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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Only limited genotoxicity data are available for petrolata. Adequate read-across data are available however on structural analogues/surrogates to predict the properties of petrolata.

Multiple studies were available to evaluate the in vitro and in vivo genetic toxicity potential of sufficiently refined and insufficiently refined petrolatum. Insufficiently refined petrolatum was found to be mutagenic to Salmonella typhimurium in vitro but was not found to be clastogenic or toxic in vivo. Insufficiently refined petrolatum was also observed to be mutagenic in the presence of metabolic activation when tested in the mouse lymphoma assay. Sufficiently refined petrolatum was not found to be mutagenic or clastogenic in vitro or in vivo.

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

Additional information

Additional information from genetic toxicity in vivo:

Limited genotoxicity data are available for petrolata but adequate read-across data are available on structural anlogues/surrogates to predict the properties of petrolata.

In Vitro Genetic Toxicity

Insufficiently Refined Petrolatum (Carcinogenic or Unknown Feed-stock)

No in vitro genetic toxicity studies have been reported for insufficiently refined petrolatum, but data have been reported for unrefined/acid treated oils, materials similar to the oil entrained in insufficiently refined petrolatum.

One key (Blackburn, 1984) and one supporting (Blackburn, 1986) read across study was identified to evaluate the in vitro genotoxic toxicity potential of insufficiently refined petrolatum 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 another 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 (ARCO, 1985a) was identified to evaluate the in vitro cytogenicity of insufficiently refined petrolatum in mammalian cells.

In a mammalian cell cytogenetics chromosome aberration assay (ARCO, 1985a), Chinese Hamster Ovary cells were exposed to L-06 (Light Hydrotreated Feedstock) in dimethylsulphoxide at concentrations of 0.2, 0.1, 0.05, or 0.02μl/ml under metabolically activated conditions for a 2 -hour exposure period and at test concentrations of 0.3, 0.15, 0.08, or 0.03μL/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 the in vitro gene mutation potential of insufficiently refined petrolatum in mammalian cells.

In a mouse lymphoma test (API, 1986c), under nonactivation 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.

 

Sufficiently Refined Petrolatum (Non-carcinogenic Feed-stock)

Only one in vitro genetic toxicity study in bacteria has been reported for severely refined petrolatum. Data have also been reported for paraffin and hydrocarbon waxes which are similar to constituents of severely refined petrolatum.

In a 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.

Supporting data from a gene mutation assay in bacteria (Petro Labs, Inc. 2000) also demonstrates that oxidized petrolatum is not mutagenic when tested in Salmonella typhimurium. In another supporting read-across reverse gene mutation assay in bacteria (BIBRA, 1993i), strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium were exposed to Paraffin wax at concentrations of 0, 0.1, 0.33, 1.0, 3.3 and 10 mg/plate in the presence and absence of mammalian metabolic activation using the plate-incorporation method. There was no evidence of induced mutant colonies over background.

One key read across study (TNO, 2005b) was identified to evaluate the in vitro cytogenicity of sufficiently refined petrolatum in mammalian cells.

In a chromosome aberration 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 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. In both chromosomal aberration tests, the positive control substances mitomycin C (in the absence of a metabolic activation system) and cyclophosphamide (in the presence of a metabolic activation system) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations.

One key read across study (TNO, 2005c) was identified to evaluate the in vitro gene mutation potential of sufficiently refined petrolatum in mammalian cells.

In a mammalian cell gene mutation assay of the TK-locus (TNO, 2005c), mouse lymphoma L5178Y cells were exposed to Sasolwax 5203 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.

Sasolwax 5203 was tested up to 10 mmol/L. The positive controls methyl methanesulphonate (MMS) and 3 -methylcholanthrene (MCA) induced the appropriate response. The negative control (DMSO) was within acceptable range. 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

Insufficiently Refined Petrolatum (Carcinogenic or Unknown Feed-stock)

No in vivo genetic toxicity data has been reported for insufficiently refined petrolatum or similar materials such as crude slack waxes. However, an in vivo mouse micronucleus assay was conducted on an untreated distillate aromatic extract (UDAE). A UDAE can be considered a “worst case” by comparison to unrefined/acid treated lubricant base oils, materials similar to crude slack wax production, in that the UDAE contains higher concentrations of or biologically active components than do the unrefined / acid treated lubricant base oils.

Two key read-across studies (Mobil Environmental and Health Science Laboratory, 1987a and Pryzgoda et al. 1999) were identified to evaluate the in vivo genetic toxicity potential of insufficiently refined petrolatum.

In a key read-across mammalian cell micronucleus assay (Mobil, 1987a), rats were dermally exposed to 30, 125, or 500 mg/kg/day of 318 Isthmus Furfural Extract for 90 days. The micronucleus test was performed to determine if 318 Isthmus Furfural Extract caused a significant increase in micronucleated red blood cells harvested from bone marrow when rats are dermally exposed. Based on statistical analysis (general linear model) there was no difference between the observed responses and the negative controls. The ANOVA F test found no significant difference in the number of micronucleated PCEs of the 318 Isthmus Furfural Extract-treated animals in comparison to each other or to the negative controls. 318 Isthmus Furfural Extract was not cytotoxic to red blood cell formation nor did it induce significant increase in the formation of micronucleated PCEs or NCEs in bone marrow of treated rats. 318 Isthmus Furfural Extract does not cause chromosome damage to rats dermally exposed.

In another key read-across study (Pryzgoda et al., 1999), four separate bone marrow micronucleus assays were conducted using two types of petroleum-derived materials: catalytically cracked clarified oil (CCCO) and an unrefined lubricating oil (ULO). In the test (experiment 3) with neat or DMSO-extracted ULO, material 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 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 the fourth 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. 

In a supporting read-across mammalian cell micronucleus assay (Mobil, 1987a), rats were orally exposed by gavage to 125, or 500 mg/kg/day of 318 Isthmus Furfural Extract for 90 days. The micronucleus test was performed to determine if 318 Isthmus Furfural Extract caused a significant increase in micronucleated red blood cells harvested from bone marrow when rats are dermally exposed.318 Isthmus Furfural Extract was not cytotoxic to red blood cell formation nor did it induce significant increase in the formation of micronucleated PCEs or NCEs in bone marrow of treated rats. 318 Isthmus Furfural Extract does not cause chromosome damage to rats orally exposed.

Sufficiently Refined Petrolatum (Non-carcinogenic Feed-stock)

 

No in vivo genetic toxicity studies have been reported for sufficiently refined petrolatum or constituents of these materials, paraffin and hydrocarbon waxes and white mineral oils. However, studies have been reported (McKee et al., 1990) for lubricant base oils and serve as the basis for a worst case assessment

 

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 lubricant base oils (IP 346 < 3%) 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 following information is taken into account for any hazard / risk assessment:

Multiple studies were available to evaluate the in vitro and in vivo genetic toxicity potential of sufficiently refined and insufficiently refined petrolatum. Insufficiently refined petrolatum was found to be mutagenic to Salmonella typhimurium in vitro but was not found to be clastogenic or toxic in vivo. Insufficiently refined petrolatum was also observed to be mutagenic in the presence of metabolic activation when tested in the mouse lymphoma assay. Sufficiently refined petrolatum was not found to be mutagenic or clastogenic in vitro or in vivo.

Justification for selection of genetic toxicity endpoint

One of 14 available in vitro and in vivo genetic toxicity studies.

Justification for classification or non-classification

The potential genetic toxicity of petrolatum is associated with the biologically available / active constituents such as polycyclic aromatic compounds (PAC) found in the entrained oil of the wax material. Sufficiently refined petrolatums, produced from refined feedstocks which contain significantly reduced amount of PAC and other constituents, are not genotoxic. Insufficiently refined petrolatums may be genotoxic, depending on the degree of refining severity of feedstocks and resulting PAC content. Based on the data available, sufficiently refined and insufficiently refined petrolatums do not meet EU CLP Regulation (EC No. 1272/2008) criteria for in vitro or in vivo mutagenicity.