Tall oil, reaction products with diethylenetriamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation assay (bridging study) is available for the submission substance DTO_DETA. No mammalian cell mutation are clastogenicity studies are available for DTO_DETA; therefore available data for the major components (AAI_DETA and Rosin) are used as read across.

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

Study period:

18 March 2015 to 13 April 2015

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

Target gene:

Point mutation - reversion to histidine dependence

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.

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.

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

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

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

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

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

It is considered appropriate to address the data requirements for DTO_DETA by read-across to the available studies on the main components of DTO_DETA: AAI_DETA and Rosin.
DTO_DETA contains comparatively lower levels of imidazolines and higher levels of resin acids than AAI_DETA and therefore consideration of data for resin acids is also considered necessary. The main resin acid in DTO_DETA is abietic acid, but abietic acid derivatives and other acids, such as pimaric acid, are also found in notable quantities, and the resin acids collectively are known as ‘rosin’. DTO_DETA contains up to 25% unreacted rosin, and taking into account the compositional information available for the rosin in DTO_DETA and Rosin (CAS# 8050-09-07, EC# 232-475-7), the latter was considered appropriate for read-across to DTO_DETA.
A bacterial reverse mutation assay (Ames test; OECD 471) has been conducted with the target substance, DTO_DETA (Thompson, 2015), to act as a bridging study in support of the read across approach. DTO_DETA was not mutagenic under the conditions of this study. AAI_DETA was found to be non-mutagenic in bacterial and mammalian cell systems in vitro. Similarly, Rosin was found to be non-mutagenic in bacterial and mammalian cell systems in vitro. On this basis, read across from AAI_DETA and Rosin to the target substance DTO_DETA is considered appropriate for in vitro genotoxicity in mammalian cells. The negative Ames test conducted with the target, DTO_DETA, supports this approach and the conclusion that DTO_DETA is not mutagenic.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human periperal

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

It was noted that Tall oil diethylenetriamine imidazoline increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the presence of S9-mix in the second cytogenetic assay. This may indicate that Tall oil diethylenetriamine imidazoline has the potential to inhibit mitotic processes.

 

No effects of Tall oil diethylenetriamine imidazoline on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

Conclusions:

Based on read across, DTO_DETA is not clastogenic in human lymphocytes.

Executive summary:

A bacterial reverse mutation assay (Ames test; OECD 471) has been conducted with the target substance, DTO_DETA (Thompson, 2015), to act as a bridging study in support of the read across approach. DTO_DETA was not mutagenic under the conditions of this study. A guideline (OECD 473) in vitro mammalian cell chromosome aberration assay (NOTOX, 2009) is available with the read across (source) substance, AAI_DETA; AAI_DETA was non-mutagenic in this assay. Rosin was also found to be non-mutagenic in a guideline (OECD 473) in vitro mammalian cell chromosome aberration assay (Harlan, 2010); this study is presented as supporting information. Read across to the AAI_DETA and Rosin data sets are considered appropriate since these substances are the major components of the target substance, DTO_DETA. In addition, the Ames test conducted with the target substance confirms the validity of the read across approach. In conclusion, DTO_DETA is not considered to be clastogenic in vitro in mammalian cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

It is considered appropriate to address the data requirements for DTO_DETA by read-across to the available studies on the main components of DTO_DETA: AAI_DETA and Rosin.
DTO_DETA contains comparatively lower levels of imidazolines and higher levels of resin acids than AAI_DETA and therefore consideration of data for resin acids is also considered necessary. The main resin acid in DTO_DETA is abietic acid, but abietic acid derivatives and other acids, such as pimaric acid, are also found in notable quantities, and the resin acids collectively are known as ‘rosin’. DTO_DETA contains up to 25% unreacted rosin, and taking into account the compositional information available for the rosin in DTO_DETA and Rosin (CAS# 8050-09-07, EC# 232-475-7), the latter was considered appropriate for read-across to DTO_DETA.
A bacterial reverse mutation assay (Ames test; OECD 471) has been conducted with the target substance, DTO_DETA (Thompson, 2015), to act as a bridging study in support of the read across approach. DTO_DETA was not mutagenic under the conditions of this study. AAI_DETA was found to be non-mutagenic in bacterial and mammalian cell systems in vitro. Similarly, Rosin was found to be non-mutagenic in bacterial and mammalian cell systems in vitro. On this basis, read across from AAI_DETA and Rosin to the target substance DTO_DETA is considered appropriate for in vitro genotoxicity in mammalian cells. The negative Ames test conducted with the target, DTO_DETA, supports this approach and the conclusion that DTO_DETA is not mutagenic.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Based on read across, DTO_DETA is not mutagenic in mouse lymphoma L5178Y cells.

Executive summary:

A bacterial reverse mutation assay (Ames test; OECD 471) has been conducted with the target substance, DTO_DETA (Thompson, 2015), to act as a bridging study in support of the read across approach. DTO_DETA was not mutagenic under the conditions of this study. A guideline (OECD 476) in vitro mammalian cell gene mutation test (NOTOX, 2009) is available with the read across (source) substance, AAI_DETA; AAI_DETA was non-mutagenic in this assay. Rosin was also found to be non-mutagenic in a guideline (OECD 476) in vitro mammalian cell gene mutation test (Harlan, 2010); this study is presented as supporting information. Read across to the AAI_DETA and Rosin data sets are considered appropriate since these substances are the major components of the target substance, DTO_DETA. In addition, the Ames test conducted with the target substance confirms the validity of the read across approach. In conclusion, DTO_DETA is not considered to be mutagenic in vitro in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The mutagenic potential of 'Tall oil reaction products with diethylenetriamine' (DTO_DETA) was evaluated in a bacterial reverse mutation assay (Ames test) according to OECD 471 (Thompson, 2015). DTO_DETA was found to be non-mutagenic under the conditions of this test. The read across substance, AAI_DETA, was found to be non-mutagenic in an Ames test (CIT, 2008), in an in vitro chromosome aberration test (NOTOX, 2009) and in an in vitro mammalian cell gene mutation test (NOTOX, 2009). Similarly, Rosins were found to be non-mutagenic in a series of Ames tests (Vivotecnia, 2010 a-c; Safepharm, 2004), in an in vitro chromosome aberration test (Harlan, 2010) and in an in vitro mammalian cell gene mutation test (Harlan, 2010). On this basis, read across from AAI_DETA and Rosin to the target substance DTO_DETA is considered appropriate. Therefore, based on read across and available data with the target substance it can be concluded that DTO_DETA is not mutagenic.

Justification for classification or non-classification

Based on the results of the available studies, DTO_DETA is not considered to be mutagenic and classification according to Regulation (EC) No. 1272/2008 is not required.