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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

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

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

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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th January 2017 to 7th March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced

Test concentrations with justification for top dose:

Preliminary Toxicity Assay to Select Dose Levels
The preliminary toxicity assay was used to establish the dose-range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.
Mutagenicity Assay
The mutagenicity assay was used to evaluate the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and seven to eight dose levels of test substance, in triplicate, in the presence and absence of Aroclor-induced rat liver S9.

Vehicle / solvent:

Vehicle: Ethanol
CAS: 64-17-5
Supplier: Sigma-Aldrich
Lot Number: SHBH3634V
Purity: 99.91%
Expiration date: July 2019

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.
One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50.0 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. 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 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.
Scoring
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 3333 μg per plate with all conditions. Toxicity was observed beginning at 66.7, 333, 667 and 1000 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 333 μg per plate without S9 activation and 1000 μg per plate with S9 activation.

In the mutagenicity assay, the dose levels tested were 3.33, 6.67, 10.0, 33.3, 66.7, 100 and 333 μg per plate without S9 activation and 6.67, 10.0, 33.3, 66.7, 100, 333, 667 and 1000 μg per plate with S9 activation. No precipitate was observed. Toxicity was observed beginning at 66.7, 100, 333, 667 and 1000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)