Tris(2-hydroxyethyl)ammonium salts of tall-oil fatty acids

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone for enzyme induction.

Test concentrations with justification for top dose:

Range-finding test: 0; 10; 31.6; 100; 316; 1000; 2500 and 5000 μg/plate
Experiment 1: 0; 1.581; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Experiment 2: 0; 1.581; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Complementary to Experiment 2: 0; 0.1581; 0.5; 1.581; 5; 15.81; 50; 158.1 and 500 μg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)
Justification for choice of solvent/vehicle:
In a solubility trial, DMSO and acetone were suitable vehicles for exposure to the test substance up to the maximum guideline recommended test substance concentration of 5000 μg/plate, whereas in distilled water the test material was insoluble. DMSO was selected as vehicle, because of its better biocompatibility with the test system than acetone.

Untreated negative controls:

yes

Remarks:

without and with S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine; sodium azide; 9-aminoacridine; methyl-methanesulfonate.

Remarks:

Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.

Untreated negative controls:

yes

Remarks:

without and with S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; Activity of S9 was additionally tested with Benzo[a]pyrene

Remarks:

Positive control substances for tests with metabolic activation (S9 mix). All of them are well established reference mutagens.

Details on test system and experimental conditions:

Range-finding test & Experiment 1: Standard Plate Incorporation Tests
Experiment 2 & Complementary to Experiment 2: Pre-incubation Test

All tests, except the Complementary to Experiment 2, were performed without and with metabolic activation (S9 mix).
The Complementary to Experiment 2, was performed without metabolic activation.
Proportion of S9 fraction in the S9 mix was 10% v/v and of S9 fraction in the final medium ca. 2% v/v in all tests with metabolic activation.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

4-nitro-1,2-phenylene-diamine: - 4 μg/plate, dissolved in DMSO: - strain: TA 98

Sodium azide: - 2 μg/plate, dissolved in distilled water: - strains: TA 100, TA 1535

9-Aminoacridine: - 50 μg/plate, dissolved in DMSO: - strain: TA 1537

Methyl-methanesulfonate: - 2 μL/plate, dissolved in distilled water: - strain: WP2 uvrA


With metabolic activation (S9 mix):

2-Aminoanthracene: - 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 1537, TA 98, TA 100

2-Aminoanthracene: - 50 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

In addition, adeqaute biological activity of the S9 batch used in the present study was confirmed by use of two mutagens, Aminoanthracene and Benzo(a)pyrene, that require metabolic activation by microsomal enzymes.

Evaluation criteria:

The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
- a dose–related increase in the number of revertants and/or;
- a reproducible, biologically relevant positive response for at least one of the dose groups in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if in at least one of the five tested strains the number of reversion was more than twice higher than the reversion rate of the vehicle (solvent) control.

A test substance is considered non-mutagenic in this test if:
Neither a dose-related increase in the number of revertants nor a reproducible, biologically relevant positive response is evident in any of the dose groups, with or without metabolic activation.

The study was considered valid if:
- the number of revertant colonies of the vehicle (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Statistics:

The data were not statistically analysed. The study result was unequivocal.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

but confined to 5000 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative 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:

cytotoxicity

Remarks:

but confined to 5000 µg/plate (+S9) and 2500 & 5000 µg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Confined to Experiment 2 to 500, 1581 & 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced background lawn down to 158.1 µg/plate

Vehicle controls validity:

valid

Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Viability checked by a plating experiment in each test was satisfactory.

Remarks on result:

other: tables of results attached as background material

At a test material concentration of 5000 µg/plate, precipitate was observed in the Range-finding test in both bacterial strains (TA98 and TA100) with metabolic activation, and in Experiment 2 in all tested strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) with and without metabolic activation.

Conclusions:

negative without and with metabolic activation (S9 mix)

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium (DMEM),
supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25µg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium).
During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, induction of rat liver enzymes with Phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
Assay 1
3h treatment (harvesting after 20h) without metabolic activation: 300, 250, 200, 150, 100, 50, 25 and 12.5 µg/mL
3h treatment (harvesting after 20h) with metabolic activation 350, 300, 250, 200, 150, 100, 50 and 25 µg/mL
Assay 2
20h treatment (harvesting after 28h) without metabolic activation: 150, 125, 100, 75, 50, 25, 12.5 and 6.25 µg/mL
3h treatment (harvesting after 28h) with metabolic activation: 350, 300, 250, 200, 150, 100, 50 and 25 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO is compatible to the test system.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Dissolved in DMEM (culture medium), final concentration 0.4 µL/mL (28-hour harvesting time) or 1.0 µL/mL (20-hour harvesting time)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Dissolved in sterile physiological saline solution (0.9% NaCl infusion); final concentration: 6.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Incubation time: 24h at 37°C
- Exposure duration: 3h, 20h
- Fixation time (start of exposure up to fixation or harvest of cells): 20h, 28h (1,5 - 2 normal cell cycles)
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.2 µg/mL) 2-2.5 hours prior to harvesting
STAIN (for cytogenetic assays): 5 % Giemsa solution

NUMBER OF REPLICATIONS: two independend cultures

DETERMINATION OF CYTOTOXICITY
- Method: surviving cells determined using a haemocytometer
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps are considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

Statistics:

Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps. (Gap:small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s).)

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Excessive cytotoxitcity: Assay 1: 3h with S9 mix: ≥ 300 µg/mL, 3h without S9 mix: ≥ 150 µg/mL; Assay 2: 3h with S9 mix: >350 µg/mL, 20h without S9 mix: ≥ 100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
In Assay 1 and 2, no insolubility was detected at the end of the treatment period in the final treatment medium. There were no large changes in the pH and osmolality.

PRELIMINARY TEST: Test with one single culture, no positive controls included; eight test material concentrations up to 5000 μg/mL. Performance in other respects of the test corresponding to the main tests (Assay 1 and 2): 3h/20h (treatment/sampling) without and with S9 mix, 3h/28h with S9 mix and 20h/28h without S9 mix. The preliminary test showed discolouring of the medium at concentrations ≥ 312,5 µg/mL , and precipitationat concentrations ≥ 625 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:The spontaneous aberration frequencies of the vehicle (solvent) controls in the performed experiments were within the historical control range of the testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assay 1:
3h without S9 mix: 150 µg/mL (0 % relative survival), 100 µg/mL (45%)
3h with S9 mix: 350 µg/mL (0%), 300 µg/mL (2%), 250 µg/mL (16%)
Assay 2:
20h without S9 mix: 150 µg/mL (0%), 125 µg/mL (1%), 100 µg/mL (44%)
3h with S9 mix: 350 µg/mL (24%), 300 µg/mL (43%)

Remarks on result:

other: Tables with results attached as background material

Occurrence of polyploid and endoreduplicated metaphases: Polyploid metaphases (1-4) or endoreduplicated metaphases (1-4) were found in some cases in the vehicle (solvent) control, positive control or test material treated samples in the performed experiments. There was no evidence for effects related to the test material.

 

Conclusions:

Interpretation of results : negative

WS400102 did not induce chromosome aberrations in Chinese hamster V79 cells, with or without metabolic activation, up to and including 350 and 300 µg/mL respectively. Therefore, WS400102 is considered not clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (tk) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: 3 types of RPMI 1640 medium [Antibiotic-antimycotic solution 0,01 mL/mL, Pluronic-F68 0,5 mg/mL, Pyruvic acid 0,2 mg/mL, NaHCO3 2 mg/mL, L-glutamine 0,3 gm/mL] - with three conc. of horse serum (5, 10 and 20 % v/v, respective RPMI-5, -10, -20), medium with the highest conc. of horse serum RPMI-20 without Pluronic-F68.
Growth medium: RPMI-20
Expression-medium: RPMI-10
Selection-medium: RPMI-5
- Properly maintained: yes
- Checked for Mycoplasma contamination: yes
- "Cleansed" against high spontaneous background: yes
After thawing, the cells were subcultured no more than 5 times before use on the study.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix; rat liver induced with Phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Based on the results of the preliminary toxicity experiment with doses up to 5000 μg/mL, the following test material concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation:
90; 85; 80; 75; 70; 65; 60; 55; 50; 45; 40; 35; 30; 15; 7.5 and 3.75 μg/mL
Assay 1, 3-hour treatment without metabolic activation:
80; 75; 70; 65; 60; 55; 50; 45; 40; 35; 30; 15; 7.5 and 3.75 μg/mL
Assay 2, 3-hour treatment with metabolic activation:
90; 85; 80; 75; 70; 65; 60; 55; 50; 45; 40; 35; 30; 15; 7.5 and 3.75 μg/mL
Assay 2, 24-hour treatment without metabolic activation:
80; 75; 70; 65; 60; 55; 50; 45; 40; 35; 30; 15; 7.5 and 3.75 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO) - solvent for the test material and the positive control chemicals
- Justification for choice of solvent/vehicle: DMSO is compatible to the test system.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Dissolved in DMSO. Final concentration 0.15 μg/mL for 3-hour treatment and 0.1 μg/mL for 24-hour treatment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Dissolved in DMSO. Final concentration: 4 μg/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3h with and without metabolic activation, 24h without activation
- Expression time: 3 days
- Selection time: 2 weeks

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT), final concentration of 3 μg/mL

NUMBER OF REPLICATIONS: 2 cultures at each concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, plating for viability

OTHER:
Scoring of large and small colonies war performed to obtain information on the mechanism of action of the test material (gene or chromosome mutation).

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met (based on M. Moore 2006):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle (solvent) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding vehicle (solvent) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel 2000 software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Excessive cytotoxicity: Assay 1 with S9 mix > 75 μg/mL; Assay 1 without S9 mix > 65 μg/mL. Assay 2 with S9 mix > 40 μg/mL; Assay 2 without S9 mix > 70 μg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
In both assays (1and 2), no insolubility was detected in the final treatment medium at the beginning and end of the treatment.There were no large changes in pH or osmolality after treatment.

The vehicle (solvent) controls and the positive controls were within the range given by historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assay 1, with S9 mix: evaluated assays ≤ 65 μg/mL (relative survival value of 12 %)
Assay 1, without S9 mix: evaluated assays ≤ 60 μg/mL (relative survival value of 33%) - rel. survival value at 65 μg/m: 0%
Assay 2, with S9 mix: evaluated assays ≤ 35 μg/mL (relative survival value of 11%)
Assay 2 without S9 mix: evaluated assays ≤ 65 μg/mL (relative survival value of 21%)

Remarks on result:

other: see Tables of results attached as background material

Conclusions:

Interpretation of results : negative

No mutagenic effect of WS400102 was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test material WS400102 does not necessitate classification regarding mutagenicity according to EU classification rules [REGULATION (EC) 1272/2008].