Tall oil, reaction products with diethylenetriamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2015 to 13 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, reaction products with diethylenetriamine

EC Number:

268-776-5

EC Name:

Tall oil, reaction products with diethylenetriamine

Cas Number:

68140-14-7

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

Tall Oil, reaction products with diethylenetriamine

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: the test material (tall oil reaction products with diethylenetriamine) was provided by Baker Hughes Ltd., no batch number was available
- Expiration date of the lot/batch: 31 January 2016
- Purity test date: not supplied; purity >95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: assumed stable for the study duration
- Solubility and stability of the test substance in the solvent/vehicle: the test material was not miscible in DMSO at 50 mg/mL, but was fully miscible in acetone at 100 mg/mL and dimethyl formamide at 50 mg/mL. Acetone was therefore selected as the vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None, but acetone is toxic to bacterial cells at 0.1 mL (see further information below)

OTHER SPECIFICS: Formulated concentrations were adjusted to allow for the stated water/impurity content (5%) of the test item

Method

Target gene:

Point mutation - reversion to histidine dependence

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction prepared from homogenised livers of male rats induced with Phenobarbitone/β-Naphthoflavone

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. Due to excessive toxicity observed in TA1535 and TA1537, an amended experiment was conducted in these strains at 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate
Experiment 2: Salmonella strains: 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate; E.coli strain 0.5, 1.5, 5, 15, 50, 1510, 500 and 1500 µg/plate

The maximum dose level in the first experiment was selected as the maximum recommended level - 5000 µg/plate. Excessive toxicity (evidenced by thinning of the background lawns) was observed in strains TA1535 and TA1537 therefore part of the test was repeated employing the toxic limit as the maximum dose (500 µg/plate). Weakened background lawns were observed in E. coli at 1500 µg/plate and above therefore this dose was employed as the highest dose in the second experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test material was not miscible in DMSO at 50 mg/mL, but was fully miscible in acetone at 100 mg/mL and dimethyl formamide at 50 mg/mL.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Master strains were checked for characteristics, viability and spontaneous reversion rate (all found to be satisfactory) before the start of the study.

Test item preparation:

Experiment 1 - plate incorporation method.
0.1 mL of the appropriate test item concentration, vehicle or positive control was added to 2 mL of molten trace amino acid supplemented media containing 0.1 mL of the bacterial strain culture and 0.5 mL of phosphate buffer (without metabolic activation) or S9 mix (with metabolic activation). The items were mixed and overlayed on a Vogel-Bonner agar plate. Negative controls were performed on the same day as the mutation test. Each test concentration and control was tested in triplicate. All plates were incubated at 37±3°C for approx. 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were performed due to interference and bubbles in the base agar.

Experiment 2 - pre-incubation method
0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer (without metabolic activation) or S9 mix (with metabolic activation) and 0.05 mL of the test item concentration, vehicle or 0.1 mL positive control were incubated at 37±3°C for 20 minutes with shaking prior to addition of 2 mL molten amino acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative controls were performed on the same day as the mutation test. Each test concentration and control was tested in triplicate. All of the plates were incubated at 37±3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

As specified by the OECD guideline

Evaluation criteria:

A dose-related increase in mutant frequency over the dose range tested; a reproducible increase at one or more concentrations; biological relevance against in-house historical control ranges; statistical analysis of data as determined by UKEMS; fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response). A test item will be considered non-mutagenic (negative) if the criteria are not met.

Statistics:

Not required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

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:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The maximum dose level in the first experiment was selected as the maximum recommended level - 5000 µg/plate. Excessive toxicity (evidenced by thinning of the background lawns) was observed in strains TA1535 and TA1537 therefore part of the test was repeated employing the toxic limit as the maximum dose (500 µg/plate). Weakened background lawns were observed in E. coli at 1500 µg/plate and above therefore this dose was employed as the highest dose in the second experiment. In the second experiment, toxicity (weakened background lawn) was observed in the absence of S9 mix from 5 µg/plate (TA100 and TA1537) and 50 µg/plate (TA1535, TA98 and WP2uvrA). In the presence of S9 toxicity was observed from 15 µg/plate (TA100 and TA1537), 50 µg/plate (TA1535 and TA98) and 150 µg/plate (WP2uvrA). A test item film (cream coloured in appearance) was noted at 5000 µg/plate, this observation did not prevent colony scoring.

There was no significant increase in the frequency of revertant colonies for any of the bacterial strains at any dose level, with or without S9 mix in either of two independent experiments.

All positive controls induced marked increases in the frequency of revertant colonies.

Any other information on results incl. tables

There was no significant increase in the frequency of revertant colonies for any of the bacterial strains at any dose level, with or without S9 mix in either of two independent experiments. All positive controls induced marked increases in the frequency of revertant colonies.

Applicant's summary and conclusion

Conclusions:

DTO_DETA was considered to be non-mutagenic in the bacterial reverse mutation assay (Ames test).

Executive summary:

The mutagenic potential of 'Tall oil reaction products with diethylenetriamine' was evaluated in a bacterial reverse mutation assay (Ames test) according to OECD 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test substance in two independent assays, according to the plate incorporation and pre-incubation methods. Eight concentrations were tested up to the maximum 5000 µg/plate, both in the presence and absence of metabolic activation (S9 mix). The test substance induced excessive toxicity in TA1535 and TA1537 in the first experiment, therefore part of the experiment was repeated using an amended dose range (0.15 to 500 µg/plate). Based on the results observed in experiment 1, the dose range was amended for experiment 2 and ranged between 0.05 and 1500 µg/plate.

The vehicle (acetone) control plate gave revertant colony counts within the normal range. All of the positive controls induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. The maximum dose level in the first experiment was selected as the maximum recommended level - 5000 µg/plate. Excessive toxicity (evidenced by thinning of the background lawns) was observed in strains TA1535 and TA1537 therefore part of the test was repeated employing the toxic limit as the maximum dose (500 µg/plate). Weakened background lawns were observed in E. coli at 1500 µg/plate and above therefore this dose was employed as the highest dose in the second experiment. In the second experiment, toxicity (weakened background lawn) was observed in the absence of S9 mix from 5 µg/plate (TA100 and TA1537) and 50 µg/plate (TA1535, TA98 and WP2uvrA). In the presence of S9 toxicity was observed from 15 µg/plate (TA100 and TA1537), 50 µg/plate (TA1535 and TA98) and 150 µg/plate (WP2uvrA). A test item film (cream coloured in appearance) was noted at 5000 µg/plate, this observation did not prevent colony scoring.

There was no significant increase in the frequency of revertant colonies for any of the bacterial strains at any dose level, with or without S9 mix in either of two independent experiments. The test substance is therefore considered to be non-mutagenic under the conditions of this test.