Amines, polyethylenepoly-, triethylenetetramine fraction

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Amines, polyethylenepoly-, triethylenetetramine fraction

EC Number:

292-588-2

EC Name:

Amines, polyethylenepoly-, triethylenetetramine fraction

Cas Number:

90640-67-8

Molecular formula:

C6H18N4, C8H20N4

IUPAC Name:

(2-aminoethyl)({2-[(2-aminoethyl)amino]ethyl})amine; tris(2-aminoethyl)amine

Test material form:

liquid

Method

Target gene:

HGPRT

Species / strain

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

Controls

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.