Reaction products of tetraethylenepentamine and maleic anhydride

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:

2014-12-31 to 2015-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study performed according to OECD guideline 476, in compliance with GLP. No deviations were noted.

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

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): XTA 823- Substance type: clear yellow liquid- Physical state: liquid- Analytical purity: 50% (provided by the sponsor) - Purity test date: no data- Lot/batch No.: BLW0010057- Expiration date of the lot/batch: no data- Stability under test conditions: not determined. The study conclusion was based on the test substance as supplied.- Storage condition of test material: 2 to 8°C, protected from light- other: -molecular weight: 350-390 g/mol

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: L5178Y/TK+/- mouse lymphoma cells, clone 3.7.2C, received from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes, each batch of frozen cells was tested using agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination- Periodically checked for karyotype stability: no data- Periodically "cleansed" against high spontaneous background: yes, prior to use in th assay, L518Y/TK+/- cells were cleansed to reduce the frequency of spontaneously occurring TK+/- cells.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 from Sprague-Dawley rat

Test concentrations with justification for top dose:

preliminary toxicity test: 15.2, 30.5, 60.9, 122, 244, 489, 975, 1950, 3900 µg/mlmouse lymphoma assay: - 4-hour treatment, with and without metabolic activation: 244, 488, 975, 1950, 2930, 3900 µg/ml- 24-hour treatment, without metabolic activation: 7.62, 15.2, 30.5, 60.9, 91.4, 122, 183, 224 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water- Supplier: Gibco- Lot number: 1420169- CAS number: 7732-18-5- Purity/grade: sterile, distilled- Expiration date: 2015-08-30- Justification for choice of solvent/vehicle: water was selected as the vehicle 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, distilled water at a concentration of ~50 mg/ml (the maximum evaluated).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

15 and 20 µg/ml for the 4-hour treatment and 5.0 and 7.5 µg/ml for the 24-hour treatment (diluted in same vehicle as test substance), without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

1.0 and 1.5 µg/ml (in DMSO as vehicle), with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumPRELIMINARY TOXICITY TEST FOR SELECTION OF DOSE LEVELS:- L5178Y/TK+/- cells were exposed to the vehicle in duplicate cultures and nine concentrations of test substance using single cultures. The test substance was evaluated at the maximum concentration of 10 mM. The pH of cultures at a concentration of 3900 µg/ml was adjusted within 6.5 to 7.5 using 1N sodium hydroxide (NaOH, CAS no. 1310-73-2); lot no. RNBD0654, expiration 2017-06-30, supplier: Sigma-Aldrich). No pH adjustment was made for the remaining concentrations. The osmolality of the vehicle control and the highest treatment condition also was measured at the beginning of treatment. Dose levels for the definitive assay were based upon post-treatment cytotoxicity (growth inhibition relative to the vehicle control). DURATION- Exposure duration: 4 hours in the presence and absence of S9, 24 hours in the absence of S9- Expression time (cells in growth medium): 10-14 days- Fixation time (start of exposure up to fixation or harvest of cells): at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 ml F10P on day 1 and in 10 ml F10P on day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10E05 cells/ml were made as follows: 4 hour treatment: 1 and 2 days after treatment; 24 hour treatment: immediately after test substance removal, and 2 and 3 days after treatment. pH: the pH of cultures at a concentration of 3900 µg/mL again were adjusted to within 6.5 to 7.5 using the same 1N NaOH as above. No pH adjustment was made for the remaining concentrations. SELECTION AGENT (mutation assays): 2-4 µg TFT/mL at a density of 1 x 1E06 cells/100mm plate in cloning medium containing 0.22 to 0.24% agarNUMBER OF REPLICATIONS: duplicate cultures for all dose levels and vehicle, single culture for positive controlNUMBER OF CELLS EVALUATED: 3 x 1E06DETERMINATION OF CYTOTOXICITY- Method: relative total growthOTHER EXAMINATIONS:- Induced mutant frequency (IMF)- The precipitation was determined with the unaided eye at the beginning and conclusion of treatment

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency which occured only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data:- A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibit induced mutant frequencies of ≥90 mutants per 10E06 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants per 10E06 clonable cells, a doubling of mutant frequency over the vehicle would also be required (Mitchell et al., 1997). - A result was considered negative if the treated cultures exhibit induced mutant frequencies of less than 90 mutants per 10E06 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency. There are some situations in which a chemical would be considered negative when there was no culture showing between 10 to 20% survival:- There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10E06) in a series of data points within 100 to 20% survival and there was at least one negative data point between 20% and 25% survival. - There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10E6) in a series of data points between 100 to 25% survival and there was also a negative data point between 10% and 1% survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.

Statistics:

no data

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: pH adjustment was required at a concentration of 3900 μg/ml to maintain neutral pH- Effects of osmolality: test substance did not have an adverse impact on the osmolality of the cultures [251 and 258 mmol/kg for the vehicle control and the highest treatment concentration (3900 µg/mL), respectively]. (preliminary toxicity test)- Evaporation from medium: no data- Water solubility: no data- Precipitation: no visible precipitate was observed at the beginning or end of treatment. PRELIMINARY TOXICITY ASSAY: - No visible precipitate was observed at the beginning or end of treatment, and the test substance did not have an adverse impact on the osmolality of the cultures [251 and 258 mmol/kg for the vehicle control and the highest treatment concentration (3900 µg/ml), respectively]. Relative suspension growth (RSG) was 80, 51 and 10% at concentrations of 3900 µg/ml (4-hour treatments with and without S9) and 60.9 µg/ml (24-hour treatment without S9), respectively. RSG was or approximated 0% at concentrations ≥122 µg/ml (24-hour treatment without S9). COMPARISON WITH HISTORICAL CONTROL DATA: all positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. ADDITIONAL INFORMATION ON CYTOTOXICITY:- Cultures treated at concentrations of 488, 975, 1950, 2930 and 3900 µg/mL (4-hour treatments with and without S9), and 30.5, 60.9, 91.4 and 122 µg/mL (24-hour treatment without S9) exhibited 78 to 115%, 22 to 92%, and 23 to 99% RSG, respectively.- Cultures treated at concentrations of 224 µg/ml (24-hour treatment without S9) were discarded prior to cloning due to excessive toxicity.- Cultures treated at concentrations 182 µg/ml (24-hour treatment without S9) were excluded from evaluation due to excessive toxicity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenesis assay:

- Cultures treated at concentrations of 488, 975, 1950, 2930 and 3900 µg/ml (4-hour treatments with and without S9), and 30.5, 60.9, 91.4 and 122 µg/ml (24-hour treatment without S9) exhibited 78 to 115%, 22 to 92%, and 23 to 99% RSG, respectively, and were cloned (cultures at a concentration of 488 µg/ml with S9 were lost due to technical error; cultures treated at other lower concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available; cultures treated at other higher concentrations were discarded prior to cloning, or excluded from evaluation of mutagenicity, due to excessive toxicity). Relative total growth of the remaining cloned cultures ranged from 73 to 110% (4-hour treatment with S9), 20 to 97% (4-hour treatment without S9) and 13 to 86% (24-hour treatment without S9). No increases in average induced mutant frequency ≥90 mutants per 10E06 clonable cells were observed under any treatment condition.

- The trifluorothymidine-resistant colonies for the positive and solvent control cultures in the presence and absence of S9 were sized according to diameter, over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Applicant's summary and conclusion

Conclusions:

The results indicate that the substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay under the conditions, and according to the criteria, of the test protocol.