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EC number: 701-299-7 | CAS number: -
The substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-7] (see Chapter 1) was not mutagenic when tested in vitro in bacterial reverse mutation assays. Furthermore, data from an in vitro mammalian cell gene mutation test and an in vitro mammalian chromosome aberration assay provided only results indicating that the substance was negative for genotoxicity. Overall, genetic toxicity in vitro was tested negative based on results from in vitro genotoxicity studies mandatory under REACH.
threshold = number of mutant colonies per 106cells of each solvent control plus 126
# Culture was not continued since a minimum of four concentrations is required by the guidelines.
(p) phase separation visible to the unaided eye
The study was performed to investigate the potential of Novares TL 10 (EC no. 615 -276 -3) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.
The main experiments were evaluated at the following concentrations:
without S9 mix: 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL with S9 mix: 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL
without S9 mix: 156.3; 312.5; 625.0; 1250; and 2500 µg/mL with S9 mix: 39.1; 78.1; 156.3; 312.5; 625.0 µg/mL
Phase separation of the test item visible to the naked eye was noted in experiments I and II at 312.5 µg/mL and above with metabolic activation (4 hours treatment) and without metabolic activation (4 and 24 hours treatment).
No toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation in the first experiment. In the second experiment relevant toxic effects were observed in the presence of metabolic activation at 156.3 µg/mL and above in culture II.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the first experiment up to the maximum concentration with and without metabolic activation. In the second experiment the threshold of 126 plus each solvent control count and the historical range of solvent controls were exceeded in culture I at 78.1 µg/mL and 625 µg/mL. However, no comparable increase was noted in the parallel culture under identical conditions; so this isolated increase was judged as irreproducible artefact. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTAT11 statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of experiment II with metabolic activation. However, a small increase of the mutation frequency at toxic concentrations is common in this assay system and does not indicate a possible mutagenic potential provided that the mutation frequency does not exceed the threshold of 126 colonies per 10E+6 cells above the corresponding solvent control. Since the mutation frequency neither exceeded the historical range of solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.
In this study the range of the solvent controls was from 99 up to 200 mutant colonies per 10E+6 cells; the range of the groups treated with the test item was from 112 up to 310 mutant colonies per 10E+6 cells. The highest solvent control value (200, 172, and 198 mutant colonies per 10E+6 cells) exceeded the recommended 50 - 170E+6 control range as stated under acceptability criteria of the assay of this report. The data are acceptable however, since the parallel culture remained within the recommended range. The cloning efficiency exceeded the upper limit of 120 % in the culture I of the second experiment with metabolic activation (170 %) and in culture II of the second experiment with and without S9 metabolic activation (130 %). The data are acceptable however, since cloning efficiency values above 100 % occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. As well, these values for the solvent controls remained well within the range of 50-200 colonies per 10E+6 cells, originally recommended by the IWGT. Therefore, the aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.
MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.
Summary of results of the chromosomal aberration study with Novares TL 10 (EC no. 615 -276 -3)
Exposure period 4 hrs without S9 mix
Exposure period 22 hrs without S9 mix
Exposure period 4 hrs with S9 mix
* Including cells carrying exchanges # Evaluation of 50 metaphases per culture
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significantly higher than corresponding control values
1 Acetone 0.5 % (v/v)
2 EMS 825.0 µg/mL
3 EMS 770.0 µg/mL
4 CPA 15.0 µg/mL
The test item Novares TL 10 (EC no. 701 -299 -7), dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:
Without S9 mix
With S9 mix
Exp. I & II
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored.
The highest applied concentration in the pre-test on toxicity (5000.0 µg/mL of the test item) was chosen with respect to the current OECD Guideline 473.
Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The following treatment concentrations were chosen for cytogenetic evaluation:
without S9 mix: 6.1, 10.6 and 18.6 µg/mL, with S9 mix: 3.5, 6.1 and 10.6 µg/mL,
without S9 mix: 0.7, 1.1, 18.6 and 32.5 µg/mL, with S9 mix: 7.0, 12.2 and 21.3 µg/mL.
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (47.1% of control).
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
In vivo genetic toxicity testing is not required, because in all tests on in vitro genetic toxicity (see above) negative results were observed.
The genotoxic potency of NAF-AO was tested in vitro in three bacterial reverse mutation assays (Ames tests), in an in vitro mammalian gene mutation study, and in an in vitro chromosomal aberration study.
Bacterial reverse mutation assays
Three bacterial reverse mutation assays (Ames, OECD 471) were conducted according to GLP standards using S. typhimurium and E. coli strains with and without metabolic activation and concentrations up to 5000 µg/plate. No mutagenicity was noted.
Mammalian cell gene mutation test
An in-vitro mammalian cell gene mutation test (OECD 476) was conducted according to GLP standards using mouse lymphoma cells with and without the presence of metabolic activation. NAF-AO did not induce gene mutations at any concentration.
Mammalian chromosome aberration test
An in-vitro mammalian chromosome aberration test (OECD 473) was conducted according to GLP standards using human lymphocytes with and without the presence of metabolic activation. NAF-AO did not induce chromosomal aberration at any concentration.
All study results from three different in vitro genotoxicity tests did not provide any evidence of a positive genotoxic response. The substance did not produce any adverse effects in in vitro genotoxicity studies mandatory according to REACH regulation Annex VII and VIII.
Therefore, in vivo genotoxicity testing can be waived.
The substance NAF-AO did not provide any positive response when tested in three different types of in vitro mutation/genotoxicity assays (see above). Overall, no adverse effects were observed in all the genotoxicity tests performed. Thus, classification according to Regulation (EC) No 1272/2008 (CLP regulation) is not required.
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