Poly(oxy-1,2-ethanediyl), .alpha.-sulfo-.omega.-hydroxy-, C8-10-alkyl ethers, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to genetic toxicity will be updated once all ongoing studies have been finalised.

Additional information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to genetic toxicity will be updated once all ongoing studies have been finalised.

There are three studies available addressing genetic toxicity of AES (C8-10, 1-2.5 EO) Na. However, the in vitro chromosomal aberration study was disregarded due to unsuited dose levels resulting in cytotoxicity. Therefore this endpoint is covered by read-across from structurally related AES, i.e. AES (C12-14, 1-2.5 EO) Na. The AES reported within the AES category show similar structural, physico-chemical, environmental and toxicological properties. The approach of grouping different AES for the evaluation of their effects on human health and the environment was also made by the Danish EPA (2001) and HERA (2003), supporting the read across approach between structurally related AES. For further details on the suitability of the read-across please refer to the AES Category Approach Justification.

In general a lack of mutagenic activity for the AES category is predictable based on structural and mechanistic considerations. Mutagens are chemicals that either 1) contain highly reactive electrophilic centers capable of interacting with nucleophilic sites on DNA (direct acting agents) or 2) can be metabolized to highly reactive electrophiles. The chemical structures represented by this chemical class do not contain electrophilic functional groups or functional groups capable of being metabolized to electrophiles. AES are readily absorbed in the gastrointestinal tract in human and rat and excreted principally via the urine or faeces depending on the length of the ethoxylate chain but independently of the route of administration. Once absorbed, AES are extensively metabolized by beta- or omega oxidation. The EO-chain seems to be resistant to metabolism. Thus, AES with fully saturated carbon chains are not metabolized to reactive electrophiles.

 

The mutagenicity of AES (C8-10, 2 EO) Na in bacteria was assessed in a study performed according to OECD Guideline 471 with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 102 and TA 100 (Wallner, 2012). The tester strains were treated using the plate incorporation and the pre incubation method both with and without S9-mix. The concentrations for both testing methods was 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Results achieved with vehicle (distilled water) and positive controls were valid. Cytotoxicity was seen in presence and absence of metabolic activation while no genotoxicity was observed under both circumstances for AES (C8-10, 1-2.5 EO) Na.

 

The mutagenicity of AES (C8-10, 2 EO) Na in a mammalian cell line was investigated according to OECD guideline 476 using the mouse lymphoma L5178Y cells with and without metabolic activation (Trenz, 2012). The test concentrations were 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM without metabolic activation as well as 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM with metabolic activation in the first experiment (4 h incubation). In the second experiment the cells were incubated with concentrations of 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM in the presence of metabolic activation for 4 h and at concentrations of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM in the absence of metabolic activation for 24 h. Results achieved with the vehicle (RPMI medium) and positive controls were valid. Cytotoxicity was seen in presence and absence of metabolic activation. No genotoxicity was observed for AES (C8-10, 2 EO) Na, except for the highest dose level in experiment I with metabolic activation where a significantly increased number of mutants and a dose dependency were seen at 0.48 mM. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was only seen in the highly cytotoxic range (relative total growth below 10%) and was not seen in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.

The clastogenicity potential of AES (C8-10, 2 EO) Na in a mammalian cell line was investigated according to OECD guideline 473 using the Chinese hamster lung fibroblasts (V79) with and without metabolic activation (Hofman-Huether, 2013). The concentrations used for the microscopic analyses were 0.25, 0.50 and 0.60 mM without metabolic activation and 0.60, 1.20 and 1.50 mM with metabolic activation in the first experiment (4 h incubation).

 

The aberration frequencies in Experiment I without metabolic activation were 3.5, 5.5, 10.5, 5.2 and 6.5% at 0, 0.25, 0.50, 0.60 mM and for the positive control. Thus, results achieved with the vehicle (MEM medium) were high but within the historical control (0-4%). The result achieved with the positive control was below the historical control. The highest concentration exerted high cytotoxicity (cytotoxicity parameters like mitotic index and cell growth well below 50%). Consequently the highest concentration is not analyzable. In addition the mid concentration showed a high variability between the slides (mitotic index (MI) 59 and 42 and cell density (CD) 63 and 26 in slide 1 and 2, respectively). Thus also slide 2 of the mid dose was not analyzable. Thus the biological relevance of the above findings was doubtful and a confirmatory experiment without metabolic activation was performed.

The aberration frequencies in Experiment I with metabolic activation were 3.5, 1.0, 4.5, 1.5 and 14.0% at 0, 0.60, 1.20, 1.50 mM and for the positive control. The highest concentration exerted high cytotoxicity (cytotoxicity parameters like mitotic index and cell growth well below 50%). Consequently the highest concentration is not analyzable. In addition the mid concentration showed a high variability in chromosomal aberration rates between the analyzed slides. Analysis of spare specimens did still result in a high variability (aberration frequency of 2.5 and 6.5 of slide 1 and 2, respectively). In addition, the aberration frequency of the negative control was at the upper limit (3.5%). The increased aberration rate (4.5%) was close to the aberration rate of the negative control and only slightly above the historical control range (0 - 4%). Therefore this isolated increase in aberration frequency was expected to be of no biological relevance and a confirmatory experiment with metabolic activation was performed.

In Experiment II with metabolic activation the concentrations used were lowered by a factor of two as a consequence of the high cytotoxicity observed in Experiment I. The aberration rates were 1.0, 2.0, 2.0 and 2.5% at 0, 0.30, 0.60 and 1.20 mM. The variation between slides of the highest concentration was high regarding the mitotic index but low regarding the cell density and the aberration frequencies. Thus this concentration can be regarded borderline with regard to cytotoxicity of the test substance. No increased aberration frequency was observed in the confirmatory experiment. Thus the test substance can be considered to be not clastogenic with metabolic activation at suited concentration level. Increased aberration frequencies observed in Experiment I seem to be an artefact due to the cytotoxicity.

In Experiment II without metabolic activation the concentrations used were not modified due to unknown reasons. The aberration rates were 1.5, 4.0, 7.5 and 7.6% at 0, 0.25, 050 and 0.60 mM. The highest dose level chosen exerted again high cytotoxicity (reflected in the decreased mitotic index, cell density and analyzable cells). Thus only two of the three concentrations were analyzable. At the mid concentration a high variability in aberration rates between the two slides was observed. The variation of cytotoxicity indicators (MI and CD) was low in experiment II without metabolic activation and the relative MI and the relative CD of the mid concentration was above 50%. However, in Experiment I this concentration was not analyzable due to the high variability between the slides. Taking into account the concentrations chosen for the Experiments without metabolic activation, cytotoxicity is to be expected at the mid concentration. The mid concentration is only 0.1 mM below the highest concentration (i.e. still 83% of the top concentration) where prominent cytotoxicity was observed. Thus the increased aberration rate at the mid concentration is presumably a result of the cytotoxicity. Also, one of the evaluation criteria for a positive response is a concentration related increase in the number of chromosomal aberrations. Although cytotoxicity was observed at the highest concentration no clear dose-response relationship was observed in Experiment II without metabolic activation. Taken together, the test cannot be regarded as valid according to OECD test guideline 473.

 

To support the conclusion that increased aberration rates observed within the OECD Guideline 473 study are artefacts of the high cytotoxicity and to close the data gap regarding clastogenicity of AES (C8-10, 1-2.5 EO) Na a read-across from structurally related AES, i.e. AES (C12-14, 1-2.5 EO) Na was performed.

The in vivo clastogenic potential of AES (C12-14) Na (CAS 68891-38-3, analytical purity 27-29%, no data on grade of ethoxylation) was assessed in a mammalian bone marrow chromosomal aberration test with CD-1 mouse according to OECD Guideline 475 (Ciliutti, 1995). The test substance was administered via gavage at doses of 1000 and 2000 mg/kg bw to five animals per sex per dose. Distilled water was used as vehicle. The post exposure period were 10, 24 and 48 h for the test group including the vehicle control and 26 h for the positive control group. Results achieved with the negative (distilled water) and positive controls were valid. No signs of toxicity and no increased number of chromosome aberration were seen at 1000 and 2000 mg/kg bw. Thus the test substance did not show clastogenicity at 1000 and 2000 mg/kg bw based on the test material and 270 to 290 and 540 to 580 mg/kg bw based on the active ingredient.

 

In conclusion, AES and their metabolites lack the structural properties which confer mutagenic properties. This is reflected by the lack of positive genotoxicity studies within the whole AES category (for details refer to the AES Category Approach Justification). The false positive result observed within an OECD Guideline 473 study is presumably related to the cytotoxicity of AES (C8-10, 2 EO) Na. The lack of clastogenicity of AES was confirmed within the in vivo Chromosomal Aberration test with AES (C12-14) Na

This is further supported by the conclusions of the HERA report for AES were it is stated that: “In all available in vitro and in vivo genotoxicity assays, there is no indication of genetic toxicity of AES.”

(HERA report, 2003);
http://www.heraproject.com/files/1-HH-04-HERA%20AES%20HH%20web%20wd.pdf

Justification for selection of genetic toxicity endpoint
No study selected as outcome is negative.

Short description of key information:
In vitro gene mutation:
Bacterial reverse mutation assay (Ames test / OECD guideline 471): negative
In vitro mammalian cell gene muatation assay (MLA / OECD guideline 476): negative
In vivo mammalian chromosome aberration assay (CA / OECD guideline 475): negative

Endpoint Conclusion: No adverse effect observed (negative)