Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th January 2017 to 18th May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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, C8H20N4, C6H18N4

IUPAC Name:

Amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 2

Reference substance name:

reaction product of Amines, polyethylenepoly-, triethylenetetramine fraction and 2,3-epoxypropyl o-tolyl ether

IUPAC Name:

reaction product of Amines, polyethylenepoly-, triethylenetetramine fraction and 2,3-epoxypropyl o-tolyl ether

Constituent 3

Reference substance name:

TOFA-TETA adducts

IUPAC Name:

TOFA-TETA adducts

Constituent 4

Reference substance name:

TOFA-TETA-CGE adducts

IUPAC Name:

TOFA-TETA-CGE adducts

Constituent 5

Reference substance name:

TOFA-TETA-TOFA

IUPAC Name:

TOFA-TETA-TOFA

Constituent 6

Reference substance name:

Higher molecular weight oligomeric adducts

IUPAC Name:

Higher molecular weight oligomeric adducts

Specific details on test material used for the study:

Identification: Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether
Batch No.: Ei 3065
EC No.: 919-898-9
Purity: Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials
Molecular Weight: Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials
Description: Blackish orange clear viscous liquid
Storage Conditions: Room temperature, protected from light
Receipt Date: 28 November 2016

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination.).

Metabolic activation:

with and without

Metabolic activation system:

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 at a dose of 500 mg/kg,five days before sacrifice.

Test concentrations with justification for top dose:

Preliminary Toxicity Test for Selection of Dose Levels
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the solvent, the highest dose, lowest precipitating dose and the highest soluble dose was measured. Doses for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control).
Chromosomal Aberration Assays
Based on the results of the preliminary toxicity test, the doses selected for testing in the chromosome aberration assay were:
Non-activated:
4 hour treatment, 16 hour recovery:
5,7,9,11,12,13,14,15,20
20 hour treatment, 0 hour recovery:
1,2.5,5,7,8,9,10,12
S9-activated:
4 hour treatment, 16 hour recovery
2.5,5,7,8,9,10,12,15,20

Vehicle / solvent:

The vehicle used to deliver the test substance to the test system was:
Vehicle: Ethanol
Supplier: Sigma-Aldrich
CAS No : 64-17-5
Lot No: SHBG4978V
Exp. Date: Aug 2018

Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Sterile water

Details on test system and experimental conditions:

Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.

Frequency and Route of Administration
Target cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.
Preliminary Toxicity Test for Selection of Dose Levels
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the solvent, the highest dose, lowest precipitating dose and the highest soluble dose was measured. Doses for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control).

After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium was aspirated, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator under standard conditions.
For the definitive assay only, two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL.

For the preliminary toxicity test and chromosomal aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data were presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008).
The cell counts and percent viability were used to determine cell growth inhibition (CGI) relative to the vehicle control (% cytotoxicity = 100 – RICC).
For the definitive assay only, cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2 to 8°C. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by the BioReliance study number, dose, treatment condition, harvest date, activation system, test phase, and replicate tube design.
The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells were examined under oil immersion without prior knowledge of treatment groups. . Whenever possible, a minimum of 300 metaphase spreads containing 20 ± 2 centromeres from each dose (150 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990).

Evaluation criteria:

The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether was concluded to be negative for the induction of structural chromosomal aberrations and positive for the induction of numerical chromosomal aberrations in the non-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether was concluded to be negative for the induction of structural and numerical chromosome aberrations in the S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

The test substance, Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL, which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 15 μg/mL in the non-activated 4 and 20-hour exposure groups, and at doses ≥ 5 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 150 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 5 to 20 μg/mL for the non-activated 4-hour exposure group, from 2.5 to 20 μg/mL for the S9-activated 4-hour exposure group, and from 1 to 12 μg/mL for the non-activated 20-hour exposure group.

In the initial chromosomal aberration assay, cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 9 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 8 μg/mL in the S9-activated 4-hour exposure group; and observed at doses ≥ 7 μg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 5, 7, and 9 μg/mL for the non-activated 4-hour exposure group; 2.5, 5, and 9 μg/mL for the S9-activated 4-hour exposure group; and 2.5, 5, and 7 μg/mL for the non-activated 20-hour exposure group.

In the non-activated 4-hour exposure group, a statistically significant increase (3.7%) in structural aberrations was observed at 9 μg/mL (p ≤ 0.05; Fisher’s Exact). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). Statistically significant and dose-dependent increases in numerical (polyploid and/or endoreduplicated cells) aberrations were observed at doses 7 and 9 μg/mL (p ≤ 0.01; Fisher’s Exact test and p ≤ 0.05; Cochran-Armitage trend test).

In the S9-activated 4-hour exposure group, a statistically significant increase (3.3%) in structural aberrations was observed at 9 μg/mL (p ≤ 0.05; Fisher’s Exact). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). No significant or dose-dependent increases in numerical aberrations were observed at any dose.

In the non-activated 20-hour exposure group, no significant or dose-dependent increases in structural or numerical aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage trend tests).

In order to confirm the positive results in numerical aberrations, the chromosomal aberration assay was repeated in the non-activated 4-hour exposure group at doses ranging from 1 to 11 μg/mL.

In the repeat chromosomal aberration assay, cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 9 μg/mL in the non-activated 4-hour exposure group. The doses selected for evaluation of chromosome aberrations were 3, 7, 8, and 9 μg/mL.

In the repeat trial of non-activated 4-hour exposure group, no significant or dose-dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage trend tests). The statistically significant increase in structural aberrations observed in the initial assay was not confirmed in the repeat assay. Statistically significant and dose-dependent increases (8.3%, 10.3%, and 17.7%) in numerical (polyploid and/or endoreduplicated cells) aberrations were observed at doses 7, 8, and 9 μg/mL, respectively (p ≤ 0.05 or p ≤ 0.01; Fisher’s Exact and Cochran-Armitage trend tests).

These results indicate Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether was negative for the induction of structural chromosomal aberrations and positive for the induction of numerical chromosomal aberrations in the absence of the exogenous metabolic activation system. Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether was negative for the induction of structural and numerical chromosomal aberrations in the presence of the exogenous metabolic activation system.