Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no in vitro genetic toxicity data available for Undecane. However, data is available for structural analogues Hydrocarbons, C10-C12, isoalkanes, <2% aromatics, Hydrocarbons, C10-C13, isoalkanes, <2% aromatics, and Hydrocarbons, C11 -C14, n-alkanes, isoalkanes, cyclics, <2% aromatics. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

All read across genetic toxicity tests listed below had negative results for Undecane.

Genetic Toxicity in vitro– Bacterial reverse mutation assay (OECD 471)

Genetic Toxicityin vitro– Mammalian Chromosome Aberration Test (OECD TG 473)

Genetic Toxicity in vitro- Mammalian Cell Gene Mutation Test (OECD TG 476)

Genetic Toxicityin vitro– Genetic Toxicology:In VitroSister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998/11/11-1999/06/11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 471: GLP.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions from Aroclor exposed rats
Test concentrations with justification for top dose:
Tests (done in triplicate) with and without Metabolic Activation: Acetone (vehicle control), 0,128, 320, 800, 2000, 5000 ug/plate
Vehicle control: 0.1 ml/plate acetone
Positive controls: 2-nitrofluorene (2NF): 5ug/plate (TA98; -S9); Sodium azide - 2ug/plate (TA100, TA1535; -S9); 9-aminoacridine (9AA): 50ug/plate (TA1537; -S9); Glutaraldehyde (GLU): 25.0ug/plate (TA102; -S9); 2-aminoanthracene (2AA): 5ug/plate (TA98; +S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
0.1 ml/plate Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (2NF): 5ug/plate (TA98; -S9); Sodium azide - 2ug/plate (TA100, TA1535; -S9); 9-aminoacridine (9AA): 50ug/plate (TA1537; -S9); Glutaraldehyde (GLU): 25.0ug/plate (TA102; -S9); 2-aminoanthracene (2AA): 5ug/plate (TA98; +S9)
Details on test system and experimental conditions:
These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46°C:

0.1 mL bacterial culture
0.025 mL test article solution or control
0.5 mL 10% 8-9 mix or buffer solution

followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
Evaluation criteria:
The test article was considered to be mutagenic if: 1) the assay was valid, 2) Dunnett’s test gave a significant response (p<0.01), and the data set(s) showed a significant dose-correlation, and 3) the positive responses described in 2) were reproducible.
Statistics:
The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett’s test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
not cytotoxic up to 5000ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not cytotoxic up to 5000ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The test to assess the genotoxicity of the test material was negative. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

No SHELLSOL TD treatments of any of the test strains, either in the absence or in the presence of S-9, resulted in a statistically significant increase in revertant numbers, when the data were analysed at the 1% level using Dunnetts test. This study was therefore considered to have provided no indication of any SHELLSOL TD mutagenic activity. The test to assess the genotoxicity of the test material was negative. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982/08/13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 471.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions from Aroclor exposed rats
Test concentrations with justification for top dose:
Tests (done in triplicate) with and without Metabolic Activation: DMSO (vehicle control), 0, 41.2, 123.5, 310.4, 1111.1, 3333.3, 10000 ug/plate
Negative Controls: DMSO
Positive controls: 2-nitrofluorene (2NF): 50ug/plate; 9-aminoacridine (9AA): 75ug/plate; MNNG: 5 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (2NF): 50ug/plate; 9-aminoacridine (9AA): 75ug/plate; MNNG: 5 ug/plate
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
not cytotoxic up to 10000ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not cytotoxic up to 10000ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The test to assess the genotoxicity of the test material was negative. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

No treatments of any of the test strains, either in the absence or in the presence of S-9, resulted in a statistically significant increase in revertant numbers. This study was therefore considered to have provided no indication of any test material mutagenic activity. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: “Acceptable, well-documented study report equivalent or similar to OECD guideline 473: GLP
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
primary culture, other: human lymphocytes from two male and one female donor
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 without S9 (40.36, 57.66, 82.34 ug/ml);
Experiment 1 with S9 for 3 hours followed by 17 hour recovery (490, 700, 1000 ug/ml)
Experiment 2 without S9 20h treatment 0h recovery (22.52, 28.15, 35.18 ug/ml)
Experiment 2 with S9 for 3 hours followed by 17 hours recovery (640, 800, 1000 ug/ml)
Experiment 2 with S9 for 3 hours followed by 41 hours recovery (1000 ug/ml)
Experiment 3 without S9 for 20 hours treatment and 0 hours recovery (28.15, 35.19, 43.99 ug/ml)
Experiment 3 without S9 for 44 hours and 0 hours recovery (43.99 ug/ml)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline, cyclophosphamide
Evaluation criteria:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and 2) the proportion of cells with structural aberrations at such doses exceeded normal range, and 3) the results confirmed in the second experiment. A positive result only at delayed harvest in Experiment 2 was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as equivocal. Full assessment of the biological importance of such increases is likely to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance.
Species / strain:
primary culture, other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The mammalian chromosomal aberration test to assess the genotoxicity of SHELLSOL D70 was negative. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

The potential of SHELLSOL D70 to cause chromosome aberration was investigated in cultured human lymphocytes with and without the metabolic activation S9 system. Negative and positive control substances were include in both experiments to confirm the activity and sensitivity of the test systems.  In the first experiment, the maximum dose levels selected for chromosome analysis were 82.34 ug/ml and 1000 ug/ml, in the absence and presence of S9 respectively.  These dose levels caused inhibitions of the mitotic index of 57% and 30% respectively.  In the second experiment, the highest concentration used for chromosome analysis were 35,18 ug/ml and 1000 ug/ml in the absence and presence of S9 respectively, these gave a reduction in the mitotic index of 52% and 12% respectively.  In both Experiments 1 and 2 in the presence of S9; and in Experiment 2 in the absence of S9 only there were no significant increases in the frequency of the cells with structural aberrations in cultures treated with SHELLSOL D70.  Following treatment in Experiment 2 in the absence of S9 there was a significant increase in the frequency of structural aberrations at the lowest dose analyzed (22.52ug/ml).  Additional doses from Experiment 1 were analyzed (19.79 and 28.25 ug/ml) to confirm whether this effect was only apparent at low concentrations.  No increase in the frequency of structural aberrations was apparent at these concentrations.  In order to further clarify the findings seen in the initial experiments, a third experiment was performed in which there were no significant increases in the frequency of cells with structural aberrations in all cultures treated with SHELLSOL D70.  Since the increase in structural aberrations seen at 22.52 ug/ml in Experiment 2 was not apparent in other experiments at similar or higher concentrations, the effect was considered to be non-reproducible and of no biological importance.  Based on these results, it is concluded that SHELLSOL D70 did not induce chromosome aberrations in cultured lymphocytes when tested to its limit of toxicity in both the absence and presence of S9.  This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983/01/13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 479.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Tests with and without Metabolic Activation: (media control), 0, 0.5, 1.7, 5.0, 17.0, 50.0 ug/plate
Negative Controls: DMSO, Cyclohexane
Positive controls: EMS - Ethylmehanesulfonate (620 ug/mL) without activation; cyclophosphamide (1.4 ug/mL) with activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: EMS - Ethylmehanesulfonate (620 ug/mL) without activation; cyclophosphamide (1.4 ug/mL) with activation
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not cytotoxic up to 50 uL/mL (maximum dose tested)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The test to assess the genotoxicity of the test material was negative. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

No treatments in either the absence or in the presence of S-9 resulted in a statistically significant increase in revertant numbers. This study was therefore considered to have provided no indication of any test material mutagenic activity. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to sound scientific principles.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK+/ phenotype
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
TK+/ phenotype of L5178Y mouse lymphoma cells from subline 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Aroclor
Test concentrations with justification for top dose:
up to was 1000 ug/mL in dimethylsulfoxide (maximum dose)
Vehicle / solvent:
dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
This assay was performed with the TK+/ phenotype of L5178Y mouse lymphoma cells from subline 3.7.2C using a minimum of eight test compound doses with and without metabolic activation by an Aroclor induced rat liver microsomal fraction. Appropriate negative, solvent, and positive controls were included with each assay. The test compound dose levels were determined by a preliminary multidose ranging study with the highest dose targeted to give approximately fifty to ninety percent inhibition of suspension cell growth depending on the solubility of the compound. C10-C13 isoalkanes achieved a homogeneous mixture at approximately 100 mg/ml in dimethylsulfoxide. The maximum dose selected for the mutagenicity test was 1000 ug/ml because it represents the limits of solubility of the test material.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Exposure to eight graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative with and without metabolic activation

Exposure to eight graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.
Executive summary:

Exposure to eight graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.

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

Genetic toxicity in vivo

Description of key information

There is no in vivo genetic toxicity data available for Undecane. However, data is available for structural analogue Hdyrocarbons, C10-C12, isoalkanes, <2% aromatics. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics were non-mutagenic when tested in both an in vivo germ cell chromosome aberration test and in an in vivo DNA damage and/or repair study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline followed
Principles of method if other than guideline:
P32-postlabeling of DNA adducts developed by Randerath. (K. Randerath, MV Reddy, RC Gupta (1981) 32P-labeling test for DNA damage. Proc. Nat. Acad. Sci. (USA), 78, 6126-6129. This procedure is one of the most sensitive procedures for detecting DNA adducts and has been used to demonstrate the absence of genotoxicity in vivo for a number of compounds
GLP compliance:
not specified
Type of assay:
other: DNA adduct
Species:
mouse
Strain:
C3H
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 8-11 weeks
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
dermal
Details on exposure:
The dorsal region of animals were shaved and only animals in resting hair phase were used. Animals were treated cutaneously with varying amounts of undiluted n-decane (C10) and n-dodecane (C12) in a series of short term exposures designed to permit comparisons with skin carcinogencity studies and to determine whether there was a treatment related dose response for genotoxicity. Groups of animals (4 per dose group) were treated at 3 dose levels of either n-decane, or n-dodecane. The three treatment groups were treated with either i) 25ul for 24h, ii) 25ul for 24h then 25ul for a further 24h, iii) 25ul for 24h, 25ul for a further 24h and then 25 ul for a final 24 h. For a positive control, animals were treated with 25ul n-dodecane containing 1ug of benzo(a)pyrene as a representative genotoxic carcinogenic PAH. An untreated group of mice that were handled in identical conditions to treated group served as negative controls.

The dose was applied to the shaved dermal area of each mouse in either single or multiple applications. A volume of 25ul was applied per animal per application. This volume covered most of the shaved area. Care was taken during application of the dose to avoid loss of the dose into surrounding unshaved areas. Positive control animals were treated with 1 ug benzo(a)pyrene per animals in 25ul n-dodecane.
Control animals:
yes, concurrent no treatment
Positive control(s):
animals were treated with 1ug benzo(a)pyrene per animal in 25ul n-dodecane
Tissues and cell types examined:
The area of the treated skin was dissected and stored individually in glass tubes at -80°C until DNA extraction. The skins were trimmed of any extraneous subcutaneous fat and peripheral hair, then treated with depilatory cream for 5 min at room temperature. The cream was removed, the treated skin sample was placed in warm water at 532°C for 30 seconds, followed by quenching in ice cold water for 15 seconds. The epidermal layer was removed by scraping. The epidermal samples were pooled, homogenized in 2.0ml PBS/lysis buffer. DNA was extracted: 2-mercaptoethanol (60l) and Proteinase K (60 units) were added and the samples incubated for 2 hours at 37°C. DNA was extracted by phenol chloroform extraction using an automated nucleic acid extracted. The recovered DNA was dissolved in water.
Details of tissue and slide preparation:
Enzymatic digestion of DNA to 3’-monophosphate nucleotides:
DNA was treated with micrococcal nuclease, spleen phosphodiesterase, 10x sodium succinate/calcium chloride buffer pH 6.0, and incubated at 37°C for 3.5 hours. Dephosphorylation of normal nucleotides was effected with nuclease P1, 1mM ZnCl2, 400mM sodium acetate pH5.0 (0.7l), H2O (1.2l) and incubation at 37°C for 40 minutes. 1M Bicine buffer pH9.6 was added and the samples evaporated to dryness.
32P-Post-labelling of DNA:
Analyses were carried out at least in triplicate. Digested dephosphorylated DNA samples were labeled using the methods of Raderath (1981) and Reddy and Randerath (1994). To each sample 10x Kinase buffer (200mM Bicine, 100mM MgCl2*6 H2O, 100mM dithirothreitol, 10mM spermidine pH 9.6), H2O, poynucleotide kinase, unlabelled ATP and [32P]-ATP were added, mixed thoroughly and then incubated at 37°C for 45 minutes. Labled samples were applied to PEI-cellulose plates fitted with wicks and developed in direction D1 (2.3M sodium phosphate pH5.3) for 16 hours. After elution in the D1 direction the sheets were trimmed and the wick, discarded. The sheets were washed in water with occasional agitation and then air dried. The samples were chromatographed in direction D3 (4.5M lithium formatted and 8.5M urea pH 3.5) and D4 (0.8M lithium chloride 0.5M tris-HCL and 8.5M urea pH 8.0) for 7-8 hours with a short wick attached to the leading edge of the TLC plate. After thoroughly washing in distilled water, the TLC plates were dried, a wick attached and a final elution in D5 performed (1.7M sodium phosphate pH 6.0).

Radioactive areas were idenfied and quantified using a Packard instant Imager with Packard Imager for Windows V2.01 software, Packard Instrument Compannty. Autoradiography was performed using Lightning Plus Intensifying Screens and BioMax film. Exposure was for at least 72 hours.

The labeling efficiency of the postlabelling assay was determined by adding a known amount of the reference DNA adduct BP dG-3’-monophosphate to the enzymic digest of epidermal DNA immediately prior to carrying out the 32P-labelling step.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results:: negative
32P-Postlabelling analysis of the epidermal DNA from mice treated with either n-decane or n-dodecane at each of the exposure levels and durations showed an absence of radioactive spots or diagonal radioactive zones which could have corresponded to adducts arising from decane. The absences of adducts indicates that n-decane and n-dodecane do not possess genotoxic activity in vivo.
Executive summary:

C3H mice were treated cutaneously with a range of doses (1x25ml to 3x25ml per animal; ca. 900-2750mg/kg) of either n-decane or n-dodecane for periods ranging from 24 to 72 hours.  The DNA was isolated for the epidermis of the treated skin and subjected to 32P-postlabeling analysis.  A group of untreated control animals was untreated and maintained under the same conditions as the exposed groups.  For a positive control, a group of mice was treated with benzo(a)pyrene in 25ul dodecane at a level of 1ug per animal, a dose which is about 2 orders of magnitude lower than a single carcinogenic dose of benzo(a)pyrene for this species.  For a positive control test of the 32P-postlabelling assay, DNA from mice treated with benzo(a)pyrene in THF was used.  32P-Postlabelling analysis of the epidermal DNA from mice treated with either n-decane or n-dodecane at each of the exposure levels and durations showed an absence of radioactive spots or diagonal radioactive zones which could have corresponded to adducts arising from decane.  The positive control adduct was easily detectable.  The limit of detection of the procedure was about 1 adduct in 109 nucleotides using 10mg samples of DNA.  The absences of adducts with this level of sensitivity of adduct detection indicates that n-decane and n-dodecane do not possess genotoxic activity in vivo.  

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 478.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: Males (7-8 weeks); females pre-treatment mating period (8-9 weeks); females post treatment mating period (7-8 weeks)
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: males were house individiually during the treatment period and hosed with two females per week during the 2 week pretreatment mating period and the 6 week post-treatment mating period. Females were housed individually during the pre-mating and post-mating periods and housed with males in a 2:1 ratio during mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum


Route of administration:
inhalation: vapour
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
MRD-77-44 was transferred from a reservoir using a metering pump into a heated flask and flash evaporated. A stream of clean air was also passed through the flask and the vapor laden air transferred to a port in the chamber air inlet where it was diluted with normal chamber intake air to give the desired concentration.
- Exposure apparatus: inhalation chamber
- Rate of air: 125 liters/minute



- Air flow rate: 125 liters/minute
- Air change rate: 8 minutes
- Method of particle size determination:
- Treatment of exhaust air:


TEST ATMOSPHERE
- Brief description of analytical method used: Wilks Scientific Copr, Miran IA Ambient Air Analyzer (long path infrared)
- Samples taken from breathing zone: no
Duration of treatment / exposure:
Six hours /day
Frequency of treatment:
five days
Triethylenemelamine was administered by intraperitoneal injection (normal saline) as a single dose.
Post exposure period:
Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks. The females were sacrificed 12 days after the last day of cohabitation
Remarks:
Doses / Concentrations:
900 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Negative control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
Positive control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
300ppm MRD-77-43: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
900ppm MRD-77-43: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
Control animals:
yes
Positive control(s):
triethylenemelamine

- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.5mg/kg/bw
Tissues and cell types examined:
Males: seminal vesicle, epididymides, prostate, and any abnormal lesion or tissue masses, testes.
Females: reproductive tissues examined (uterine horns preserved, implantation sites, resorption sites)
Statistics:
Comparisons were made during the treatment and post-treatment periods between negative control, positive control and test substance-treated groups by the chi-square method where applicable. Absolute data were compared using the F-test and Students t-test. When variances differed significantly, Students T-test was appropriately modified using Cochran’s approximation.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
When administered by vapor inhalation, MRD-77-43 is not mutagenic by the dominant lethal test. This finding does not warrant classification of (the test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

In a dominant lethal assay,  MRD-77-43 was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to MRD-77-43 produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, no treatment related effects were observed on the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resoprtions.  Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, MRD-77-43 when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test.  This finding does not warrant the classification of MRD-77-43 as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.  

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

Additional information

There is no in vitro or in vivo genetic toxicity data available for Undecane. However, data is available for structural analogues Hydrocarbons, C10-C12, isoalkanes, <2% aromatics, Hydrocarbons, C10-C13, isoalkanes, <2% aromatics, and Hydrocarbons, C11 -C14, n-alkanes, isoalkanes, cyclics, <2% aromatics. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

In Vitro

 

In vitro gene mutation study in bacteria

In a key study (Shell, 1999), no test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) treatments of any of the test strains, either in the absence or in the presence of S-9, resulted in a statistically significant increase in revertant numbers, when the data were analysed at the 1% level using Dunnetts test. This study was therefore considered to have provided no indication of any Hydrocarbons, C10-C12, isoalkanes, <2% aromatics mutagenic activity. The test to assess the genotoxicity of the test material was negative.

In a second key study (Chvevron, 1982) no test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) treatments of any of the test strains, either in the absence or in the presence of S-9, resulted in a statistically significant increase in revertant numbers. This study was therefore considered to have provided no indication of any test material mutagenic activity.

In Vitro Chromosome Aberration in Mammalian Cells

In a key OECD Guideline 473 study (Shell, 1998), the potential of the test material (Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics) to cause chromosome aberration was investigated in cultured human lymphocytes with and without the metabolic activation S9 system. Negative and positive control substances were include in both experiments to confirm the activity and sensitivity of the test systems.  In the first experiment, the maximum dose levels selected for chromosome analysis were 82.34 ug/ml and 1000 ug/ml, in the absence and presence of S9 respectively.  These dose levels caused inhibitions of the mitotic index of 57% and 30% respectively.  In the second experiment, the highest concentration used for chromosome analysis were 35,18 ug/ml and 1000 ug/ml in the absence and presence of S9 respectively, these gave a reduction in the mitotic index of 52% and 12% respectively.  In both Experiments 1 and 2 in the presence of S9; and in Experiment 2 in the absence of S9 only there were no significant increases in the frequency of the cells with structural aberrations in cultures treated with the test material.  Following treatment in Experiment 2 in the absence of S9 there was a significant increase in the frequency of structural aberrations at the lowest dose analyzed (22.52ug/ml).  Additional doses from Experiment 1 were analyzed (19.79 and 28.25 ug/ml) to confirm whether this effect was only apparent at low concentrations.  No increase in the frequency of structural aberrations was apparent at these concentrations.  In order to further clarify the findings seen in the initial experiments, a third experiment was performed in which there were no significant increases in the frequency of cells with structural aberrations in all cultures treated with the test material.  Since the increase in structural aberrations seen at 22.52 ug/ml in Experiment 2 was not apparent in other experiments at similar or higher concentrations, the effect was considered to be non-reproducible and of no biological importance.  Based on these results, it is concluded that Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics did not induce chromosome aberrations in cultured lymphocytes when tested to its limit of toxicity in both the absence and presence of S9.

In a key OECD Guideline 479 study (Chevron, 1983), no test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) treatments in either the absence or in the presence of S-9 resulted in a statistically significant increase in revertant numbers. This study was therefore considered to have provided no indication of any test material mutagenic activity.

 

In vitro Gene Mutation study in Mammalian Cells

In a key study (Chevron, 1982), exposure to eight graded doses of the test material (Hydrocarbons, C10-C13, isoalkanes) in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.

 

In Vivo

 

In a key dominant lethal assay (Exxon, 1978), the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to the test material produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, no treatment related effects were observed on the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resoprtions.  Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.

In a supporting study (Shell, 1998), C3H mice were treated cutaneously with a range of doses (1x25ml to 3x25ml per animal; ca. 900-2750mg/kg) of either n-decane or n-dodecane for periods ranging from 24 to 72 hours.  The DNA was isolated for the epidermis of the treated skin and subjected to 32P-postlabeling analysis.  A group of untreated control animals was untreated and maintained under the same conditions as the exposed groups.  For a positive control, a group of mice was treated with benzo(a)pyrene in 25ul dodecane at a level of 1ug per animal, a dose which is about 2 orders of magnitude lower than a single carcinogenic dose of benzo(a)pyrene for this species.  For a positive control test of the 32P-postlabelling assay, DNA from mice treated with benzo(a)pyrene in THF was used.  32P-Postlabelling analysis of the epidermal DNA from mice treated with either n-decane or n-dodecane at each of the exposure levels and durations showed an absence of radioactive spots or diagonal radioactive zones which could have corresponded to adducts arising from decane.  The positive control adduct was easily detectable.  The limit of detection of the procedure was about 1 adduct in 109 nucleotides using 10mg samples of DNA.  The absences of adducts with this level of sensitivity of adduct detection indicates that n-decane and n-dodecane do not possess genotoxic activity in vivo.

Justification for classification or non-classification

The negative results using in vitro and in vivo genotoxicity assays from structural analogues do not warrant the classification of Undecane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).