Reaction products of tetraethylenepentamine and maleic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity: Ames

A K1, GLP-compliant Ames-test was performed according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100 with the following Salmonella typhimurium strains: TA 1535, TA 1537, TA 98, TA 100 and Escherichia Coli strain WP2 uvr A (Thompson, 2014). The substance was tested with and without metabolic activation system, according to the plate incorporation method and pre-incubation method. The test article was miscible in sterile distilled water at 50 mg/ml. 

In the first mutation test (plate incorporation method), the dose levels tested were 1.5, 5, 15, 50, 150, 500 and 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. 

In the second mutation test (pre-incubation method), the dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation. No test item precipitate was observed on the plates at any of the doses tested in either the presence of absence of S9 -mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation.

All vehicle, untreated and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical data.

Under the conditions of this study, the substance was considered to be non-mutagenic in the Bacterial Reverse Mutation Assay.

Genetic toxicity: mammalian chromosome aberration test

A K1, GLP-compliant mammalian chromosome aberration test was performed in the absence and presence of a metabolic activation system (Shambhu, 2015). The preliminary toxicity assay was used to establish the concentration range for the main study. 

Water was used as vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test, the test substance formed a soluble and clear solution in water at a concentration of approximately 50 mg/ml.

Toxicity of the substance (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 9% at 3900 µg/ml, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 3900 µg/ml, was 14% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 1500, 3000, and 3900 µg/ml. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p>0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (33.0%) was statistically significant (p≤0.01, Fisher's Exact test).

Toxicity of the substance (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was not observed at 3900 µg/ml, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 3900 µg/ml, was 8% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 1500, 3000, and 3900 µg/ml. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p>0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (34.0%) was statistically significant (p≤0.01, Fisher's Exact test). 

Due to excessive cytotoxicity in the positive control (MMC) group, the chromosome aberration assay was repeated again at dose levels of 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 2500, 3000, 3200, 3500, and 3900 µg/ml. 

A second repeat chromosome aberration assay was performed. Toxicity of the substance (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 50% at 1000 µg/ml, the highest test dose level evaluated for chromosome aberrations.

The mitotic index at the highest dose level evaluated for chromosome aberrations, 1000 µg/ml, was 55% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 200, 600, and 1000 µg/ml. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (22.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

The frequency of cells with structural chromosome aberrations in the vehicle control group was within the historical range for vehicle controls. The percentage of cells with chromosome aberrations in the positive control group was statistically increased (p ≤ 0.05, Fisher's Exact test), relative to the vehicle control. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.  

Under the conditions of the assay described in this report, the substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. 

Genetic toxicity: mammalian cell gene mutation test

The substance was tested in a K1, GLP-compliant mammalian cell gene mutation (L5178Y/TK +/- Lymphoma assay) test in the absence and presence of a metabolic activation system (K1, GLP, Stankowski, 2015). The preliminary toxicity assay was used to establish the concentration range for the initial mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test article. 

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test, the test substance formed a clear solution in sterile, distilled water at a concentration of approximately 50 mg/ml (the maximum evaluated). 

Cultures treated at concentrations of 488, 975, 1950, 2930 and 3900 µg/ml (4-hour treatments with and without S9), and 30.5, 60.9, 91.4 and 122 µg/ml (24-hour treatment without S9) exhibited 78 to 115%, 22 to 92%, and 23 to 99% relative suspension growth (RSG), respectively, and were cloned (cultures at a concentration of 488 µg/ml with S9 were lost due to technical error; cultures treated at other lower concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available; cultures treated at other higher concentrations were discarded prior to cloning, or excluded from evaluation of mutagenicity, due to excessive toxicity). Relative total growth of the remaining cloned cultures ranged from 73 to 110% (4-hour treatment with S9), 20 to 97% (4-hour treatment without S9) and 13 to 86% (24-hour treatment without S9). No increases in average induced mutant frequency ≥ 90 mutants per 10E06 clonable cells were observed under any treatment condition.

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. 

The results indicate the substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay under the conditions, and according to the criteria, of the test protocol.

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

Based on the results of the three key in vitro genetic toxicity studies (bacterial reverse mutation test (K1, Thompson, 2015), mammalian chromosome aberration test (K1, Shambhu, 2015), mammalian cell gene mutation test (K1, Stankowski, 2015)) and according to the criteria of the CLP Regulation, the substance should not be classified as a mutagenic substance.