE values for the negative and solvent controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCEs were produced by the DM' positive control which indicated that the metabolic activation system was suitably active.

The proportion of cells at various mitotic stages of cell division following exposures to triethylenetetramine are often helpful to determine the extent of biological effects of the test agent or to identify doses which cause severe mitotic inhibition. Observation of increases in the numbers of first division cells would indicate cytotoxic inhibition of the cell progression of the cell population. In the data from the test with S9, the proportion of 1st division cells decreased commensurate with increasing test concentration. The 0.5 mg/ml dose had approximately 13% of the cells which only completed one round of cell division in comparison to 2% for the control culture. In the test without S9 activation, no remarkable increases in 1st division cells were evident indicating absence of cytotoxic effects. The data from these two tests demonstrate that the doses evaluated for SCEs were in a biologically effective range of concentrations, but did not produce excessive cytotoxicity. Also, the inverse dose-response effect in the test with S9 was similar to the SCE increases at only the lowest dose. The reason for the inverse relationship in absence of overt toxicity is not known.

Historical control data:
Negative controls: n = 30; mean = 0.509 SCEs/chromosome; S.D. = 0.087; median = 0.498 SCEs/chromosome; 95 percentile range = 0.227 - 0.666 SCEs/chromosome
Conclusions:
Interpretation of results: positive without metabolic activation

Triethylenetetramine - Sample A was evaluated for potential genotoxic activity using the Sister Chromatid Exchange (SCE) test in Chinese hamster ovary (CHO) cells in vitro. The results indicated that the test chemical produced a dose-related, statistically significant genotoxic effect in SCE test conducted without addition of a rat-liver S9 metabolic activation system. With metabolic activation, the test material produced a statistically elevated SCE incidence only at the lowest test dose examined for SCEs. Triethylenetetramine - Sample A was considered to be a weakly-active genotoxic agent in the absence but not in the presence of a metabolic activation system.
Executive summary:

Selection of Test Concentrations - Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would produce moderate cytotoxicity but still allow sufficient numbers of cells in the second division (M2) for determination of SCEs. Test results with triethylenetetrarnine - sample A indicated that concentrations of 1.0 mg/ml or higher were excessively cytotoxic to CHO cells in the tests with and without S9 activation and the treated cells were detached from the culture flask. For the SCE test without S9 activation, a maximum concentration of 0.5 mg/ml was tested. With S9 activation, a slightly lower degree of growth inhibition was noted and a maximum dose of 0.8 mg/ml was used for the definitive SCE test, In the SCE test without S9 activation, the 0.5 mg/ml doses produced excessive mitotic inhibition and too few cells were available for SCE scoring.

SCE Test- Triethylenetetramine - Sample A produced dose-related and statistically significant increases in SCEs in the test without addition of a rat-liver S9 metabolic activation system. With S9 activation, an inverse dose response relationship was observed and a significant response was obtained only at the lowest test concentration. The highest increases in SCEs above the combined solvent control values were approximately 1.4 fold without S9 and 1.3 fold with S9 activation. No remarkable degree of cell-cycle inhibition was produced by the test chemical by determination of the ratio of numbers of cells in the first and second cycle of division. The test chemical was considered to be a positive but weakly-active genotoxic agent in the SCE test system.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 17, 1980 - July 10, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Principles of method if other than guideline:
- The test was not performed with duplicate cultures for each concentration.
- 15 cells were evaluated per culture instead of 25.
GLP compliance:
no
Remarks:
pre-GLP, inhouse QA in place
Type of assay:
sister chromatid exchange assay in mammalian cells
Target gene:
interchanges between sister chromatids
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells are maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine.
For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 µg/ml streptomycin and 5% (v/v) of dialyzed bovine serum (F12-D5) is used.
For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
For determination of mutant frequencies, F12-D5 medium containing 2.0 µg/ml TG (6-thioguanine) is used as a "selective medium."
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
40.0, 20.0, 10.0, 5.0, 2.5, 1.25 x 10E-2% without S9 mix
40.0, 20.0, 10.0, 5.0, 2.5 x 10E-2% with S9 mix
Top dose was chosen based on cytotoxicity data from the CHO mutation test. Higher concnetrations were expected to produce delays in the mitotic cycle and to decrease the number of cells with SCE staining.
Vehicle / solvent:
water, dsmo
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water, dmso
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix EMS 100 µg/ml, +S9 mix DMN 500µg/ml
Details on test system and experimental conditions:
Production of SCE's following exposure to various concentrations of TETA was studied in CHO cells both with and without the incorporation of an S9 metabolic activation system. Selection of a maximum dose level which would permit survival of at least 50% of the treated cells was based on the prescreening test for cytotoxicity performed as part of the CHO Mutation test. Dilutions of TETA for testing were prepared either by direct addition of various aliquots into the culture medium or by making sequential one-half dilutions of the stock solution for the maximum dose level using sterile H20. For determination of direct mutagenic action, CHO cells were exposed to TETA and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU) required to differentiate between the individual "sister" chromatids by SCE staining, was present at a concentration of 3 µg/ml in the growth medium during treatment and during the culture period following exposure. A total of 20 cells/dose level and 5 dose levels, tested either with or without metabolic activation, were examined.

Statistics:
Data from the SCE test were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center.
Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 >20 x 10E-2%
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
A highly statistically significant increase in the SCE frequency was produced by three out of five dose levels of TETA tested for direct action in the absence of a metabolic activation system. The higest dose (0.4%) was cytotoxic and thus not considered for evaluation. All five of the five concentrations of TETA tested produced an increased frequency of SCE in CHO cells and the effect was directly related to the treatment dose. A positive dose-related effect is considered a strong indication for the existence of a biologically significant mutational effect.
The test without S9 activation was considered a positive indication for potential direct mutagenic action of the test agent. The number of SCE produced by the concurrent EMS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the test system comparable to our historical positive control data. The numbers of SCE obtained with the solvent and controls were also in an acceptable range of values included in the variability encountered in our historical control values for this test.
A statistically significant increase in the SCE values was observed at two of the five tested concentrations of TETA in presence of S9 mix. However, there was no evidence for dose-response effect of TETA treatments, in contrast to the findings in the test without addition of S9. The production of a highly significant effect at the highest dose level suggested that additional higher concentrations should be tested to determine the existence of a reproducible, biologically significant response. However, these tests were not considered necessary to adequately evaluate the mutagenic potential of TETA which was clearly a positive mutagen i n the other tests of the mutation test battery; including the positive effect upon SCE in the test without S9 activation. The SCE values for the negative and solvent controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCE were produced by the DMN positive control which indicated that the metabolic activation system was suitably active.

Cytotoxicity: The highest dose of 0.4% was toxic to the cells.

For detailed information see attached tables.
Conclusions:
Interpretation of results: positive

The pattern of positive responses produced by TETA in the 3 -test battery to determine potential mutagenicity indicated that TETA was active in comparison to known mutagenic agents, such-as the positive controls tested concurrently with the test chemical. The consistent observation of a positive effect in each of the three tests employed indicates that TETA (or one of its many components) should be considered to be an active mutagenic agent
Executive summary:

TETA was evaluated for potential mutagenic activity with a battery of three in vitro tests, which were: the Chinese Hamster Ovary (CHO) Mutation test , the -- Sister Chromatid Exchange (SCE) test and an assay for induction of Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that TETA produced a statistically significant mutagenic effect in all of the three in vitro tests performed. Increases in the mutation index were observed both with and without metabolic activation. These positive results are consistent with literature reports on positive effects of TETA in the Ames (Salmonella) mutation assay.

RESULTS AND INTERPRETATION

Selection of Test Concentrations - Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of a proximately 10% of the treated cells. A maximum concentration of 80x 10E-2% (by volume) was chosen for the highest dose-level and a total of seven concentrations of TETA were tested for mutation induction because a steep dose response was suggested from prescreening data.

CHO Mutation Test - TETA produced a statistically significant increase in the frequency of mutations of CHO cells at several concentrations between 80 x 10E-2% to 2.5 x 10E-2% (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a definite dose-related effect of treatment suggested that the alkaline effect of the test agent may have interfered with the tests. With S9 metabolic activation, the acidic S9 liver homogenate may have somewhat buffered the alkaline effect and a dose related trend in the mutation index was observed for treatments between 10 x 10E-2% and 40 x 10E-2%.

SCE Test - TETA produced a highly statistically significant and dose-related increase in the frequency of SCE in CHO cells in tests without the incorporation of an S9 metabolic activation system. With metabolic activation, the effect on the frequency of SCE was decreased with respect to the results without S9, but the highest dose level produced a highly statistically significant effect . An overall range of concentrations between 40 x 10E-2% to 1.25 x 10E-2% (by volume) was used. The highly positive effects observed in the test without S9 metabolic activation indicated that TETA was an active mutagenic agent in the production of SCE in CHO cells.

UDS Test - TETA produced statistically significant increases in the amount of UDS activity in evaluations of concentrations between 100 x 10E-2% to 0.1 x 10E-2% (by volume). TETA was considered to be active in the present test with the hepatocyte test system and positive effects were observed in tests using both nuclei and DNA to detect increases in UDS.

Comparative Mutagenicity - The pattern of positive responses produced by TETA in the 3 -test battery to determine potential mutagenicity indicated that TETA was active in comparison to known mutagenic agents, such-as the positive controls tested concurrently with the test chemical. The consistent observation of a positive effect in each of the three tests employed indicates that TETA (or one of its many components) should be considered to be an active mutagenic agent.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 29, 1992 to December 30, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
yes
Remarks:
Analytical information on the test substance was left out. Draft version (1983) of OECD guideline cited.
GLP compliance:
yes
Type of assay:
other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
hepatocytes: obtained from male F344 rats
Metabolic activation:
without
Test concentrations with justification for top dose:
0.1, 0.5, 1, 5, 10, 25, 50, 100, 150 and 200 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Remarks:
see below
Untreated negative controls:
yes
Remarks:
untreated and deionized water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-acetamidofluorene- 1.0 E-7 M
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 18-20 hours (10 uC/ml of 3H-thymidine, specific activity of 50-80 Ci/mM, was added during exposure)


NUMBER OF REPLICATIONS: 3 coverslips per group


NUMBER OF CELLS EVALUATED: A total of 150 cells/group were counted


DETERMINATION OF CYTOTOXICITY
- Method: Slides were screened for toxicity by visual inspection under a microscope (reduction in number of hepatocytes and nuclei without cytoplasma).



OTHER: After exposure, the cultures were washed three times with phosphate buffered saline by aspiration. The cells on coverslips were swelled in 1 % sodium citrate for 10-15 minutes and fixed in three 10 minute changes of 100 % ethanol:glacial acetic acid (3:1). The fixed cultures were then washed twice with deionized water. The coverslips were air dried and mounted cell surface up on glass slides with Permaslip. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored in light proof slide boxes containing desiccant for one week. Seven days later, autoradiographs were developed.
Evaluation criteria:
A positive response was defined as a mean net nuclear grain count of >/= 5.
Species / strain:
hepatocytes: rat
Metabolic activation:
not applicable
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:

COMPARISON WITH HISTORICAL CONTROL DATA: Positive and negative controls fell within historical data ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Prior to scoring, coverslips from each dose were prescreened for toxicity by visual inspection under a microscope. The analysis of the coverslips indicated that 200 ug/ml induced some toxic effects to the hepatocyte culture by reducing their number and by the presence of nuclei without cytoplasma. However, there were sufficient numbers of scorable hepatocytes for a relative analysis. Therefore, 200 ug/ml was selected as the highest dose to be scored. Three lower doses of 25, 100 and 150 ug/ml were also selected for evaluation.

Analysis of the data for the test substance did not produce mean net nuclear grain counts >= 5 at any of the doses scored. In addition, the percentage of hepatocytes in repair ranged from 4 -8 %. The negative and positive control values were 13.5 +/- 11.1 and 22.8 +/- 12.8 with 4 and 92.7% hepatocytes in repair, respectively. These values were within the criteria for a valid test.

Conclusions:
Interpretation of results: negative

Under the conditions of this assay, the test substance did not induce unscheduled DNA synthesis (repair) in rat primary hepatocytes at concentrations up to 200 µg/ml.
Executive summary:

An UDS test was carried out with TETA in rat hepatocytes. Analysis of the data for the test substance did not produce mean net nuclear grain counts >= 5 at any of the doses scored. In addition, the percentage of hepatocytes in repair ranged from 4 -8%. The negative and positive control values were 13.5 +/- 11.1 and 22.8 +/- 12.8 with 4 and 92.7% hepatocytes in repair, respectively. These values were within the criteria for a valid test. Under the conditions of this assay, the test substance did not induce unscheduled DNA synthesis (repair) in rat primary hepatocytes at concentrations up to 200 µg/ml.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 28, 1980 - September 11, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
no
Remarks:
pre-GLP, inhouse QA in place
Type of assay:
other: assay for induction of Unscheduled DNA Synthesis (UDS) in rat liver cells.
Specific details on test material used for the study:
A commercial sample of TETA ws hydrogenated with Raney Nickel catalyst to determine if treatment would alter the effects of this chemical observed in previous studies.
Target gene:
incorporation of tritiated thymidine into DNA
Species / strain / cell type:
hepatocytes: rat
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
100, 30, 10, 3, 1, 0.1 x 10E-2%
Test concentrations were selected based on preliminary cytotoxicity studies. Selection of suitable range of concentrations for testing was based upon an estimate of the doses which would not kill over 50% of the treated cells.
Vehicle / solvent:
water, dsmo
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water, dmso
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: DMN, 5000, 3000, 1000 µg/ml, 4-NQO 3.0, 1.0, 0.3 µg/ml
Details on test system and experimental conditions:
Induction of primary DNA damage in rat liver cells (hepatocytes), was studied at a minimum of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with TETA for 2 hours in culture medium containing 3H-thymidine, hydroxyurea and appropriate dilutions of TETA prepared i n DMSO. Determination of UDS activity was performed by analyses of incorporation of 3H-thymidine into isolated hepatocyte nucleior in DNA (precipitated from aliquots of the isolated nuclei) using a Searle Analytic Model 81 or Packard Model 2650 scintillation spectrometer. Data
are presented in tabular form with an indication of the level of statistical significance above the concurrent solvent control values.
Statistics:
Data from the UDS test were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center.
Species / strain:
hepatocytes: rat
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
With hepatocytes treated with TETA-RNT, two concentrations tested for potential activity induced a statistically significant increase in the amount of 3H-thymidine incorporation in comparison to the concurrent control. Although these two values were obtained at non-consecutive concentrations, the level of the increases was greater than produced by some of the concentrations of the positive control agents. Four of the six concentrations, from 0.1 x 10-2% to 10 x 10-2% produced UDS values greater than the upper limit of the 95% confidence interval of the historical negative controls. The results in this test indicated that TETA-RNT produced a low level of primary DNA damage in rat hepatocytes and should be considered weakly active in this in vitro test system.
Both of the positive control agents, NQO and DMN, induced numerically elevated and dose-related increases in UDS over values obtained with the solvent control. With both positive controls, the highest concentration produced UDS levels which were highly statistically different from the concurrent control.

For hepatocytes treated with TETA-RNT, two of the test concentrations induced levels of UDS which were statistically different from the concurrent solvent control. In agreement with the data obtained with nuclei, four of the six concentrations 0.1 x 10-2% to 10 x 10-2%) produced levels of UDS which were greater than the variability expected from the historical negative controls for this test system. The same four concentrations produced UDS levels higher than those obtained with some of the doses of the positive control agents. These comparisons indicated that TETA-RNT was effective in producing a low level of DNA damage but, at most, it could be considered only weakly active as a potential mutagenic agent. The pattern of UDS activity produced by varying concentrations of the positive control agents NQO and DMN were similar and consistent with the data obtained in the assessment of 3H-thymidine incorporated into nuclei. All doses of the positive control agents produced a numerical elevation in the amount of UDS and the effects were dose elated and higher than values expected from the historical negative control data for this test system.


Conclusions:
Interpretation of results: positive

TETA-RNT was evaluated for potential mutagenic activity using the Sister Chromatid Exchange (SCE) test and an assay to detect the production of
Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that TETA-RNT produced a statistically significant effect upon SCE frequency in CHO cells and weakly positive effects in the UDS test. Thus, TETA-RNT was considered to be an active, but weak, mutagenic agent in the in vitro screening tests employed.
Executive summary:

An appropriate range of test concentrations was chosen from data of previous studies of TETA in our laboratory (BRRC Report Numbers 43-127, 44-11 and 44-12). For the SCE test, a range of concentrations was chosen which would not produce excessive cytotoxicity and inhibition of cell division. For the UDS test, a wide range of concentrations spanning cytotoxic to non-cytotoxic doses were tested.

SCE: TETA-RNT produced a statistically significant and dose-related effect upon the frequency of SCE in CHO cells in tests both with and without the incorporation of an S9 metabolic activation system. An overall range of concentrations betwen 2.5 x 10E-2% to 50 x 10E-2% (by volume) was tested and

the effects on the SCE frequency were determined with the highest five concentrations which allowed adequate cell division. The results indicated that TETA-RNT was an active agent in this test and should be considered a probable positive mutagenic agent for production of DNA damage in animal cells in culture.

UDS Test: TETA-RNT produced slight increases in the amount of UDS activity in evaluations of concentrations between 100 x 10E-2% to 0.1 x 10E-2% ( by volume) . TETA-RNT was considered to be weakly active in the present test with the hepatocyte test system because a majority of the UDS levels were significantly greater than historical negative control values for this test system.

Comparative Mutagenicity: The pattern of responses produced by TETA-RNT in the screening tests to determine potential mutagenicity indicated that TETA-RNT was weakly active in comparison to known mutagenic agents, such as the positive controls tested concurrently with the test chemical. The observation of positive effects in the SCE test and weak effects in the UDS test indicated that TETA-RNT was a weakly active mutagen in the in vitro- assays employed.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Principles of method if other than guideline:
- 50 cells per culture should be evaluated 30 were evaluated.
GLP compliance:
no
Remarks:
inhouse QA in place
Type of assay:
other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
incorporation of tritiated thymidine into DNA
Species / strain / cell type:
hepatocytes: rat
Metabolic activation:
without
Test concentrations with justification for top dose:
10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6, 10 -7 or 10 -8 M
Vehicle / solvent:
Williams Medium or DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 18 hours
- Expression time (cells in growth medium): 18 hours
- Selection time (if incubation with a selection agent): none
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: 15 on each of 2 slides

DETERMINATION OF CYTOTOXICITY
- Method: appearance of the cells

Statistics:
The net number of nuclear grains in treated cells are compared to the appropriate control by analysis of variance and Dunnett's t-test (Steel and Torrie, 1960). A mean of 6 or more net grains per nucleus (this may vary with experimental conditions) and statistical significance from control at p ≤ 0.05 is required for a positive result.
Species / strain:
hepatocytes: rat
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 10-3 M
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Neither TETA nor distilled-TETA elicited significant UDS at any concentration tested compared to media control cultures. On the contrary, 2-AAF elicited a significant dose-related increase in UDS compared to 0.1% DMSO control cultures. The positive response of 2-AAF, a known genotoxic chemical, demonstrates the responsiveness of the assay in the present study.
Conclusions:
Interpretation of results: negative

The inability of TETA or distilled-TETA to elicit DNA repair over the wide spectrum of concentrations tested indicates a lack of genotoxicity under the conditions of the present assay.
Executive summary:

The genotoxic potential of product grade triethylenetetramine (TETA) and distilled-TETA was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay. Neither TETA nor distilled-TETA elicited significant UDS at concentrations of 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6, 10 -7 or 10 -8 M. Alterations in the hepatocyte cultures indicative of toxicity were observed with both TETA and distilled-TETA at a concentration of 10 -1M. The appearance of the cultures improved with decreasing concentrations of TETA and distilled-TETA so that cultures exposed to 10 -3 to 10 -8 appeared comparable to control cultures. The inability of TETA or distilled-TETA to elicit DNA repair over the wide spectrum of concentrations tested indicates a lack of genotoxicity under the conditions of the present assay.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 17, 1980 - July 10, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
no
Remarks:
pre-GLP, inhouse QA in place
Type of assay:
other: assay for induction of Unscheduled DNA Synthesis (UDS) in rat liver cells.
Target gene:
incorporation of tritiated thymidine into DNA
Species / strain / cell type:
hepatocytes: rat
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
100, 30, 10, 3, 1, 0.1 x 10E-2%
The maximum dose level was selected with consideration of the cytotoxicity data obtained in the CHO mutation test which indicated that higher values would result in excessive cell killing.
Vehicle / solvent:
water, dsmo
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water, dmso
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: DMN, 1000, 300, 100 µg/ml, 4-NQO 3.0, 0.9, 0.3 µg/ml
Details on test system and experimental conditions:
Induction of primary DNA damage in rat liver cells (hepatocytes), was studied at a minimum of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with TETA for 2 hours in culture medium containing 3H-thymidine, hydroxyurea and appropriate dilutions of TETA prepared i n DMSO. Determination of UDS activity was performed by analyses of incorporation of 3H-thymidine into isolated hepatocyte nucleior in DNA (precipitated from aliquots of the isolated nuclei) using a Searle Analytic Model 81 or Packard Model 2650 scintillation spectrometer. Data are presented in tabular form with an indication of the level of statistical significance above the concurrent solvent control values.
Statistics:
Data from the UDS test were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center.
Species / strain:
hepatocytes: rat
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In hepatocytes treated with TETA, three concentrations tested for potential activity induced a statistically significant increase in the amount of 3H-thymidine incorporation and four of six concentrations induced a numerical elevation in the amount of UDS. The level of the increases were substantially outside the confidence interval of the historical data with the solvent controls for this assay but the effects were not clearly dose-related. The results in this test were interpreted as a probable positive effect on primary DNA damage in rat hepatocytes.
Both of the positive control agents, NQO and DMN, induced numerically elevated and statistically significant increases in UDS over values obtained with the solvent control. With both positive controls, all dose levels produced a numerical elevation in UDS, but the highest two dose levels of DMN appeared to be cytotoxic and the responses were not significant with respect to the concurrent control.

For hepatocytes treated with TETA, three of the test concentrations induced levels of UDS which were statistically significant from the solvent control. However, we did not observe a definite dose-related effect upon 3H-thymidine incorporation following treatments with TETA. Positive results on 3H-thymidine incorporation into DNA were consistent with the data obtained in the assessment of UDS measured using nuclei from cells treated with the same range of concentrations. TETA was considered to be active in the induction of DNA- damage discernible by UDS activity in the present study.
The pattern of UDS activity produced by varying concentrations of the positive control agents NQO and DMN were similar and consistent with the data obtained in the assessment of 3H-thymidine incorporated into nuclei. All doses of the positive control agents produced a numerical elevation in the amount of UDS but the variability was lower (than with nuclear measurements) and with both NQO and DMN all dose-levels produced statistically significant increases from the solvent control.

For further detail see attached tables.
Conclusions:
Interpretation of results: positive

The pattern of positive responses produced by TETA in the 3 -test battery to determine potential mutagenicity indicated that TETA was active in comparison to known mutagenic agents, such-as the positive controls tested concurrently with the test chemical. The consistent observation of a positive effect in each of the three tests employed indicates that TETA (or one of its many components) should be considered to be an active mutagenic agent
Executive summary:

TETA was evaluated for potential mutagenic activity with a battery of three in vitro tests, which were: the Chinese Hamster Ovary (CHO) Mutation test , the -- Sister Chromatid Exchange (SCE) test and an assay for induction of Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that TETA produced a statistically significant mutagenic effect in all of the three in vitro tests performed. Increases in the mutation index were observed both with and without metabolic activation. These positive results are consistent with literature reports on positive effects of TETA in the Ames (Salmonella) mutation assay.

RESULTS AND INTERPRETATION

Selection of Test Concentrations - Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of a proximately 10% of the treated cells. A maximum concentration of 80x 10E-2% (by volume) was chosen for the highest dose-level and a total of seven concentrations of TETA were tested for mutation induction because a steep dose response was suggested from prescreening data.

CHO Mutation Test - TETA produced a statistically significant increase in the frequency of mutations of CHO cells at several concentrations between 80 x 10E-2% to 2.5 x 10E-2% (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a definite dose-related effect of treatment suggested that the alkaline effect of the test agent may have interfered with the tests. With S9 metabolic activation, the acidic S9 liver homogenate may have somewhat buffered the alkaline effect and a dose related trend in the mutation index was observed for treatments between 10 x 10E-2% and 40 x 10E-2%.

SCE Test - TETA produced a highly statistically significant and dose-related increase in the frequency of SCE in CHO cells in tests without the incorporation of an S9 metabolic activation system. With metabolic activation, the effect on the frequency of SCE was decreased with respect to the results without S9, but the highest dose level produced a highly statistically significant effect . An overall range of concentrations between 40 x 10E-2% to 1.25 x 10E-2% (by volume) was used. The highly positive effects observed in the test without S9 metabolic activation indicated that TETA was an active mutagenic agent in the production of SCE in CHO cells.

UDS Test - TETA produced statistically significant increases in the amount of UDS activity in evaluations of concentrations between 100 x to 10E-2% 0.1 x 10E-2% (by volume). TETA was considered to be active in the present test with the hepatocyte test system and positive effects were observed in tests using both nuclei and DNA to detect increases in UDS.

Comparative Mutagenicity - The pattern of positive responses produced by TETA in the 3 -test battery to determine potential mutagenicity indicated that TETA was active in comparison to known mutagenic agents, such-as the positive controls tested concurrently with the test chemical. The consistent observation of a positive effect in each of the three tests employed indicates that TETA (or one of its many components) should be considered to be an active mutagenic agent.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 were not included
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate of Aroclor-induced rats
Test concentrations with justification for top dose:
0, 8, 370, 40, 200, 1000 and 5000 µg/plate without S9 mix
0, 8, 40, 200, 1000, 50000 µg/plate with S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: hycanthone methanesulphonate (HMS) and 2/aminoanthrace (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

DURATION
- Exposure duration: 3 days


NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background lawn of bacterial growth
Evaluation criteria:
Test substance is considered positive if revertants surpassing more than twice the control value and show a dose-dependency.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
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:
not examined
Positive controls validity:
valid
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
Remarks:
in the highest dose without S9 mix reduced background lawn
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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:
not valid
Positive controls validity:
valid
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:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
in the highest dose without S9 reduced background lawn
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
TETA was evaluated for mutagenic activity in the Ames test, using a set of five histidine requiring mutants of S. typhimurium (TA1535, TA1537, TA1538, TA98, TA100) and liver homogenate of Aroclor-induced rats. Incorporation of TETA with the bacteria induced a dose-related, reproducible increase in the numbers of his+ revertants with strains TA1535 with S9 mix and TA100 with and without S9 mix. From the present results it was concluded that TETA showed mutagenic activity in bacterial test system.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 6, 1987 - January 9, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was performed similar to methods of OECD471 but strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was not included. Negative results were not confirmed by an independent repeat.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- The study was performed according to methods similar to OECD471 but strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is not included.
- Negative results were not confirmed by an independent repeat.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: r-factor (ampilicin resistance) (TA100 and TA98), uvrB-, rfa-
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: uvrB-, rfa-
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
without S9 0.03, 0.1, 0.3, 1 and 2 mg/plate
with S9 0.1, 0.3, 1, 3 and 5 mg/plate
Selection of concentration is based on preliminary cytotoxicity tests.
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplcate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative, If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.
Statistics:
none
Species / strain:
S. typhimurium, other: TA100, TA98, TA1537, TA1538
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
increases in number of revertant colonies: TA98 6.6-fold, TA100 4.3-fold, TA1538 2.6-fold, TA1537 3-fold
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2mg/plate in all strains except TA100
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
maximum 5-fold increase
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1537, TA1538
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Range finding study:
In preliminary tests to determine cytotoxicity, ten concentrations of TETA-Sample B ranging from 0.01 to 98 mg/plate were tested with and without the presence of an S9 metabolic activation system. Cytotoxicity was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn. Dose levels ranging from 3.0 to 98 mg/plate produced complete absence of growth of the background lawn in the test without S9. A lower dose of 1.0 mg/plate produced no evidence of cytotoxicity, allowing confluent growth of the background lawn. In addition the 1.0 mg/plate dose level produced a 4.3-fold increase in relative numbers of revertant colonies indicating that a biologically effective dose level had been attained. In the preliminary test performed with activation, dose levels ranging from 10 to 98 mg/plate produced absence of growth of the background lawn and a dose of 5 mg/plate produced cytotoxicity evident by sparse growth of the background lawn. In addition, this dose produced a 5.3-fold increase in revertant colonies while a lower dose of 1 and 3 mg/plate produced a 7.1-fold and 6.2-fold increase above control levels, repsectively.
Based on the results of these preliminary toxicity tests, 5 doses ranging from 0.03 to 2.0 mg/plate were tested without S9 and 5 doses ranging from 0.1 to 5 mg/plate were tested in the presence of S9 in definitive mutagenicity experiments using triplicate cultures at each dose level.
Conclusions:
Interpretation of results: positive

TETA-Sample A was considered to be mutagenic in this vitro bacterial assay.
Executive summary:

Triethylenetetramine - Sample A (TETA-Sample A) was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test doses for the Ames test were chosen from data obtained in a preliminary study with strain TA100. Tests without a rat liver S9 activation system indicated that a concentration of 3.0 mg/glate was cytotoxic and produced absence of growth of the bacterial lawn. A slightly lower dose of 1.0 mg/plate allowed confluent growth of the background lawn. In the test with S9, a dose of 5 mg/plate produced cytotoxicity evident by sparse growth of the bacterial lawn. Higher doses produced complete absence of the background lawn. Based on these results, five doses ranging from 0.03 to 2.0 mg/plate were tested in the definitive test without S9 and a slightly higher range of 0.1 to 5 mg/plate were tested in the presence of the S9 metabolic activation system, These concentrations were rested with five different strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) using triplicate cultures at each dose level for each strain.

In the test without S9, dose-related mutagenic activity was observed with all five strains except TA1535. In tests performed in the presence of a

rat-liver S9 metabolic activation system, strains TA98, TA100 and TA1535 had highly positive and dose related increases in numbers of revertant colonies. Thus, TETA-Sample A was considered to be mutagenic in this in vitro bacterial assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 3, 1987 - March 6, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was performed similar to OECD471 but strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is not included.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- The study was performed according to methods similar to OECD471 but strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is not included.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: r-factor (ampilicin resistance) (TA100 and TA98), uvrB-, rfa-
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: uvrB-, rfa-
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.01, 0.03, 0.1, 0.3, 3 mg/plate
A preliminary toxicity test was performed using strain TA100 to determine the level of toxicity of the test substance.
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplcate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative, If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.
Statistics:
none
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+S9 5 mg/plate, -S9 1/3 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+S9 5 mg/plate, -S9 1/3 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Range finding study:
In preliminary tests to determine cytotoxicity, ten concentrations of TETA-Sample B ranging from 0.01 to 98 mg/plate were tested with and without the presence of an S9 metabolic activation system. Cytotoxicity was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn. Dose levels ranging from 5.0 to 98 -/plate produced complete absence of growth of the background lawn in the test without S9. A lower dose of 1.0 mg/plate produced no evidence of cytotoxicity, allowing confluent growth of the background lawn, and an intermediate dose of 3.0 mg/plate allowed only sparse growth of the bacterial lawn, In addition the 3.0 mg/plate dose level produced a 3.5-fold increase in relative numbers of revertant colonies indicating that a biologically effective dose level had been attained. In the preliminary test performed with activation, dose levels ranging from 10 to 98 mg/plate produced absence of growth of the background lawn and a dose of 5 &plate produced cytotoxicity evident by sparse growth of the background lawn. In addition, this dose produced a 3-fold increase in revertant colonies while a lower dose of 3 mg/plate produced a 4.2-fold increase above control levels. Based on the results of these preliminary toxicity tests, 5 doses ranging from 0.03 to 3 mg/plate were tested without S9 and 5 doses ranging from 0.1 to 5 mg/plate were tested in the presence of S9 in definitive mutagenicity experiments using triplicate cultures at each dose level.
Conclusions:
Interpretation of results: positive

TETA-Sample B was considered to be mutagenic in this vitro bacterial assay.
Executive summary:

Triethylenetetramine - Sample B (TETA-Sample B) was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test doses for the Ames test were chosen from data obtained in a preliminary study with strain TA100. Tests without a rat liver S9 activation system indicated that a concentration of 5.0 mg/plate was cytotoxic and produced absence of growth of the bacterial lawn. A slightly lower dose of 3.0 mg/plate allowed sparse growth of the background lawn. In the test with S9, a dose of 5 mg/plate produced cytotoxicity evident by sparse growth of the bacterial lawn. Higher doses produced complete absence of the background lawn. Based on these results, five doses ranging from 0.03 to 3.0 mg/plate were tested in the definitive test without S9 and a slightly higher range of 0.1 to 5 mg/plate were tested in the presence of the S9 metabolic activation system, These concentrations were tested with five different strains of Salmonella tv~himurium (TA98, TA100, TA1535, TA1537, and TA1538) using triplicate cultures at each dose level for each strain. In the test without S9, positive and dose-related mutagenic activity was observed with all five strains tested. Although TA1535 showed an increase which was slightly less than the two fold increase required for a positive response by the criteria of this test system, the maximum increase was 1.7 times the concurrent control value and the response was dose-related which is indicative of biological significance. In tests performed in the presence of a rat-liver S9 metabolic activation system all strains except TA1538 showed positive and dose related increases in numbers of revertant colonies. Thus, TETA-Sample B was considered to be mutagenic in this vitro bacterial assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In three in vivo Micronucleus Tests no genotoxic potential of Amines, polyethylenepoly-, triethylenetetramine fraction were seen. In a Drosophila SLRL test Amines, polyethylenepoly-, triethylenetetramine fraction showed ambiguous results after feeding exposure and negative results after test substance injection.

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:
March 10, 1987 - April 17, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No information of substance purity or composition. In appendix I of the report analytical information has been added but that is an MS printout, purity 68.5%? The study followed GLP and was performed according to methods similar to OECD 474. 1000 instead of 2000 immature erythocytes were examined per animal. No bone marrow toxicity was observed; however, at higher dose levels as administered in this test mortality occurred.
Qualifier:
according to guideline
Guideline:
other: Environmental Protection Agency - Health Effect Test Guidelines, EPA Report 560/6-83-001
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
- 1000 instead of 2000 immature erythocytes were examined per animal.
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles Rivers Laboratories, Portage, MI
- Age at study initiation: 5 weeks
- Weight at study initiation: male 23.6 g to 26.6 g, female 20.5 g to 23.5 g.
- Assigned to test groups randomly: yes, under following basis: randomized by weight and animals outside a range of two standard deviations from the mean were not used.
- Fasting period before study: not applicable
- Housing: Five mice/sex/cage were housed in shoe-box type plastic cages, measuring 30 x 20 x 12.5 cm.
- Diet (e.g. ad libitum): ad libitum with a basic diet of Agway PROLAB@ Animal Diet
- Water (e.g. ad libitum): Municipal Authority of Westmoreland County (Greensburg, PA) and was available ad libitum.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): - Humidity (%): In the opinion of the study director, there were no unacceptable variations in temperature or humidity during the testing periods which would adversely affect the quality or integrity of the study.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: March 10, 1987 - April 17 1987
Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
None
Duration of treatment / exposure:
na
Frequency of treatment:
single i.p. injection.
Post exposure period:
72 hours
Dose / conc.:
185 mg/kg bw/day (nominal)
Dose / conc.:
370 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): standard positive control
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw
Tissues and cell types examined:
Blood from the tail fo the mice
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Three dose levels of approximately 80%, 50% and 25% of the pooled LD50 value were evaluated for effects upon the incidence of micronuclei. A single i.p. injection was given. Blood samples were taken at 3 time periods at approximately 30, 48 and 72 hr after dosing.

DETAILS OF SLIDE PREPARATION: One or two blood smear slides were prepared for each animal sampling time. Micronuclei in peripheral blood, polychromatic erythrocytes were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded by animal number only and read blindly to prevent bias.

METHOD OF ANALYSIS:
A minimum of 1000 polychromatic erythocytes was examined microscopically for each animal per sample time, unless cytotoxicity of the test material prevented this goal. The polychromatic:normochromatic erythrocyte ratio for approximately 1000 total cells was calculated and recorded and these data are summarized in the final report as an estimate, of cytotoxicity of the test agent.
Micronuclei were identified as darkly-stained, spherical, inclusions in polychromatic erythrocytes. Polychromatic, erythrocytes were identified by the pale-bluish staining of the cytoplasm in contrast to the lack of blue stain for normochromatic erythrocytes.
Statistics:
Data were compared for significant differences from the vehicle control frequencies using the Fisher's Exact Test (Sokal and Rohlf, 1981). Data for
males and female mice at each sample perfod were combined for statistical analyses because Analysis of Variance tests showed that there was.no
significant difference in micronuclei frequencies between sexes at each sample period. A positive result in the micronucleus test was concluded if at least one statistically significant (p ≤0.01) increase above the vehicle control was .observed with an indication of a dose-related effect of treatment. A test was considered to be inconclusive if only one dose produced effects statistically different from the control (0.05 ≥ p ≥ 0.01) and a dose-effect relationship was apparent. A test result was considered to be negative if no statistically significant differences were apparent between the vehicle control and groups of animals treated with TETA.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
No bone marrow toxicity was observed however at higher dose levels as administered in this test mortality occurs.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Analysis of variance testing indicated that there was no significant difference between the mortality response of male and female mice treated with TETA, Thus, a combined LD50 value of approximately 740 mg/kg was calculated and used to determine the test doses for the definitive micronucleus test. The lower and upper 95% fiducial limits for the LD50 value were 651 mg/kg and 877 mg/kg, respectively.
The PCE/NCE ratio of the vehicle control and the highest test dose with adequate numbers of survivors (≥ 3) was quantified and compared to determine possible bone marrow cytotoxicity. At 48 hrs after dosing; the PCE/NCE ratios of both the male and female mice were similar to the vehicle control values, thus indicating the absence of bone marrow toxicity. Since no bone marrow toxicity was evident at this sample period, no additional blood smears were obtained at later time intervals

RESULTS OF DEFINITIVE STUDY
Micronucleus determinations were conducted using a minimum of 5 animals/sex/group. Additional animals were added to some groups because deaths were expected at the higher dosages. However, extra animals were assessed for micronucleus frequencies only as needed to assure that a total of five animals are evaluated. Three dose levels of 600 mg/kg, 370 mg/kg and 185 mg/kg were selected for testing in the definitive micronucleus test at approximately 80%, 50% and 25% of the combined male-female LD50 value, respectively.

No remarkable decreases in the PCE/NCE ratios relative, to the control values were observed in this study at any of the three sampling periods. PCE/NCE ratios of the male animals treated with TEM were lower than the concurrent negative control values which is an expected and typical finding because of the cytotoxicity and clastogenicity of this agent.

Analysis of variance (AOV) testing indicated that the data for male and female mice sampled at 30 hr, 48 hr or 72 hr after dosing were not statistically different; thus, values were pooled for Fisher's Exact analyses. No statistically significant or treatment-related increases in the numbers of micronuclei were observed with any of the treatment groups sampled at any of the sample intervals following injection of the test chemical.

TEM, used as a positive control agent for this study, produced highly significant increases in numbers of. micronuclei demonstrating the appropriate sensitivity of the test system. Numbers of micronuclei in the vehicle control animals were in a low and acceptable range for this test system at all sampling times.
Conclusions:
Interpretation of results: negative
Test results for this study showed that TETA was not an active agent in producing treatment-related increases in micronuclei in male and female Swiss-Webster mice. Relatively high dosage levels of TETA were evaluated no treatment-related clastogenic activity was observed. TETA was considered to be inactive as a clastogenic agent in vivo under the conditions of the micronucleus test.
Executive summary:

Triethylenetetramine (TETA) was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female Swiss-Webster mice. Test doses for the micronucleus test were chosen from data obtained in a preliminary toxicity study with mice. Five doses of TETA ranging from 434 mg/kg bw to 900 mg/kg bw were administered as a single intraperitoneal (i .p.) injection. The LD50 dose was calculated from the cumulative mortality observed during a three day period after dosing. To select dose levels for the definitive micronucleus test, a combined LD50 value of approximately 740 mg/kg bw (651 to 877; 95% fiducial limits) was calculated by pooling the total number of deaths for males and females.For the definitive micronucleus test, doses of 185 mg/kg bw, 370 mg/kg bw and 600 mg/kg bw were tested with both male and female Swiss-Webster mice. Concurrent positive (triethylenemelamine) and negative (water) control agents, administered by i.p, injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that TETA did not produce positive or dose-related increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals at any of the sample periods tested. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test. The absence of positive effects of TETA upon the incidence of micronuclei indicates that TETA does not possess clastogenic activity in vivo under the conditions of the micronucleus test system.

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

Additional information

In vitro:

Gene mutation in bacteria:

Amines, polyethylenepoly-, triethylenetetramine fraction was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test) similar to OECD guideline 471 (Stankowski, 1992). S.typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 showed all dose-dependent increase in revertant frequencies compared to control with and without metabolic activation. Based on those results the test substance is considered to induce gene mutation in bacteria.

No increase (compared to the negative control cultures) in revertant frequencies was seen in strain TA1538. A dose-dependent increase in revertant frequencies to approx. 1.9- to 13- fold of the control values, were observed in strain TA1538, TA1537, TA98 and TA100 without metabolic activation. Thus, Amines, polyethylenepoly-, triethylenetetramine fraction was considered to be mutagenic in this in vitro bacterial assay.

This result was supported by seven further studies which all show similar results.

 

Gene mutation in mammalian cells:

A reliable study similar to OECD guideline 476 with the source substance Amines, polyethylenepoly-, triethylenetetramine fraction is available (Slesinki, 1981). Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of a proximately 10% of the treated cells. A maximum concentration of 0.8% (by volume) was chosen for the highest dose-level and a total of seven concentrations of the test substance were tested for mutation induction because a steep dose response was suggested from prescreening data. The test substance produced a statistically significant increase in the frequency of mutations of CHO cells at several concentrations between 0.8% to 0.025% (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a definite dose-related effect of treatment suggested that the alkaline effect of the test agent may have interfered with the tests. With S9 metabolic activation, the acidic S9 liver homogenate may have somewhat buffered the alkaline effect and a dose related trend in the mutation index was observed for treatments between 0.1% and 0.4%.

DNA damage/repair:

Three reliable studies are available similar to OECD 479 (in vitro sister chromatid exchange assay in mammalian cells (Slesinki 1981 and 1987). Amines, polyethylenepoly-, triethylenetetramine fraction produced a highly statistically significant and dose-related increase in the frequency of SCE in CHO cells in tests without the incorporation of a metabolic activation system. With metabolic activation, the frequency of SCE was decreased, was found to be lower than without metabolic activation, but the highest dose level produced a highly statistically significant effect. An overall range of concentrations of 0.4% to 0.0125% (by volume) was used. The high frequency of SCE observed in the test without metabolic activation indicates that the test substance is mutagenic in CHO cells (Slesinski, 1981). TETA- Sample A (Slesinski, 1987), which is considered to be Amines, polyethylenepoly-, triethylenetetramine fraction, produced dose-related and statistically significant increases in SCEs in the test without addition of a rat-liver S9 metabolic activation system. With S9 activation, an inverse dose response relationship was observed and a significant response was obtained only at the lowest test concentration. The highest increases in SCEs above the combined solvent control values were approximately 1.4 fold without S9 and 1.3 fold with S9 activation. No remarkable degree of cell-cycle inhibition was produced by the test chemical by determination of the ratio of numbers of cells in the first and second cycle of division. The test chemical was considered to be a positive but weakly-active genotoxic agent in the SCE test system. TETA-RNT, which is also considered to belong to the substance identity of Amines, polyethylenepoly-, triethylenetetramine fraction, produced a statistically significant and dose-related effect upon the frequency of SCE in CHO cells in tests both with and without the incorporation of an S9 metabolic activation system. An overall range of concentrations between 0.025% to 0.5% (by volume) was tested and the effects on the SCE frequency were determined with the highest five concentrations which allowed adequate cell division. The results indicated that TETA-RNT was an active agent in this test and should be considered a probable positive mutagenic agent for production of DNA damage in animal cells in culture (Slesinski, 1981, Nickel).

In conclusion, Amines, polyethylenepoly-, triethylenetetramine fraction is considered to have a potential to induce DNA damage in mammalian cells.

Four reliable studies are available similar to OECD 482 (unscheduled DNA synthesis in mammalian cells, Slesinski 1981, Schumann, 1979, Pharmakon, 1992). Amines, polyethylenepoly-, triethylenetetramine fraction produced statistically significant increases in the amount of UDS activity in evaluations of concentrations between 1% and 0.001% (by volume). The test substance was considered to be active in the present test with the hepatocyte test system and positive effects were observed in tests using both nuclei and DNA to detect increases in UDS (Slesinksi, 1981). Triethylenetetramine Raney Nickel Treated (TETA-RNT) produced slight increases in the amount of UDS activity in evaluations of concentrations between 1% and 0.001% (by volume). TETA-RNT was considered to be weakly active in the present test with the hepatocyte test system because a majority of the UDS levels were significantly greater than historical negative control values for this test system (Slesinski, 1981). The genotoxic potential of product grade triethylenetetramine (TETA) and distilled-TETA was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay (Schumann, 1979). Neither TETA nor distilled-TETA elicited significant UDS at concentrations of 0.1, 0.01, 0.001, 0.00001, 10E-5, 10E-6, 10E-7 or 10E-8 M. Alterations in the hepatocyte cultures indicative of toxicity were observed with both TETA and distilled-TETA at a concentration of 0.1M. The appearance of the cultures improved with decreasing concentrations of TETA and distilled-TETA so that cultures exposed to 10E-3 to 10E-8 M appeared comparable to control cultures. The inability of TETA or distilled-TETA to elicit DNA repair over the wide spectrum of concentrations tested indicates a lack of genotoxicity under the conditions of the present assay. A further UDS test was carried out with Amines, polyethylenepoly-, triethylenetetramine fraction in rat hepatocytes (Pharmakon, 1990). Analysis of the data for the test substance did not produce mean net nuclear grain counts >= 5 at any of the doses scored. In addition, the percentage of hepatocytes in repair ranged from 4 -8%. The negative and positive control values were 13.5 +/- 11.1 and 22.8 +/- 12.8 with 4 and 92.7% hepatocytes in repair, respectively. These values were within the criteria for a valid test. Under the conditions of this assay, the test substance did not induce unscheduled DNA synthesis (repair) in rat primary hepatocytes at concentrations up to 200 µg/mL.

In conclusion, Amines, polyethylenepoly-, triethylenetetramine fraction is considered to have a potential to induce unscheduled DNA synthesis (repair) in mammalian cells.

 

In vivo:

A reliable study similar to OECD guideline 474 with Amines, polyethylenepoly-, triethylenetetramine fraction is available (Guzzie, 1987). The test substance was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female Swiss-Webster mice. Test doses for the micronucleus test were chosen from data obtained in a preliminary toxicity study with mice. Five doses of the test substance ranging from 434 mg/kg bw to 900 mg/kg bw were administered as a single intraperitoneal (i .p.) injection. The LD50 dose was calculated from the cumulative mortality observed during a three day period after dosing. To select dose levels for the definitive micronucleus test, a combined LD50 value of approximately 740 mg/kg bw (651 to 877; 95% fiducial limits) was calculated by pooling the total number of deaths for males and females.

For the definitive micronucleus test, doses of 185 mg/kg bw, 370 mg/kg bw and 600 mg/kg bw were tested with both male and female Swiss-Webster mice. Concurrent positive (triethylenemelamine) and negative (water) control agents, administered by i.p, injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that the test substance did not produce positive or dose-related increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals at any of the sample periods tested. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test. The absence of positive effects of the test substance upon the incidence of micronuclei indicates that the test substance does not possess clastogenic activity in vivo under the conditions of the micronucleus test system.

Similar results were observed in a supporting study similar to OECD guideline 474 (SanSebastian, 1992). Amines, polyethylenepoly-, triethylenetetramine fraction was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female CD-1 mice. In a preliminary test 50, 100, 250, 500 and 1000 mg/kg bw were tested via the intraperitoneal route. Pharmacotoxic signs were observed in the 100 mg/kg bw group. Mortality occurred in the 250 mg/kg bw group (1/4). All animals but one, died within 2 hours post-dose in the 500 and 1000 mg/kg bw dose groups. Therefore, 150 mg/kg bw was selected as test dose for the main study. Concurrent positive (triethylenemelamine) and negative (water) control agents, administered by i.p, injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that the test substance did not produce an increase in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals at any of the sample periods tested. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test. The absence of positive effects of the test substance upon the incidence of micronuclei indicates that the test substance does not possess clastogenic activity in vivo under the conditions of the micronucleus test system.

Further, an in vivo micronucleus test was carried out with Amines, polyethylenepoly-, triethylenetetramine fraction in mice using the intraperitoneal route (at levels of 130, 190 and 250 mg/kg bw) or the oral route (at levels of 1500, 3000 and 6000 mg/kg bw) which was described just briefly in published literature (Heinz, 1981). TETA was not mutagenic in the micronucleus test in vivo using both the oral and ip route.

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sexlinked recessive lethal (SLRL) assay among those also Amines, polyethylenepoly-, triethylenetetramine fraction (Foureman, 1994). As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. The test substance was ambiguous after feeding and negative after injection.

 

In conclusion, based on the above study results sufficient evidence is available to conclude that the Amines, polyethylenepoly-, triethylenetetramine fraction is not mutagenic in vivo.

Justification for classification or non-classification

Reliable data on genetic toxicity indicates that the registration substance does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and the available data are therefore conclusive but not sufficient for classification.