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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The potential genetic toxicity of slack waxes is expected to be associated with the biologically available / active impurities such as polycyclic aromatic compounds (PAC) found in the entrained oil of the wax material.  Slack waxes (non-carcinogenic feed-stock), produced from refined feedstock's which contain significantly reduced amount of PAC and other impurities, are not genotoxic.  In contrast, the genotoxic potential of slack waxes (carcinogenic or unknown feed-stock) may vary depending on the degree of refining severity of the feed stocks from which they are derived. Slack waxes (carcinogenic or unknown feed-stock) were found to be mutagenic when tested on S. typhimurium in vitro (Blackburn 1984 and 1986) and on mouse lymphoma cells (API 1986c) in the presence of metabolic activation. Crude slack waxes were observed to be non clastogenic when tested (Microbiological Associates Inc., 1987a; 1987b) on Chinese hamster ovary (CHO) cells in vitro and did not appear to be mutagenic when tested in vivo in a CD-1 mouse bone marrow micronucleus assay (McKee et al. 1990).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Slack waxes are waxes with entrained oils. Since paraffin and hydrocarbon waxes are non-hazardous the category hazard profile is determined by the entrained oils. Since the entrained oils are a minor portion of the slack waxes, this is considered to be a worst-case approach.

Slack Waxes (Carcinogenic or Unknown Feed-stock)

In Vitro Genetic Toxicity:

Read across justification

No in vitro genetic toxicity study has been reported for waxes, but data have been reported for unrefined / acid treated oils, materials similar to the oil entrained in slack waxes (carcinogenic or unknown feed-stock).

One key read-across study (Blackburn, 1984) was identified to evaluate the in vitro genetic toxicity potential in bacteria.

In a modified in vitro mutagenicity assay (Blackburn, 1984), S. typhimurium strain TA98 was exposed to a DMSO-extracted light paraffinic distillate at a concentration of 50 microlitres per plate with metabolic activation (Aroclor 1254 -induced rat or hamster liver S9) using the standard pre-incubation method. The test substance was considered mutagenic and produced a mutagenicity index of 17.

In a supporting modified Ames assay (Blackburn 1986), S. typhimurium strain TA 98 was exposed to six DMSO-extracted oil samples (heavy paraffinic distillate, light paraffinic distillate, three separate samples of heavy naphthenic distillate, and heavy naphthenic distillate blend) in the presence of Aroclor 1254-induced hamster liver S9 fraction. All of the test substances were classified as mutagenic, with mutagenicity indices ranging from 4.1 to 10.

One key read-across study (Microbiological Associates Inc., 1987a) was identified to evaluate the in vitro cytogenicity of slack waxes (carcinogenic or unknown feed-stock) in mammalian cells.

In a mammalian cell cytogenetics chromosome aberration assay (Microbiological Associates Inc., 1987a), Chinese Hamster Ovary cells were exposed to L-06 (Light Hydrotreated Feedstock) in dimethylsulphoxide at concentrations of 0.2, 0.1, 0.05, or 0.02 μL/mL under metabolically activated conditions for a 2 -hour exposure period and at test concentrations of 0.3, 0.15, 0.08, or 0.03 μL/mL under non-activated conditions for 10 hours. The test article was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background under activation or non-activation assay conditions.

One key read-across study (API, 1986c) was identified to evaluate in vitro gene mutation in mammalian cells.

Under nonactivated conditions, the test material API 84 -01 was analyzed for mutant induction from 400 nL/mL to 1000 nL/mL, and little or no toxicity was observed (percent relative growths, 114.0% to 95.4%). None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 61.7 x 10-6. Since there was no evidence for mutagenic activity well into the insoluble range, the test material was considered nonmutagenic without activation in this assay.  In the presence of metabolic activation, the test material was analyzed for mutant induction from 50 nl/ml to 1000 nL/mL and a wide range of toxicities was induced (percent relative growths, 72.6% to 7.3%). The test material appeared to interact with the activation mix to convert the test material to a mutagenic form or forms. In order for a treatment to be considered mutagenic in this assay, a mutant frequency exceeding 62.3 x 10-6 was required. All of the assayed treatments induced mutant frequencies that exceeded the minimum criterion and the increases ranged from 2.1-fold to 7.3-fold above the background mutant frequency (average of solvent controls). The test material was therefore considered mutagenic with activation in this assay. In the assays used in this evaluation, the average cloning efficiencies for the solvent controls varied from 86.6% without activation to 87.4% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background.

In Vivo Genetic Toxicity:

Read across justification

No in vivo genotoxicity data have been reported for slack waxes (carcinogenic or unknown feed-stock) but data have been reported for heavy fuel oils and unrefined/acid treated oils, and can be used as a worse case comparison. IP 346 data were not available for these samples.  Accordingly it was not possible to differentiate them on the basis of IP 346 levels. However unrefined and acid treated oils and heavy fuel oils were not active when tested in this assay when applied as neat or DMSO-extracted materials. Accordingly, if the aromatic constituents of these oils are not active when tested separately, it seems reasonable to assume that none of the oils entrained in the slack waxes (carcinogenic or unknown feed-stock) would be active in bone marrow assays for chromosomal mutations.

In a key read-across study (Przygoda et al. 1999) four separate bone marrow micronucleus assays were conducted using two types of petroleum-derived materials: catalytically cracked clarified oil (CCCO) and unrefined lubricating oil (ULO). In the first study, CD-1 mice (5/sex/dose) were administered CCCO in corn oil in two consecutive daily doses via oral gavage or intraperitoneal injection at dose levels of 0, 0.188, 0.375, or 0.75 g/kg. An additional high dose of 1.50 g/kg was administered to the oral gavage group only. Bone marrow cells were harvested at 24 and 48 hours after the final dose. In a second micronucleus test, CD-1 mice (2/sex/dose) were administered a DMSO extract of CCCO in two consecutive daily doses via oral gavage at dose levels of 0, 1.25, 2.5, or 5.0 g/kg. Bone marrow cells were harvested at 24 hours after the final dose. In a third test, neat or DMSO-extracted ULO was administered to CD-1 mice (2/sex/dose) in two consecutive daily doses via oral gavage at dose levels of 0, 1.25, 2.5, or 5.0 g/kg. Bone marrow cells were harvested at 24 hours following the final dose. In the fourth and final test, CCCO in corn oil was administered to CD-1 mice (2/sex/dose) in two consecutive daily doses by IP injection at dose levels of 0, 0.75, 1.5, or 3.0 g/kg. Bone marrow cells were harvested at 24 hours after the final dose.

 

There were no signs of clastogenicity in any of the four studies, even though a lethal response was observed in mice administered DMSO-extracted CCCO where one of four mice in the 2.5 g/kg group and three of four mice in the 5 g/kg group died. The positive and negative controls of all studies induced the appropriate response. 

Slack Waxes (Non-carcinogenic Feed-stock)

In Vitro Genetic Toxicity:

Read across justification

No in vitro cytogenicity or gene mutation (in mammalian cells) data have been reported for slack waxes (non-carcinogenic feed-stock), but data have been reported for refined lubricant base oils and paraffin waxes, materials similar to the oil entrained in slack waxes (non-carcinogenic feed-stock). However, one key study (Petrolabs, 2004) was identified to evaluate the in vitro genetic toxicity potential in bacteria.

DMSO extracts of 4 highly refined slack waxes were tested in the modified assay (Petrolabs, 2004) using Salmonella typhimurium TA 98 at concentrations of 0, 12, 24, 36, 48, and 60µL/plate. The S-9 microsomal fraction derived from Syrian golden hamster liver was used for metabolic activation at a level 8-fold higher than in the standard assay. The MI values were 0 for all 4 slack waxes. Thus, under the conditions of this study, highly refined slack waxes did not induce mutations in Salmonella typhimurium.

In a key read-across reverse gene mutation assay in bacteria (TNO 2005a), strains of S. typhimurium (TA 1535, TA 1537, TA 98, TA 100) and E. coli (WP 2 uvrA) were exposed to extracted Sasolwax 5203 in DMSO.  A highest dose of 100% extract was tested. In total, five dose levels ranging from 1.23 to 100% of the extract were tested, which is comparable to nominal concentrations of 62 to 5000 μg of the test substance per plate.  The positive controls induced the appropriate response in the corresponding strains.  There was no evidence of induced mutant colonies over background.

In a supporting read-across bacterial reverse mutation assay that tested paraffin wax (BIBRA, 1993e), there was no evidence of induced mutant colonies over background.

Two key read-across studies (Microbiological Associates Inc., 1987b and TNO, 2005b) were identified to evaluate the in vitro cytogenicity of slack wax (non-carcinogenic feed-stock) in mammalian cells.

In a mammalian cell chromosome aberration assay (Microbiological Associates Inc., 1987b), Chinese hamster ovary cells were exposed to L-01 (batch TA288) at concentrations of 0.02, 0.04, 0.08, or 0.15 μL/mL, -S9, for 10 hours and 0.05, 0.1, 0.2, or 0.4 μL/mL, +S9, for 2 hours.

Results from the cytotoxicity test showed a marked decrease in relative cell growth at dose 0.3 μL/mL, -S9, and 1μL/mL, +S9. There were no surviving cells at 1 μL/mL, -S9. Doses used in the chromosome aberration study were based from these results. Results from the chromosome aberration assay showed no significant structural or numerical aberrations in CHO cells at any dose level, with or without metabolic activation. Positive controls induced the appropriate response.

In another key read-across study (TNO, 2005b), two mammalian cell chromosome aberration tests were performed. In the first test, Chinese hamster ovary (CHO) cells were exposed to extracts of Sasolwax 5203 (Paraffin wax) at concentrations of 0.034, 0.069, 0.138, 0.277, 0.625, 1.25, 2.5, 5, or 10 mmol/l for four hours with or without metabolic activation at 37°C under 5% CO2. In the second chromosome aberration test, extracts of Sasolwax 5203 were tested at concentrations of 2.78, 4.17, 5.56, 6.94, 8.33, or 10 mmol/l for 18 hours continuous treatment without metabolic activation or 4 hours pulse treatment with metabolic activation at 37°C under 5% CO2. None of the extract concentrations analysed induced a statistically significant increase in the number of aberrant cells with or without metabolic activation or at pulse or continuous exposure. 

Two key read-across studies (API, 1986d and TNO, 2005c) were identified to evaluate in vitro gene mutation in mammalian cells.

In a mammalian cell gene mutation assay (API, 1986d), mouse lymphoma L5178Y cells cultured in vitro were exposed to AP 83 -15 in DMSO at concentrations of 400, 500, 600, 800, 1,000 nL/mL in the absence of mammalian metabolic activation and concentrations of 200, 400, 500, 600, 800, and 1,000 nL/mL in the presence of metabolic activation, for 4 hours. Low to moderate toxicities were observed, thus, this sample was considered to be inactive in the mouse lymphoma assay. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 73.3 x l0-6 and none of the assayed treatments induced this level of mutant action.

 

The negative control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background.

In a mammalian cell gene mutation assay of the TK-locus (TNO, 2005c), mouse lymphoma L5178Y cells were exposed to Sasolwax 5203 (paraffin wax) in DMSO at nominal concentrations of 0.018, 0.037, 0.074, 0.15, 0.29, 0.59, 1.2, 1.7, 2.4, 3.4, 4.9, 7.0, or 10 mmol/l in the presence or absence of mammalian metabolic activation by Aroclor 1254-induced male Wistar rat liver S-9 fraction for 24 hours at 37°C under 5% carbon dioxide.

 

Increased mutant frequency was observed at a single dose level of 2.4 mmol/l in the absence of S-9 mix. Study authors concluded that the reading was caused by an unaccountably low value of the cloning efficiency and is not indicative of mutagenicity. There was no evidence that Sasolwax 5203 induced mutant colonies over background.

In Vivo Genetic Toxicity:

Read across justification

No in vivo genetic toxicity studies have been reported for slack waxes (non-carcinogenic feed-stock), but data have been reported for refined lubricant base oils, materials similar to the oil entrained in slack waxes (non-carcinogenic feed-stock).

One key read-across study (McKee et al. 1990) was identified to evaluate in vivo genetic toxicity in mammalian cells.

In a CD-1 mouse bone marrow micronucleus assay (McKee et al. 1990), male and female mice were given a single intraperitoneal injection of 5 different paraffin oils in corn oil vehicle at doses of 0, 1.0, 2.5, or 5.0 g/kg.  Bone marrow cells were harvested at 24, 48, and 72 hours post-dosing.  One animal did not survive to scheduled sacrifice, but there were no gross signs of toxicity.  The micronucleus frequency was significantly greater than the concurrent negative control in bone marrow cells of male mice given 5.0 g/kg at 48 hours post-dosing, but the negative control was unusually low in this instance, and therefore this result is not considered significant. The five mineral hydrocarbons tested in the mouse micronucleus assay were not considered to be clastogenic.

Justification for selection of genetic toxicity endpoint

One of 13 available genetic toxicity studies.

Justification for classification or non-classification

Slack waxes (carcinogenic or unknown feed-stock and non-carcinogenic feed-stock) do not meet the EU CLP Regulation (EC No. 1272/2008) criteria for mutagenicity. The PAC in oil products are poorly bioavailable due to their physico-chemical properties (low water solubility and high molecular weight), making it unlikely that the genotoxic constituents can reach and cause damage to germ cells (Roy, 2007; Potter, 1999). Considering their poor bioavailability, oil products which have been classified as carcinogenic do not need to be classified as mutagenic unless there is clear evidence that germ cells are affected by exposure.