Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides, reaction products with polyethylenepolyamines and tall-oil fatty acids, humates hydrochlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following in vitro studies were conducted with organolignite: gene mutation in bacteria (OECD 471); cytogenicity in mammalian cells (OECD 473); and gene mutation in mammalian cells (OECD 476). All in vitro genetic toxicity tests were negative.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-10 to 2013-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidin and tryptophan operon

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:

Rat liver

Test concentrations with justification for top dose:

Preliminary toxicity assay (all strains): 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, or 5000 µg per plate, ± S9
Mutagenicity assay (all strains): 50, 150, 500, 1500, or 5000 µg per plate, ± S9
Mutagenicity assay (positive controls): 1 µg per plate of 2-nitrofluorene (2NF); 1 µg per plate of sodium azide (SA); 75 µg per plate of 9-aminoacridine; and 1000 µg per plate of methylmethanesulfonate (MMS)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: THF was selected based on its ability of form a workable suspension with the test material and its compatibility with the target bacteria

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

SELECTION AGENT (mutation assays): None

NUMBER OF REPLICATIONS: Three

Evaluation criteria:

A response was considered positive for the test material if a dose-related increase in mean revertants per plate in at least one tester strain was observed over a minimum of two increasing concentrations of test material. For the tester stains, a positive response was considered if an increase in mean revertants at the dose-response peak was observed at greater than or equal to 2 (strains TA98, TA100, or urA) or 3 (strains TA1525 and TA1527) times the mean vehicle control value. An equivocal response was considered to be an increase in revertant count that partially met the criteria for a positive response. A negative response was considered if the response with neither positive or equivocal.

The following must be demonstrated for a test to be considered valid:
• The presence of deep rough mutation and the deletion in the uvrB gene for all Salmonella tester stain cultures
• The deletion in the uvrA gene for all WP2 urA cultures
• The presence of pKM101 plasmid R-factor for strains TA98 and TA100
• The following spontaneous revertants in vehicle control: TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; and WP2 uvrA, 10-60.
• Tester strain cultures titers greater or equal to 0.3x109 cells/mL
• Evaluation of a minimum of three non-toxic dose levels. A dose was considered toxic if: a
> 50% reduction in the mean number of revertants per plate as compared to the vehicle control, along with an abrupt dose-dependent drop in revertant count, was observed ; and/or at lease a moderate reduction in background lawn was observed.

Statistics:

None performed.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 100, TA 98

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:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative with and without metabolic activation.

Under the conditions of this study, test article Organolignite was concluded to be negative in the Bacterial Reverse Mutation Assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 of S. typhimurium and WP2 uvrA of E. coli were exposed to organolignite in tetrahydrofuran at concentrations up to 5000 μg/plate in the presence and absence of mammalian metabolic activation using the plate-incorporation method.

 

Organolignite was not cytotoxic at any dose level. Based on these findings, the maximum dose used in the mutagenicity assay was 5000 μg/plate. There were no positive mutagenic responses for any tester strain at any dose level, both with and without metabolic activation. The positive controls induced the appropriate responses in the corresponding strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Negative responses were observed in three, OECD guideline in vitro genetic toxicity studies conducted with organolignite. Summaries of the studies are as follows.

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 of S. typhimurium and WP2uvrA of E. coli were exposed to organolignite in tetrahydrofuran (THF) at concentrations up to 5000 μg/plate in the presence and absence of mammalian metabolic activation using the plate-incorporation method.

Organolignite was not cytotoxic at any dose level. Based on the lack of cytotoxicity, the maximum dose used in the mutagenicity assay was 5000 μg/plate. There were no positive mutagenic responses for any tester strain at any dose level, both with and without metabolic activation. The positive controls induced the appropriate responses in the corresponding strains.

 

In a mammalian cell chromosome aberration assay, Chinese hamster ovary (CHO) cells were exposed to organolignite in THF at the following concentrations and test conditions: 0, 40, 60, 80, 100, 120, 140, 160, or 180 µg/mL, -S9, for 4 hr exposure with 16 hr recovery period; 0, 5, 10, 20, 30, 40, 50, or 60 µg/mL, -S9, for 20 hr exposure with no recovery period; and 0, 50, 100, 200, 400, 450, 500, 550, or 588 µg/mL, +S9, for 4 hr exposure with 16 hr recovery period. A preliminary toxicity test was conducted to assess cytotoxicity for the following concentrations and test conditions: 0.0588, 0.1764, 0.588, 1.764, 5.88, 17.64, 58.8, 176.4, or 588 µg/mL, ±S9, for 4 hr exposure with 16 hr recovery period or for 20 hr exposure with no recovery.

There was no evidence of chromosome aberration induced over background for the test material. The positive controls induced the appropriate response. Therefore, it was concluded that the test material did not increase the frequency of chromosomal damage with or without metabolic activation at any dose level.

 

In a mammalian cell gene mutation assay, mouse L5178Y cells cultured in vitro were exposed to organolignite in THF in the presence and absence of mammalian metabolic activation for either 4 hours or 24 hours. 

In a preliminary assay, organolignite was tested 375 µg/mL, ±S9, for either 4 or 24 hours of exposure. Visible precipitate was observed at ≥ 5 µg/mL for the 4-hr exposure, ±S9, and at ≥1.5 µg/mL for the 24-hr exposure, -S9. Suspension growth relative to the solvent control was < 10% at ≥ 150 µg/mL for the 4-hr exposure, -S9, 18% at 375 µg/mL for the 4-hr exposure, +S9, and < 10% at ≥ 1.5 µg/mL for the 24-hr exposure, -S9. Doses selected for the mutation assay were based on the precipitation profile. Therefore, the following dose levels of organolignite were evaluated in the mutation assay: 0.25, 0.5, 0.75, 1, 1.25 µg/mL, ±S9, for 4-hr exposure; or 0.5, 0.75, 1, 1.25, 1.50 µg/mL, -S9, for 24-hr exposure. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background for any dose level and/or test condition. Under the conditions of this study, organolignite was negative in the L5178Y/TK+/- mouse lymphoma assay.

Justification for selection of genetic toxicity endpoint

Three key studies identified that received a Klimisch score of 1 because the studies were conducted in accordance with OECD guidelines and are GLP compliant.

Justification for classification or non-classification

All in vitro genetic toxicity studies (i.e., gene mutation studies in bacteria; cytogenicity studies in mammalian cells; and gene mutation studies in mammalian cells) showed negative results. Based on the results of these studies, organolignite is unlikely to be mutagenic and does not meet the criteria for classification and labelling as such, described in CLP EU Regulation 1272/2008.