Reaction mass of trientine and trientine, mono- and di-propoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 September to 15 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test guideline study conducted in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triethylenetetramine, propoxylated (triethylene-tetramine adduct)

IUPAC Name:

Triethylenetetramine, propoxylated (triethylene-tetramine adduct)

Test material form:

other: Clear light yellow multiconstituent liquid

Details on test material:

Common name: TTriethylenetetramine, propoxylated (triethylene-tetramine adduct)
CAS: 500-055-5

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system

Test concentrations with justification for top dose:

625, 1250, 2500, 3750 and 5000 μg/mL with and without S9.

Vehicle / solvent:

Sterile, distilled water (CAS No. 7732-18-5; Lot No. 1420169, Expiration Date: 30 August 2015), obtained from Gibco.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with activation

Controls 2

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without activation

Details on test system and experimental conditions:

The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80% (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN) and the stock cells were stored frozen in liquid nitrogen.

The in-vitro mammalian cell forward gene mutation (CHO/HPRT) assay was conducted by exposing CHO-K1-BH4 cells to concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.

CHO cells were maintained in Ham's F12 medium, supplemented with 3 mM L-glutamine and 5% (v/v) heat-inactivated and dialyzed fetal bovine serum (F12FCM5) under standard conditions (37±1°C in a humidified atmosphere of 5±1% CO2 in air). Treatment medium was Ham’s F12 medium supplemented with 3 mM L-glutamine (serum free). Hypoxanthine-free F12FCM5 (Hx-F12FCM5) was used for mutant selection and to determine cloning efficiency at the time of selection. Medium for selection of mutants also contained 10 μM TG. All media contained antimycotics and antibiotics.
To reduce the frequency of spontaneous HPRT- mutants prior to use in an assay, the cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT). Frozen stock cultures were tested to confirm the absence of mycoplasma contamination and for karyotpye stability. Cells used in the mutation assay did not exceed 15 passages from frozen stock.

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3283, Expiration Date: 28 May 2016) was purchased commercially from Moltox (Boone, NC). Upon arrival, the S9 was stored at -60°C or colder until used. The lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.

Target cells were treated for 5 (±0.5) hours in the presence and absence of S9, by addition of the test and control formulations to the treatment medium (with or without S9, as appropriate).

After the 5-hour treatment, the treatment media were removed, the cultures were washed twice with CMF-HBSS and then were trypsinized and counted. Cells were subcultured at ~1 x 106 cells/75-cm2 flask in 10 mL F12FCM5 in duplicate (or all available into 1 or 2 flasks) for phenotypic expression and incubated under standard conditions (larger numbers of cells may be subcultured for phenotypic expression where decreases in cloning efficiency are observed in the DRF; i.e., there should be ~1 x 106 viable cells for phenotypic expression). An additional aliquot of cells was plated at 200 cells/60-mm plate in 5 mL F12FCM5 in triplicate for initial survival. The 60-mm plates were incubated under standard conditions for 7 to 10 days and the resulting colonies were fixed in methanol, stained with crystal violet, and counted.
The cultures were subcultured for 7 to 8 days, at 2- to 3-day intervals, to maintain logarithmic growth and permit expression of the mutant phenotype. At each subsulture, the flasks were trypsinized, counted and subcultured at ~1 x 106 cells/75-cm2 flask in 10 mL F12FCM5 in duplicate (or all available into 1 or 2 plates). If visual observation indicated there were sufficient cells in a single flask, only one was subcultured (into duplicate flasks) and the back-up was discarded; if visual observed indicated there were relatively few cells present, the culture(s) were re-fed with fresh medium and re-incubated for an additional 2 to 3 days.

At the end of the phenotypic expression period, 1 x 106 cells from each culture were plated at a density of 2 x 105 cells/100-mm plate (5 plates total) in 10 mL Hx-F12FCM5 containing 10 μM TG. Three 60-mm plates also were plated, at 200 cells/plate in 5 mL Hx-F12FCM5 in triplicate, to determine the cloning efficiency at the time of selection. The plates were incubated under standard conditions for 7 to 10 days.
After the 7- to 10-day incubation period, the colonies were fixed with methanol, stained with crystal violet and counted. Mutant frequencies were expressed as the number of TGr mutants/106 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.

Evaluation criteria:

The positive controls must induce a significant increase in mutant frequency as compared to the concurrent vehicle controls (p≤ 0.01). A significant increase in the absence of S9 indicated the test system could identify a mutagen, while a significant increase in the presence of S9 was considered to have demonstrated the integrity of the S9 mix as well as the ability of the test system to detect a mutagen.

The highest concentration evaluated was the limit dose for this assay (5000 μg/mL or 10 mM), or must have induced 10 to 20% adjusted relative survival, or must have been the highest concentration able to be prepared in the vehicle and administered (whichever was lowest). If increasing cytotoxicity was observed at precipitating concentrations, cytotoxicity was the determining factor. This latter requirement was waived if the highest concentration with acceptable cytotoxicity (>10% adjusted relative survival) was at least 75% of an excessively toxic concentration (cultures with adjusted relative survivals <10% were excluded from evaluated as excessively cytotoxic). There was no maximum concentration or toxicity requirement for test substances which clearly showed mutagenic activity.

A minimum of four acceptable concentrations was required for a valid assay. Fewer concentrations may be justified for test substances which clearly show mutagenic activity.

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Once criteria for a valid assay were met, the responses observed in the assay were evaluated as follows.
The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.05) that exceeded the 95% confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility Test
Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in sterile water at a concentration of ~50 mg/mL, the maximum tested.

Dose Range-Finding Assay
Results of the preliminary toxicity assay are presented in Table 1. The test material was prepared in distilled water and evaluated in a dose range-finding assay at concentrations of 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, but the pH of the cultures was adjusted at all concentrations, using 1N HCl, to maintain neutral pH. The test substance did not have an adverse impact on the osmolality of the cultures [252 and 261 mmol/kg for the solvent control and the highest concentration evaluated, respectively]. Adjusted relative survival was 88.18 and 86.05% at a concentration of 5000 μg/mL with and without S9, respectively.

Definitive Mutagenicity Assay
Results of the mutagenicity assay are presented in Table 2. Based on the results of the dose range-finding assay, the test material was evaluated in the definitive mutagenicity assay at concentrations of 625, 1250, 2500, 3750 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, but the pH of the cultures was adjusted at all concentrations to maintain neutral pH. The average adjusted relative survival was 95.39 and 99.43% at a concentration of 5000 μg/mL with and without S9, respectively. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

The results of the test indicate that the test was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures under the conditions, and according to the criteria, of the test protocol.

Executive summary:

The test material, triethylenetetramine, propoxylated , was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). The test material was prepared in distilled water and evaluated in a preliminary dose range-finding assay at concentrations of 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9.

No visible precipitate was observed at the beginning or end of treatment, but the pH of the cultures was adjusted at all concentrations, using 1N HCl, to maintain neutral pH. The test substance did not have an adverse impact on the osmolality of the cultures. Adjusted relative survival was 88.18 and 86.05% at a concentration of 5000 μg/mL with and without S9, respectively. Based on these results, the test material was evaluated in the definitive mutagenicity assay at concentrations of 625, 1250, 2500, 3750 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, and the pH of the cultures was adjusted at all concentrations to maintain neutral pH. The average adjusted relative survival was 95.39 and 99.43% at a concentration of 5000 μg/mL with and without S9, respectively. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

These results indicate that the test material was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures under the conditions, and according to the criteria, of the test protocol.