Isooctadecanoic acid, reaction products with tetraethylenepentamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

EC 701-204-9 was tested in the L5178Y TK+/-mutagenicity screen with and without S-9 metabolic activation at concentrations ranging from 1 ug/ml to 333 ug/ml. None of the cultures exhibited a mutant frequency which was two times that of the average mutant frequency of the negative (solvent) controls. Under the conditions tested, this material was considered to be nonmutagenic in an OECD 476 study on mammalian cells.

EC 701-204-9 was also tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, TA1535, and TA1537 at dose levels of 0.1 to 10 mg/plate with and without metabolic activation provided by Aroclor-induced Rat liver S-9. Cytoxicity was observed in TA98, TA100, and TA1535 (1.0 and 3.3 mg/plate) and TA1537 (3.3 mg/plate) with metabolic activation. However, the observed cytotoxicity was inconsistent and nonreproducible, due at least in part to interference from test material precipitation at all dose levels tested. Under the conditions tested, the test material was not mutagenic to strains TA98, TA100, TA1535, or TA1537 with or without metabolic activation.

EC 701-204-9 was evaluated for induction of structural and numerical chromosome aberrations in non-activated and S9-activated test systems via the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes. Mitotic inhibition was 53 -57% at 2.5 ug/mL in all three groups (4h activated, 4h non-activated, 20 h non-activated). The percentage of cells with aberrations in the doses selected for analysis were not significantly increased above the solvent control at any dose level for any of the treatment groups

Link to relevant study records

Reference

Reference 1

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:

16 Sep - 19 Nov, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study following GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Strain: 3.7.2C
Cells were cultured in Fischer's medium in a shaker incubator at 37°C in humidified 5% CO2 in air. Cultures were diluted daily to a cell density of approximately 3 x 10ˆ5 cells per ml. Each time a culture was used, it was checked for bacterial or fungal contamination.
Prior to use in the assay, L5178Y cells were treated with methotrexate to reduce the frequency of spontaneously occurring TK-/- cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor S-9 metabolic activation

Test concentrations with justification for top dose:

0, 3, 10, 33, 100, 333 µg/mL with S-9
0, 1, 3, 7, 10, 33 µg/mL without S-9

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

- Test Material Exposure: Based on the data derived from the toxicity test, the test material was prepared so that the highest concentration would yield a percent total growth of approximately 10%. The test material was solubilized and diluted to produce evenly spaced dose levels which would yield approximately 90% total growth at the lowest dose. The test material was added to cells, both with and without metabolic activation and incubated for approximately 4 hours. Cells were then washed and placed into suspension culture at a density of 0.3 x 10ˆ6 cells/mL.

- Expression Time: In order for induced mutations to be expressed, the cells must undergo several divisions. This period is designated as the expression time. After the initial exposure to the test material the cells were incubated for 2 days and adjusted to 0.3 x 10ˆ6 cells/mL at 24 hours.

- Cloning: At the end of the expression period, a portion of the cells were plated in
(a) restrictive medium containing 3 ug/ml trifluorothymidine (TFT) which allowed only the TK-/- cells to grow, and
(b) non-restrictive medium which indicated cell viability.
The plates were incubated at 37°C + 1 C in humidified 5% CO2 in air for 10 days.

- Accumulation of Data: After the incubation period, both the mutagenicity (TFT restrictive medium) plates and the viability (non-restrictive medium) plates were scored for the total number of colonies per plate using an Artek 880 colony counter or by hand. The frequency of mutants induced by each test material dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. Induced mutagenic activity, if any, was quantified by comparing the mutant frequency of the treated plates to that of the control plates.

DETERMINATION OF CYTOTOXICITY
The test material was checked for toxicity with and without S-9 metabolic activation at dose levels of 1 ug/ml to 5000 µg/ml. No growth of the cell population was observed at >500 ug/ml with S-9 and > 100 ug/ml without S-9. Less severe toxicity was observed at 100 (+S-9) and 10 ug/ml (-S-9). Compound precipitate was present at 5000 ug/ml.

Evaluation criteria:

Criteria for an Acceptable Mouse Lymphoma Screen: All the following criteria should be met for the mutagenicity screen to be considered valid:
- The mutant frequency of the appropriate positive control (either with or without S-9 activation) should be at least twice that of the appropriate negative (solvent) control.
- The spontaneous mutant frequency of the negative (solvents control should be in the range of 20 to 200 per 10ˆ6 cells.
- The plating efficiency of the negative (solvent) control should be at or above 50%.
- The test material should be tested to the level of approximately 10% total growth, or the limits of solubility, or to a high dose of 10 mg/ml. Test materials may be tested as slurries.

The following criteria must be met for the outcome of the Mouse Lymphoma Screen (either with or without S-9 metabolic activation) to be considered mutagenic:
The mutant frequency of one or more test material concentrations, with >10% total growth, is at least two times greater than that of the negative (solvent) control.

The following result must be met for the outcome of the Mouse Lymphoma Screen (either the with or without metabolic activation) to be considered non-mutagenic:
None of the mutant frequencies of any of the test material concentrations, with a survival of >=10% total growth, are two times greater than that of the negative (solvent) control.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No growth of the cell population was observed at >= 500 ug/ml with S-9, and > =100 ug/ml without S-9. Less severe toxicity was observed at 100 ug/ml (+S-9) and 10 ug/ml (-S-9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None of the cultures exhibited a mutant frequency which was two times that of the average mutant frequency of the negative (solvent) controls. Percent total growth ranged from 33% to 96% (with metabolic activation) and 90% to 97% (without activation). The positive contro (DMBA) responded satisfactorily.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions tested, this material was considered to be nonmutagenic in this screen.

Executive summary:

The test material was evaluated for gene mutation in the L5178Y TK+/-mutagenicity screen with and without S-9 metabolic activation at concentrations ranging from 1 ug/ml to 333 ug/ml. None of the cultures exhibited a mutant frequency which was two times that of the average mutant frequency of the negative (solvent) controls. Under the conditions tested, this material was considered to be nonmutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to an internationally approved guideline and followed Good Laboratory Practice standards.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 ug streptomycin/ml and 1% phytohemagglutinin
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced Sprague Dawley rat liver S9 and cofactor mix (magnesium chloride, potassium chloride, glucose-6-phosphate, NADP)

Test concentrations with justification for top dose:

Definitive assay:
0.313, 0.625*, 1.25*, 2.5*, 5.0, 7.5, 10 ug/mL
*Concentrations evaluated for chromosome aberrations

Vehicle / solvent:

Ethanol was used based on solubility of the test article and compatibility with the target cells.

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44-48 h
- Exposure duration: 4 and 20 h in non-activated test system; 4 h in S9-activated test system
- Expression time (cells in growth medium): 20 h
- Selection time (if incubation with a selection agent): 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): ~21 h

FIXATIVE AGENT: methanol:glacial acetic acid
SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 100/ culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The frequency of cells with structural chromosome aberrations in the solvent controls must be within the historical range for solvent controls. The percentage of cells with chromosome aberrations in the positive control must be statistically increased relative to the solvent control and must be similar to historical positive control range. Toxic effects of treatment are based upon inhibition of mitosis. The number and types of aberrations (structural and numerical) found, percentage of aberrant cells, and mean aberrations per cell are calculated for each treatment group. Chromatid and isochromatid gaps are reported but not included in the total percentage of cells with aberrations or in the average number of aberrations per cell.

Statistics:

Fishers Exact test was used to compare the percent aberrant cells of each treatment group with that of the solvent control. Cochran-Armitage test is used to measure dose-responsiveness.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic inhibition was 53-57% at 2.5 ug/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: human peripheral blood lymphocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results :
negative

Under the conditions of this assay, EC 701-204-9 was negative for induction of structural and numerical chromosome aberrations in non-activated and S9-activated test systems using human peripheral blood lymphocytes.

Executive summary:

EC 701-204-9 was evaluated for induction of structural and numerical chromosome aberrations in non-activated and S9-activated test systems via the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes. Mitotic inhibition was 53 -57% at 2.5 ug/mL in all three treatment exposure groups (4h activated, 4h non-activated, 20 h non-activated). The percentage of cells with aberrations in the doses selected for analysis were not significantly increased above the solvent control at any dose level for any of the treatment groups. Positive and solvent control group responses were as expected.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 11, 1986 - Aug 7, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study following GLP. Only 4 strains were tested. The 5th strain E. coli WP2 uvrA or S. typhimurium TA102 was not used. Otherwise, the study was a high quality study clearly demonstrating negative results in the other 4 strains, with and without metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains were tested. The 5th strain E. coli WP2 uvrA or S. typhimurium TA102 was not used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine-deficient strains

Metabolic activation:

with and without

Metabolic activation system:

Aloclor-induced rat liver S-9

Test concentrations with justification for top dose:

0, 0.1, 0.33, 1.0, 3.33, 10.0 mg/plate, with and without S-9

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: See table in materials and methods section below.

Details on test system and experimental conditions:

METHOD OF APPLICATION: All media and solutions were prepared as described by Maron and Ames (1983).

TEST PROCEDURE:
The bacterial tester strain culture was grown by inoculating nutrient broth with a tester strain and incubating with shaking at ~37°C to a density of 1-2 x 10ˆ9 cells/ml. Without metabolic activation, 100 µl of tester strain and 100 µl of solvent or test material were added to 2.5 ml of molten selective top agar at ~45°C. With metabolic activation, 100 µl of tester strain, 100 µl of solvent or test material, and 0.5 ml of S-9 mix were added to 2.0 ml of molten selective top agar at ~45°C. Due to unusual toxicity results observed in the first experiment , the amount of solvent/test material delivered to each plates was reduced to 50 ul in Experiment 2. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 hours at ~37°C. The number of revertant colonies was counted and the presence and condition of the bacterial lawn was noted. Solvent and positive controls were run concurrently. All dose levels were plated in triplicate.

Test material was not completely miscible with the top agar at >=0.3 mg/plate. Testing at the 3.3 mg/plate with 25-400 µl S-9/plate on strains TA98 and TA100 indicated that increasing the concentrations of S-9 did not signficantly increase the induction of revertants; therefore, 25 µl S-9/plate was used for the mutagenicity assay.

DETERMINATION OF CYTOTOXICITY AND OPTIMAL S-9 CONCENTRATION
- The test materials were checked for toxicity on strain TA100 with and without S-9 mix at dose levels of 0.003 to 10 mg/plate. Due to heavy precipitation of the test material at the highest dose level, cytotoxicity results were not reported at 10 mg/plate; the test material appeared to be slightly cytotoxic at 3.3 mg/plate with metabolic activation.

Evaluation criteria:

Criteria for a valid test:
The following criteria were met before the results of the mutagenicity assay were judged as positive or negative:
(1) the high dose showed some toxicity, or was 10 mg/plate or the limit of solubility;
(2) the spontaneous controls were within the laboratory's acceptable range;
(3) the positive controls produced at least a three-fold increase in the number of revertants over the values for the respective negative controls.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to heavy precipitation of test material at highest dose, cytotoxicity results were not reported at 10 mg/plate; test material appeared to be slightly cytotoxic at 3.3 mg/plate with metabolic activation. However, cytotoxicity was inconsistent &nonrepro

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to heavy precipitation of test material at highest dose, cytotoxicity results were not reported at 10 mg/plate; test material appeared to be slightly cytotoxic at 3.3 mg/plate with metabolic activation. However, cytotoxicity was inconsistent &nonrepro

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to heavy precipitation of test material at highest dose, cytotoxicity results were not reported at 10 mg/plate; test material appeared to be slightly cytotoxic at 3.3 mg/plate with metabolic activation. However, cytotoxicity was inconsistent &nonrepro

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to heavy precipitation of test material at highest dose, cytotoxicity results were not reported at 10 mg/plate; test material appeared to be slightly cytotoxic at 3.3 mg/plate with metabolic activation. However, cytotoxicity was inconsistent &nonrepro

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions tested, the test material was not mutagenic to strains TA98, TA100, TA1535, or TA1537 with or without metabolic activation.

Executive summary:

The test material was tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, TA1535, and TA1537 at dose levels of 0.1 to 10 mg/plate with and without metabolic activation provided by Aroclor-induced Rat liver S-9. The test material appeared to be completely miscible with acetone but was not completely miscible with the top agar at <0.1 mg/plate. Cytoxicity was observed in TA98, TA100, and TA1535 (1.0 and 3.3 mg/plate) and TA1537 (3.3 mg/plate) with metabolic activation. However, the observed cytotoxicity was inconsistent and nonreproducible, due at least in part to interference from test material precipitation at all dose levels tested. Under the conditions tested, the test material was not mutagenic to strains TA98, TA100, TA1535, or TA1537 with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)