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Key value for chemical safety assessment

Additional information

In vitro gene mutation study in bacteria (Ames):

In the key study Richold & Jones (1980) the potential for the test material to cause gene mutation in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 was determined in an Ames test. The five strains were exposed to the test material at concentrations ranging from 0.0001 to 10 µL/plate dissolved in DMSO both in the presence and absence of metabolic activation. The mean number of revertant colonies together with the individual plate counts were recorded for evaluation. Solvent controls, positive controls, sterility and viability tests were run concurrently. During the bacterial growth inhibition and mutation tests (0, 0.01, 0.1, 0.1, 1.0 and 10.0 µL/plate), the test material proved to be toxic to bacteria at the two highest concentrations, 10 and 1 µL/well, resulting in either absence or incomplete formation of a bacterial lawn. According to these findings repeat tests were performed at lower test concentrations. The first repeat test was conducted at concentrations of 0, 0.0001, 0.001, 0.01 and 0.1 µL/plate for TA 100, for all other strains at concentrations of 0, 0.0001 and 0.001 µL/plate. A slight increase in revertant colony numbers was observed with tester strain TA 100, in the absence of metabolic activation at the 0.01 µL/plate dose level only. As this increase was slight and restricted to one dose level a second repeat test was performed over a narrower dose range. No substantial increase in the revertant colony numbers was observed for any of the remaining four strains, regardless of the dose level or the presence/absence of metabolic activation. The second repeat test was performed to verify the TA 100 results at concentrations of 0, 0.005, 0.1, 0.02 and 0.05 µL/plate. The test material failed to produce any substantial increase in revertant colony numbers. As the results of the first repeat were slight and unrepeatable the result was not considered to be significant. Concurrently run viability test and positive, vehicle and sterility control confirm the viability of the test system. Under the conditions of the test, the test material was determined to be non-mutagenic giving a negative result. The study was performed to sound scientific principles, with a sufficient level of reporting to assess the reliability of the submitted data. A reliability score of 2 was assigned in line with the principles for assessing data quality defined in Klimisch (1997).

 

Wild et al. (1983) has been provided as supporting data, where the potential for the test material to cause gene mutation in S. typhimurium was determined in a screening study of seventy six chemicals. The study was performed to a method similar to the guideline OECD 471, where five strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA98 and TA 100) were each exposed to five test concentrations of the test material in an Ames test, with and without metabolic activation. Plates were assessed for the number of revertant colonies. Positive control results were collected over 2 years. Under the conditions of the study, the test material gave a negative (i.e. non-mutagenic) response in all strains in both the presence and absence of metabolic activation. This study was assigned a reliability score of 2 in accordance with Klimisch (1997).

 

In vitro mammalian cell gene mutation study:

In the key study Brown (2012) the potential of the test material to cause gene mutation effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L5178Y TK +/- mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived metabolic activation system S9 mix, performed in two independent experiments. In Experiment 1 the cells were treated at eight dose levels, in duplicate, together with vehicle and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and was 25 to 300 µg/mL in the absence of metabolic activation and 32.55 to 2083 µg/mL in the presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 12.5 to 300 µg/mL in the absence of metabolic activation and 12.5 to 500 µg/mL in the presence of metabolic activation. The maximum dose levels used in the mutagenicity test were limited by test material induced toxicity. A precipitate of the test material was observed at and above 1041.5 µg/mL in the presence of metabolic activation during Experiment 1. The vehicle controls had acceptable mutant frequency values that were within the normal range of the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test. The study was conducted in line with GLP and standardised guidelines with a sufficient level of reporting to assess the quality of the data. Accordingly the study was assigned a reliability score of 1 in line with Klimisch (1997).

 

In vivo cytogenicity, mammalian erythrocyte micronucleus test:

In the key study, Wild (1983), the potential for the test material to cause genetic mutation was determined as part of a screening study, in which seventy six artificial flavouring chemicals were screened using a mouse micronucleus test. The study was reported in brief, but was performed to a guideline similar to OECD 474. Two male and two female NMRI mice were treated with test material in a single exposure, at concentrations of 0, 625, 1250 and 1875 mg/kg, administered via intraperitoneal injection in olive oil. The mice were sacrificed and bone marrow smears prepared 30 hours after exposure. All slides were then stained and scored. Under the conditions of the test, the test material gave negative results and is considered to be non-mutagenic. This study was assigned a reliability score of 2 in accordance with Klimisch (1997).


Justification for selection of genetic toxicity endpoint
No single study can be selected, since both the in vitro and in vivo data are required to address this endpoint (see discussion below).

Short description of key information:
In vitro gene mutation study in bacteria (Ames): Negative, S. typhimurium, TA 1535, TA 1537, TA 1538, TA 98 and TA 100, Richold & Jones (1980).
In vitro mammalian cell gene mutation study: Negative, mouse lymphoma L5178Y cells, OECD 476, EU B.17, EPA OPPTS 870.5300, Brown (2012).
In vivo cytogenicity, mammalian erythrocyte micronucleus test: Negative, mouse micronucleus assay, equivalent to OECD 474, Wild et al. (1983).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to Regulation EC 1272/2008 and Directive 67/548/EEC, the substance does not meet the criteria for classification as a mutagenic substance.