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Genetic toxicity in vitro

Description of key information

All read across genetic toxicity tests listed below had negative results for Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics.

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

Genetic Toxicity in vitro – Mammalian chromosome aberration test (OECD 473)

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

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.
Key result
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
Key result
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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 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)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Test #1 (8, 40, 200, 1000, 5000 ug/plate)
Test #2 (1000, 2000, 3000, 4000, 5000 ug/plate)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [ethanol]
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine, 2-nitrofluorene, sodium azide, glutaraldehyde
Evaluation criteria:
The test article was considered to be mutagenic if:
1) the assay was valid
2) Dunnett's test gave a significant response (<=0.01), and the data set showed a significant dose-correlation.
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.
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 bacterial reverse mutation 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:

SHELLSOL D60 was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98 and 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver S9 fraction for metabolic activation.  Two tests were performed: Test #1 (8, 40, 200, 1000, 5000 ug/plate), Test #2 (1000, 2000, 3000, 4000, 5000 ug/plate).  The material was not cytotoxic.  In all cases, SHELLSOL D60 did not induce any significant changes in the number of revertant colonies, with or without metabolic activation.   It is concluded in this study that SHELLSOL D60 is not a mutagenic agent.   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:
1985/07/05-1985/07/15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles: 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 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 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: Acetone (vehicle control), 0, 50, 150, 500, 1500, 5000 ug/plate
Vehicle control: 0.1 ml/plate acetone
Positive controls: 0.5ug/plate 2AA, 5ug/plate MNNG, 80ug/plate 9AA, 2ug/plate 2NF
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:
yes
Remarks:
non treated
Positive controls:
yes
Positive control substance:
other: TA 1537 (+S9 2-aminoanthracene) TA 1537 (-S9 9-aminoacridine); TA 98 (-S9 2-nitrofluorene) (+S9 2-aminoanthracene); TA100 (-S9 MNNG) (+S9 2-aminoanthracene); TA1535 (-S9 MNNG) (+S9 2-aminoanthracene); TA138(-S9 2-Nitrofluorene) (+S9 2-aminoanthracene)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
DURATION
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS:
- triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants and/or clearing of the background lawn of bacterial growth
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the positive controls meet the criteria for a positive response and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increase in a concentration-related manner and/or if it is a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitution for frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic.
Statistics:
The mean plate count and standard deviation for each dose point were determined. Any test value that was equal to or greater than two times the mean value of the concurrent vehicle control was considered to be a positive dose.
Key result
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 5,000ug/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

Based on these data, classification under EU requirements for dangerous substances and preparations and under EU GHS guidelines is not required.
Executive summary:

Petrepar 120 was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 1538, 98, and 100 in the absence and presence of a liver S9 fraction for metabolic activation. The test was performed in triplicate using doses of 0, 50, 150, 500, 1500, 5000 ug/plate.  In all cases, Petrepar 120 did not induce any significant changes in the number of revertant colonies.  It is concluded in this study that Petrepar 120 is not a mutagenic agent and classification under EU requirements for dangerous substances and preparations guidelines is not required, nor is it required under EU GHS guidelines.

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
Key result
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
Key result
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 gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04-Aug-2014 to 21-Aug-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are part of the same homologous series as the constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512 and 1600 μg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
650 µg/plate in DMSO for TA100
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
Positive controls:
yes
Remarks:
10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9
Positive controls:
yes
Remarks:
10 µg/plate in DMSO for WP2uvrA
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9
Positive controls:
yes
Remarks:
5 µg/plate in saline for TA1535
Positive control substance:
sodium azide
Remarks:
without S9
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation (droplets of test substance) was observed at 1600 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Conclusions:
Interpretation of results: negative

An Ames test was performed with Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics according to OECD 471 and GLP. Strains tested were Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA, both with and without metabolic activation (Aroclor 1254-induced rat liver S9). The test was negative both with and without metabolic activation.
Executive summary:

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04-Aug-2014 to 18-Aug-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are also constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512 and 1600 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation (droplets of the test substance) was observed at dose levels of 1600 and 5000 µg/plate


RANGE-FINDING/SCREENING STUDIES:
-No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

 In the first mutation experiment, no dose level with precipitate or toxicity was tested in the tester strains TA1535, TA1537 and TA98.

Evaluation: Although precipitation of the test substance on the plates was observed at concentrations of 1600 and 5000 µg/plate in the dose range finding, no precipitation was observed at 1600 µg/plate in the first experiment anymore. Since clear negative responses were observed in all tester strain tested and in both the dose range finding test and the second mutation experiment the test substance was tested up to 5000 µg/plate, the lack of a dose level with precipitate or toxicityhad no influence on the study.

Conclusions:
Interpretation of results:
negative with and without metabolic activation

An Ames test was performed with Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics according to OECD 471 and GLP. Strains tested were Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA, both with and without metabolic activation (Aroclor 1254-induced rat liver S9). The test was negative both with and without metabolic activation.
Executive summary:

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07-Aug-2014 to 25-Aug-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are also constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Of
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 200 µg/plate.
Experiment 2:
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation (small droplets of test substance) was observed.
Dose range finding test: at the start of the incubation period at concentrations of 512 µg/plate and upwards and at the end of the incubation period at 1600 and 5000 µg/plate.
First mutation experiment: at the start and end of the incubation period at 5000 µg/plate.
Second mutation experiment: at the start of the incubation period at a concentration of 1600 and 5000 µg/plate and at 5000 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Conclusions:
An Ames test was performed with Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics according to OECD 471 and GLP. Strains tested were Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA, both with and without metabolic activation (Aroclor 1254-induced rat liver S9). The test was negative both with and without metabolic activation.
Executive summary:

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07-Aug-2014 to 25-Aug-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are part of the same homologous series as the constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation (small droplets the test substance) was observed.
Dose range finding test: at concentrations of 1600 and 5000 µg/plate.
Mutation experiment: at 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- In this study, the negative control values were within the laboratory historical control data ranges,
except the response of TA98 in the presence of S9-mix in the second experiment. However, since the value was just outside the limit of the historical control range, the validity of the test was considered to be not affected. The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Conclusions:
An Ames test is also available for Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatic, conducted according to OECD 471 and GLP. Strains tested were Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA, both with and without metabolic activation (Aroclor 1254-induced rat liver S9). The test was negative both with and without metabolic activation.
Executive summary:

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative control values were within the laboratory historical control data ranges, except the response of TA98 in the presence of S9-mix in the second experiment. However, since the value was just outside the limit of the historical control range, the validity of the test was considered to be not affected. The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

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.
Key result
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 cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August to 21 September 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Few details on test material (no certificate of analysis)
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in 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)
Deviations:
yes
Remarks:
No certificate of analysis
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A culture medium supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% C02 in air.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 from male Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range finding assay: half-log series of concentrations of 0.0835 to 2500 µg/mL
Main experiment:
- without metabolic activation: 3.13, 6.26, 9.35 and 12.5 µg/mL with 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL with 20-h harvest
- with metabolic activation: 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test material was insoluble in water and dimethylsulfoxide. A clear and homogeneous stock solution of 201 mg/mL with ethanol could be maintained.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: without metabolic activation: 7.25 and 17 h for 10 and 20 h assay, respectively; with metabolic activation: 2 h
- Expression time (cells in growth medium): with metabolic activation: 7.75 and 17.75 h for 20 and 10 h assay, respectively;
- Time in 0.1 µg/mL Colcemid: without metabolic activation: 1 and 0.5 h for 20 and 10 h assay, respectively; with metabolic activation: 2.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 10 h and 20 h without and without metabolic activation

STAIN (for cytogenetic assays): 5% Giemsa solution and BrdUrd (5-bromodeoxyuridine) at 10 µM

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 cells for test substance; at least 25 cells for positive controls

CYTOTOXICITY: visual observations based on confluence of monolayer and floating dead cells
Evaluation criteria:
Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 were analyzed.
The following factors were taken into account in the evaluation of the chromosomal aberrations data: the overall chromosomal aberration frequencies, the percentage of cells with any aberrations, the percentage of cells with more than one aberration, any evidence for increasing amounts of damage with increasing dose.
Chromatid and isochromatid gaps were not considered as they may be due to toxicity.
Statistics:
Fisher's exact test with an adjustment of multiple comparisons
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range-finding without metabolic activation:
A very unhealthy cell monolayer, -70% reduction in the cell monolayer confluence, floating dead cells, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 25.0 µg/mL. Slight reductions in the number of visible mitotic cells and -15% reduction in the cell monolayer confluence were observed in the cultures dosed with 2.50 and 8.35 µg/mL.
Range-finding with metabolic activation:
An unhealthy cell monolayer, -85% reduction in the cell monolayer confluence, floating dead cells and debris, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 835 µg/mL. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 25.0 and 83.5 µg/mL.

Chromosomal aberrations assay without metabolic activation (Table 1):
In the 10 h assay, no toxicity was observed in any of the test cultures. These cultures were not analyzed for chromosomal aberrations as four dose levels were available for analysis from the 20 h assay. In the 20 h assay, an unhealthy cell monolayer, -70% and -45 % reduction in the cell monolayer confluence, floating dead cells and debris, and a severe reduction in visible mitotic cells were observed at 75.0 and 50.0 µg/mL, respectively. Toxicity was evident on the slides prepared from these cultures by the very sparse numbers of metaphases available for analysis.

Chromosomal aberration assay with metabolic activation (Tables 2 and 3):
In the 10 h assay, slight reductions in the numbers of visible mitotic cells were observed in the cultures dosed at 563 and 751 µg/mL.
In the 20 h assay, severe toxicity was exhibited on the slides prepared from the cultures dosed with 562 and 750 µg/mL by the presence of many dead cells and the sparse numbers of metaphases available for analysis. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 99.7, 187, 281, 375, 562, and 750 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 without metabolic activation (results from pooled duplicate cultures)

 

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

Concentration (µg/mL) 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

7

1

 

 

0.0

Positive (Mitomycin C)

0.04

7

 

4

7

28.0*

Test article

25.0

15

2

 

 

0.0

37.5

7

3

1

1

0.5

50.0

8

1

 

1

0.5

75.0

19

2

4

 

0.5

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 2: Chromosome aberrations in CHO cells fixed 10 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

 

Concentration (µg/mL)

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

2

 

 

0.0

Positive (Cyclophosphamide)

25.0

1

 

8

13

44.0*

Test article

282

7

1

 

 

0.0

375

3

1

0.5

563

4

 

1

0.5

751

3

3

 

1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 3: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

 

Concentration (µg/mL)

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

7

 1

0.0

Positive (Cyclophosphamide)

12.5

1

 

17

31

80.0*

Test article

281

15

2

 

1.0

375

16

6

1

1

1.0

562

3

1

1

1.0

750

10

1

1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Conclusions:
Interpretation of results: negative

MRD-90-843 was found not to increase chromosome aberrations in CHO cells with and without metabolic activation.
Executive summary:

In an in vitro chromosome aberration test, Chinese Hamster Ovary cells were exposed to MRD-90-843 at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation.

Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, MRD-90-843 is not classified according to the criteria of Annex VI to Directive 67/548/EEC and the CLP Regulation (1272/2008).

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
Key result
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 cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-07-14 to 2014-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are part of the same homologous series as the constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 10.6, 18.5, 32.4, 56.7, 99.3, 173.8, 304.1, , 532.2, 931.4, 1630.0 µg/mL
Experiment II: 32.4, 56.7, 99.3, 173.8, 304.1, , 532.2, 931.4, 1630.0 µg/mL

Without metabolic activation:
Experiment I: 10.6, 18.5, 32.4, 56.7, 99.3, 173.8, 304.1, , 532.2, 931.4, 1630.0 µg/mL
Experiment II: 10.6, 18.5, 32.4, 56.7, 99.3, 173.8, 304.1, , 532.2, 931.4, 1630.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Phase separation occurred at the higher concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.

The highest treatment concentration in this study, 1630.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

No visible precipitation of the test item in the culture medium was observed. Phase separation was observed at the end of treatment in Experiment I at 32.4 µg/mL and above in the absence of S9 mix and at 99.3 µg/mL and above in the presence of S9 mix and in Experiment II at 56.7 µg/mL and above in the absence and presence of S9 mix.

No relevant influence on osmolarity or pH value was observed.

In this study no relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration (Table 3 – Table 4).
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Table 6, Table 7, Table 9 and Table 10). The aberration rates of the cells after treatment with the test item (0.5 – 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 – 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data (see Appendix 2).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table 2. Summary of results of the chromosomal aberration study with Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

 

incl. gaps*

excl. gaps*

carrying exchanges

 

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control2

65.8

10.0

10.0S

2.0

 

 

18.5

84.5

2.5

2.5

0.0

 

 

532.2PS

89.6

2.0

2.0

0.0

 

 

931.4PS

78.8

1.0

0.5

0.0

 

 

1630.0PS

76.3

2.5

2.0

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

1.0

1.0

0.5

 

 

Positive control2#

57.5

47.0

46.0S

21.0

 

 

32.4

98.6

0.5

0.5

0.0

 

 

532.2PS

96.2

1.0

1.0

0.0

 

 

931.4PS

116.4

1.5

1.5

0.0

 

 

1630.0PS

96.5

1.5

1.5

0.0

*   Including cells carrying exchanges

#    Evaluation of 50 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Ethanol       0.5 % (v/v)

2     EMS       660.0 µg/mL


Table 2, cont.  Summary of results of the chromosomal aberration study with Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

 

incl. gaps*

excl. gaps*

carrying exchanges

 

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control2

68.7

11.0

11.0S

1.5

 

 

56.7

86.6

0.5

0.5

0.0

 

 

532.2PS

88.3

2.0

1.5

0.0

 

 

931.4PS

110.0

2.0

2.0

0.0

 

 

1630.0PS

101.4

1.0

1.0

0.0

II

22 hrs

Solvent control1

100.0

2.0

2.0

0.0

 

 

Positive control2

25.3

14.5

14.5S

1.5

 

 

32.4

87.9

2.5

2.5

0.0

 

 

532.2PS

85.7

3.0

2.5

0.0

 

 

931.4PS

101.9

2.0

2.0

0.5

 

 

1630.0PS

90.9

1.5

1.0

0.0

*   Including cells carrying exchanges

#    Evaluation of 50 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Ethanol       0.5 % (v/v)

2    CPA          15.0 µg/mL

 

Conclusions:
Interpretation of results: negative with and without metabolic activation

A chromosome aberration study was performed for Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics according to OECD 473 and GLP. In the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration (1630.0 μg/mL). 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. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.
Executive summary:

The test item Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics, dissolved in ethanol, 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

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (1630.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised in Table 2.

In the absence and presenceof S9 mix, no cytotoxicity was observed up to the highest applied concentration.

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 in cells with structural chromosome aberrations.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-07-21 to 2014-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are also constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 12.4, 21.7, 38.0, 66.5, 116.4, 203.6, 356.4, 623.7, 1091.4, 1910.0 µg/mL
Experiment II: 12.4, 21.7, 38.0, 66.5, 116.4, 203.6, 356.4, 623.7, 1091.4, 1910.0 µg/mL

Without metabolic activation:
Experiment I: 12.4, 21.7, 38.0, 66.5, 116.4, 203.6, 356.4, 623.7, 1091.4, 1910.0 µg/mL
Experiment II: 12.4, 21.7, 38.0, 66.5, 116.4, 203.6, 356.4, 623.7, 1091.4, 1910.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Key result
Species / strain:
lymphocytes: Peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction of mitotic index to 75% of control at highest dose, 22 h exposure without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 1910.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I in the absence of S9 mix at 66.5 µg/mL and above and in Experiment II in the absence of S9 mix at 21.7 µg/mL and above. In Experiment I and II in the presence of S9 mix phase separation was observed at 38.0 µg/mL and above.
No relevant influence on osmolarity or pH value was observed.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration (Table 3 - Table 4).
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Table 6, Table 7, Table 9 and Table 10). The aberration rates of the cells after treatment with the test item (0.5 – 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 – 2.5 % aberrant cells, excluding gaps) and clearly within the range of the laboratory historical solvent control data (see Appendix 2). However, in Experiment II in the presence of S9 mix one single statistically significant increase, clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore biologically irrelevant, was observed after treatment with 623.7 µg/mL (3.0 % aberrant cells, excluding gaps).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770.0 or 660.0 µg/mL) or CPA (15.0 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table2     Summary of results of the chromosomal aberration study with
Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

100.0

2.5

2.5

0.0

 

 

Positive control2

97.1

8.5

8.5S

0.5

 

 

38.0

77.1

2.5

2.5

0.0

 

 

623.7PS

106.3

2.5

2.5

0.0

 

 

1091.4#PS

84.0

2.5

2.3

0.0

 

 

1910.0PS

91.4

0.5

0.5

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

2.0

1.5

0.0

 

 

Positive control3##

37.3

48.0

48.0S

13.0

 

 

12.4

107.8

1.5

1.5

0.0

 

 

623.7PS

117.1

2.0

2.0

0.0

 

 

1091.4PS

106.9

1.5

1.5

0.0

 

 

1910.0PS

110.1

3.0

2.5

0.0

*   Including cells carrying exchanges

#    Evaluation of 200 metaphases per culture

##  Evaluation of 50 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone         0.5 % (v/v)

2     EMS          770.0 µg/mL

3     EMS          660.0 µg/mL

4    CPA             15.0 µg/mL

5    CPA               7.5 µg/mL


Table 2, cont.  Summary of results of the chromosomal aberration study with
Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics

Exp.

Preparation interval

Test item concentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

2.5

2.0

0.0

 

 

Positive control4

63.2

16.0

16.0S

3.0

 

 

21.7

112.5

3.5

3.0

0.0

 

 

623.7PS

83.3

3.0

3.0

0.0

 

 

1091.4PS

78.5

1.5

1.5

0.5

 

 

1910.0PS

75.0

1.0

0.5

0.0

II

22 hrs

Solvent control1

100.0

1.0

0.5

0.0

 

 

Positive control5

64.9

17.0

17.0S

3.0

 

 

21.7

113.5

2.0

2.0

0.0

 

 

623.7PS

124.6

3.0

3.0S

0.0

 

 

1091.4PS

112.9

1.0

1.0

0.0

 

 

1910.0PS

114.0

1.5

1.5

0.0

*   Including cells carrying exchanges

#    Evaluation of 200 metaphases per culture

##  Evaluation of 50 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone         0.5 % (v/v)

2     EMS          770.0 µg/mL

3     EMS          660.0 µg/mL

4    CPA             15.0 µg/mL

5    CPA               7.5 µg/mL

Conclusions:
Interpretation of results: negative with or without metabolic activation

A chromosome aberration study was performed for Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics according to OECD 473 and GLP. In the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration (1910.0 μg/mL). Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II in the presence of metabolic activation mix one single statistically significant increase, clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore biologically irrelevant, was observed after treatment with 623.7 μg/mL (3.0 % aberrant cells, excluding gaps). 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 in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.
Executive summary:

The test item Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics, 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

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (1910.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II in the presence of S9 mix one single statistically significant increase, clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore biologically irrelevant, was observed after treatment with 623.7 µg/mL (3.0 % aberrant cells, excluding gaps).

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 in cells with structural chromosome aberrations.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-07-21 to 2014-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are also constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment IA:13.8, 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL
Experiment II: 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL

Without metabolic activation:
Experiment IA: 13.8, 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL
Experiment IB: 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 543.9, 692.2, 951.8, 1211.4, 2120.0 µg/mL
Experiment II: 13.8, 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2120.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. Phase separation was observed at the end of treatment at 73.8 µg/mL and above in Experiment IA and at 42.2 µg/mL and above in Experiment II in the absence of S9 mix. In Experiment IB in the absence of S9 mix and Experiment II in the presence of S9 mix phase separation was observed at 129.2 µg/mL and above. In Experiment IA in the presence of S9 mix phase separation was observed at 226.0 µg/mL and above.
No relevant influence on osmolarity or pH value was observed.
In this study no relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration (Table 3 – Table 5).
In Experiment IA in the absence of S9 mix, statistically significant increases in chromosomal aberrations were observed after treatment with 42.2 and 692.2 µg/mL (3.0 and 5.5 % aberrant cells, excluding gaps). The first value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. The second value exceeded the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) (Table 7). However, no dose-dependency was observed. In the confirmatory Experiment IB in the absence of S9 mix, in Experiment II in the absence of S9mix after continuous treatment and in Experiment IA and II in the presence of S9 mix no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 – 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 – 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data (see Appendix 2). The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Either EMS (660.0 or 770.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table2     Summary of results of the chromosomal aberration study with Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix

IA

22 hrs

Solvent control1

100.0

1.0

0.5

0.0

 

 

Positive control2

90.2

10.0

10.0S

2.0

 

 

42.2

114.0

3.5

3.0S

0.0

 

 

395.6PS

92.4

2.5

2.5

0.5

 

 

692.2#PS

96.0

5.5

5.5S

0.0

 

 

1211.4PS

96.3

2.5

2.5

0.0

 

 

2120.0PS

107.9

2.0

2.0

0.0

IB

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

Positive control3

71.6

12.0

12.0S

2.0

 

 

73.8

69.4

1.5

1.0

0.0

 

 

543.9PS

80.4

1.5

1.5

0.0

 

 

692.2PS

79.0

1.5

1.5

0.0

 

 

951.8PS

64.6

0.5

0.5

0.0

 

 

1211.4PS

76.8

1.5

1.5

0.0

 

 

2120.0PS

70.8

1.5

1.5

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

2.0

1.5

0.0

 

 

Positive control3

32.8

24.0

24.0S

6.5

 

 

24.1

91.1

2.0

2.0

0.0

 

 

692.2PS

85.0

2.0

2.0

0.0

 

 

1211.4PS

86.6

1.0

1.0

0.0

 

 

2120.0PS

79.8

0.5

0.5

0.0

*   Including cells carrying exchanges

#    Evaluation of 200 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone        0.5 % (v/v)

2     EMS       770.0 µg/mL

3     EMS       660.0 µg/mL


Table 2, cont.  Summary of results of the chromosomal aberration study with
Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control2

76.6

9.5

9.5S

1.5

 

 

129.2

106.6

1.0

1.0

0.0

 

 

692.2PS

100.0

1.0

1.0

0.0

 

 

1211.4PS

102.9

1.5

1.5

0.0

 

 

2120.0PS

102.9

1.5

1.0

0.0

II

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

Positive control3

48.7

17.0

17.0S

3.0

 

 

73.8

91.2

1.5

1.5

0.0

 

 

692.2PS

100.4

0.5

0.5

0.0

 

 

1211.4PS

101.3

2.0

2.0

0.0

 

 

2120.0PS

106.6

1.5

1.5

0.0

*   Including cells carrying exchanges

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone        0.5 % (v/v)

2    CPA            2.5 µg/mL

3    CPA            7.5 µg/mL

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.
Executive summary:

The test item Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. IA & IB

Exp. II

Exp. IA & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (2120.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In Experiment IA in the absence of S9 mix, statistically significant increases in chromosomal aberrations were observed after treatment with 42.2 and 692.2 µg/mL (3.0 and 5.5 % aberrant cells, excluding gaps). The first value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. The second value exceeded the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps). However, no dose-dependency was observed. In Experiment IB these findings could not be confirmed. In Experiment II in the absence of S9mix after continuous treatment and in Experiment IA and II in the presence of S9 mix no relevant increases in chromosomal aberrations were observed.

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 in cells with structural chromosome aberrations.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-07-21 to 2014-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
The hypothesis for the analogue approach is that the test substance, Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, contains constituents which are part of the same homologous series as the constituents of the target substance, Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. The two substances have some constituents in common and contain no functional groups that are structural alerts for genetic toxicity. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). 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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Experiment II: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL

Without metabolic activation:
Experiment I: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Experiment II: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluatedAt least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2400.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. Phase separation was observed at the end of treatment in Experiment I and II in the presence of S9 mix at 83.6 µg/mL and above. In the absence of S9 mix phase separation was observed in Experiment I at 146.2 µg/mL and above and in Experiment II at 15.6 µg/mL and above.
No relevant influence on osmolarity or pH value was observed.
No relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration (Table 3 - Table 4).
Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, one single statistically significant increase was observed after treatment with 783.7 µg/mL (3.0 % aberrant cells, excluding gaps) (Table 7). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of S9 mix, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 µg/mL (Table 10). Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 or 770.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table2     Summary of results of the chromosomal aberration study with Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics    

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

100.0

2.5

2.5

0.5

 

 

Positive control2

120.8

10.0

10.0S

2.0

 

 

83.6

102.9

2.0

2.0

0.0

 

 

783.7PS

88.2

2.5

2.5

0.0

 

 

1371.4PS

91.8

3.0

3.0

0.0

 

 

2400.0PS

124.5

2.5

2.0

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

Positive control3#

31.9

40.0

39.0S

12.0

 

 

783.7PS

106.1

2.5

1.5

0.0

 

 

1371.4PS

105.2

1.0

1.0

0.0

 

 

2400.0PS

94.8

2.0

1.5

0.0

*   Including cells carrying exchanges

#    Evaluation of 50 metaphases per culture

##  Evaluation of 200 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone         0.5 % (v/v)

2     EMS          770.0 µg/mL

3     EMS          660.0 µg/mL


Table 2, cont.  Summary of results of the chromosomal aberration study with           
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics         

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

Positive control2

31.6

9.5

9.5S

1.5

 

 

47.7

72.6

1.0

1.0

0.0

 

 

783.7PS

77.7

3.0

3.0S

0.0

 

 

1371.4PS

88.9

0.0

0.0

0.0

 

 

2400.0PS

95.2

1.5

1.5

0.0

II

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control3

35.5

18.0

18.0S

3.0

 

 

27.3

107.6

1.0

0.5

0.0

 

 

47.7##

112.7

4.3

3.8

0.3

 

 

83.6PS

104.2

3.0

2.5

0.0

 

 

783.7PS

110.9

1.0

1.0

0.0

 

 

1371.4PS

116.4

1.5

1.5

0.0

 

 

2400.0PS

113.6

2.5

1.5

0.5

*   Including cells carrying exchanges

#    Evaluation of 50 metaphases per culture

##  Evaluation of 200 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone         0.5 % (v/v)

2    CPA               2.5 µg/mL

3    CPA               7.5 µg/mL

Conclusions:
A chromosome aberration study was performed for Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics according to OECD 473 and GLP. In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration (2400 μg/mL). Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of metabolic activation, one single statistically significant increase was observed after treatment with 783.7 μg/mL (3.0 % aberrant cells, excluding gaps). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of metabolic activation, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 μg/mL. Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant. 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 in cells with structural chromosome aberrations.
Executive summary:

The test item Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2400.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data inaccordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, one single statistically significant increase was observed after treatment with 783.7 µg/mL (3.0 % aberrant cells, excluding gaps). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of S9 mix, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 µg/mL. Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant.

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 in cells with structural chromosome aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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 source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation 0.6 ug/mL; with metabolic activation 5000 ug/mL
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 with and without metabolic activation

Exposure to test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane is not considered to be mutagenic in this test system.
Executive summary:

This data is being read across from the source study that tested Isododecane based on analogue read across.

Exposure to test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane is not considered to be mutagenic in this test system.

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.
Key result
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

Read across genetic toxicity test listed below had negative results for Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics.

 

Genetic Toxicity in vivo – micronucleus assay (OECD 474)

 

Genetic Toxicity in vivo – Rodent Dominant Lethal Test (OECD TG 478)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990/10/24 - 1990/11/30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to OECD Guideline 474. 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 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Source: Charles River Breeding Laboratories, Inc.
Sex: Male (65), Female (65)
Age at study initiation: Approximately 9-10 weeks
Weight at study initiation: 23-39g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent 5002 chow (pellets), ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
Temperature (°F): 68-76
Humidity (%): 40-70%
Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
Corn oil was used. Dose volume did not exceed 1.0 ml/100 g bw.
Details on exposure:
The test material and the carrier were administered by oral gavage as a single dose. The carrier was dosed at a volume equal to the test material dose volume. The individual animal dose volumes did not exceed 1.0 ml/100 g body weight; animals were administered 1.0, 2.5, 5.0 g test material/ kg body weight. The positive control, cyclophosphamide was administered as a single dose of 40 mg/kg using water as a carrier.
Duration of treatment / exposure:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
Frequency of treatment:
One dose was given at either 1.0, 2.5, 5.0 g test material/ kg body weight. Cyclophosphamide was dosed at 40 mg/kg.
Post exposure period:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
No. of animals per sex per dose:
Male (65), Female (65) ; 5 Males and 5 Females per treatment group
Positive control(s):
The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.
Tissues and cell types examined:
Erythrocytes derived from femur bone marrow.
Details of tissue and slide preparation:
Immediately following the sacrifice of the animals, both femurs were removed and the bone marrow was removed and suspended in fetal bovine serum. After the suspension was centrifuged the pellet was resuspended and smears were prepared (two slides per animal).
Evaluation criteria:
Slides were stained using acridine orange; polychromatic erythrocytes (PCE) stained red/orange, nonchromatic erythrocytes (NCE) are unstained (dull green), and micronuclei stain bright yellow. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell’s diameter. 1000 PCE from each animal were examined for the presence of micronuclei and the ratio of PCE to NCE was determined for each animal by counting 1000 erythrocytes (PCE and NCE).
Statistics:
Calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period (ANOVA). When ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test.

A standard regression analysis was performed to test for a dose response.
Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance). Therefore nonparametric analysis was not performed.

Sexes were analyzed separately.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The positive control (cyclophosphamide) induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes, indicating that the positive control was clastogenic and was responding in an appropriate manner. Carrier control values for the mean percent of polychromatic erythrocytes and for the mean number of micronucleated polychromatic erythrocytes are within the normal range for the corn oil control. MRD-90-874 did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes which is a measure of bone marrow toxicity. MRD-90-874 did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. MRD-90-874 did not induce a significant increase in the mean number of micronucleated polychromatic erythrocytes. MRD-90-874 was not cytotoxic at doses up to 5.0 g/kg to mouse bone marrow under the conditions of this test.
Conclusions:
Interpretation of results: negative
These data indicate that MRD-90-874 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight.
Executive summary:

The test material, MRD-90-874 was tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  MRD-90-874 was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes.  Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner.  These data indicate that MRD-90-874 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 474: 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 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
According to US EPA Guideline 84-2
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: ca. 8-9 weeks
- Weight at study initiation: 21-40 grams
- Assigned to test groups randomly: [no/yes, under following basis: computer generated, body weight sorting program
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 28 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): not to exceed 1ml/100 grams bw
- Purity: assumed to be 100% pure
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighted out and on the day of dosing, mixed with the carrier to provide stock solutions such that individual animal dose volumes did not exceed 1ml/100grams body weight. The mice were administered 1.25, 2.5, or 5.0 grams of test material/kg of body weight. Corn oil served as the carrier for the test material and was dosed at the same volume as the test material.

Duration of treatment / exposure:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing.
Positive control animals were sacrificed 24 hours after injection
Frequency of treatment:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing
Post exposure period:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing
Remarks:
Doses / Concentrations:
5.0 g/kg/bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.5g/kg/bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1.25 g/kg/bw
Basis:
nominal conc.
No. of animals per sex per dose:
30 animals (5 male; 5 female)/dose; 10/timepoint
Positive control(s):
cyclophosphamide;

- Route of administration: intraperitoneal injection
- Doses / concentrations:40 mg/kg using water as the carrier
Tissues and cell types examined:
Bone marrows were collected and extracted, smear preparations made and stained. Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored for each animal.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range finding study was performed using 5.0, 2.5, and 1.0 gram of test material per kg of body weight. Two males and two females were used for each dose group. All animals survived and were sacrificed 24 hours after dosing. bome marrow was removed and slides were prepared. Slides were evaluated for percent of polychromatic erythrocytes in 1000 erythrocytes and for number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes.




DETAILS OF SLIDE PREPARATION: After sacrifice, both femurs were removed. The bone marrow was then removed and suspended in fetal bovine serum. After the suspension was centrifuged, the pellet was resuspended and smears were prepared (two slides per animal). Slides were labeled with blind coding. Slides were stained using acridine orange. 1000 polychromatic erythrocytes from each animal were examined for the presence of micronuclei, and the ratio of PCE’s to NCE’s determined


METHOD OF ANALYSIS: staining color, and circular appearance and a diameter between 1/20 and 1/5 of the cell's diameter


Statistics:
Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test. A standard regression analysis was performed to test for a dose response. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance) in more than 25% of the analyses. Therefore nonparametric analysis were not performed. Sexes were analyzed separately
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No deaths or clinical signs of toxicity were observed in animals dosed with the test material up to the maximum recommended dose of 5g/kg
Conclusions:
Interpretation of results: negative
The in vivo micronucleus assay of MRD-89-582 in mice was negative. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.
Executive summary:

MRD-89-582 was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively.  Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing.  MRD-89-582 did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations guidelines.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978
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)

- Housing: males were house individiually during the treatment period and housed 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 Corp, Miran IA Ambient Air Analyzer (long path infrared)
- Samples taken from breathing zone: no
Duration of treatment / exposure:
MRD-77-44 was administered by vapor inhalation for six hours/day for five consecutive days.
Negative control (chamber air) was administered 6 hours/day for five consecutive days
Triethylenemelamine was administered by intraperitoneal injection (normal saline) as a single dose.
Frequency of treatment:
MRD-77-44 was administered by vapor inhalation for six hours/day for five consecutive days.
Negative control (chamber air) was administered 6 hours/day for five consecutive 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-77-44: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
900ppm MRD-77-44: 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.
Key result
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-77-44 is not mutagenic by the dominant lethal test. This finding does not warrant classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.
Executive summary:

In a dominant lethal assay, MRD-77-44 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-77-44 produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resorptions were unaffected by MRD-77-44 exposure.  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-77-44 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-77-44 as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.

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.
Key result
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 are no data available for Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics. However, in vitro and in vivo data are available for structural analogues Hydrocarbons, C10-C12, isoalkanes, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, <2% aromatics; Hydrocarbons, C10 -C13, isoalkanes, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics; isododecane; Hydrocarbons; C12 -C15, n-alkanes, isoalkanes, <2% aromatics; Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics; Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics; Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics; and isohexadecane. These data are read across to Hydrocarbons, C11-C16, n-alkanes, isoalkanes, <2% aromatics 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

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

In a key Guideline OECD 471 bacterial reverse mutation test (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 mutagenic activity. The test to assess the genotoxicity of the test material was negative.

In another key Guideline OECD 471 study (Chevron Phillips, 1982), 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 test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) mutagenic activity.

Hydrocarbons, C10-C13, n-alkanes, <2% aromatics

In a key bacterial reverse mutation test (Cepsa Quimica, 1985), the test material (Hydrocarbons, C10-C13, n-alkanes, <2% aromatics) was examined for mutagenic activity using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 1538, 98, and 100 in the absence and presence of a liver S9 fraction for metabolic activation. The test was performed in triplicate using doses of 0, 50, 150, 500, 1500, 5000 ug/plate. In all cases, the test material did not induce any significant changes in the number of revertant colonies. It is concluded in this study that hydrocarbons, C10-C13, n-alkanes, <2% aromatics is not a mutagenic agent and classification under EU CLP/GHS guidelines is not required.

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a study conducted according to OECD TG 471 and in compliance with GLP (WIL Research, 2014c). No evidence of substance induced increase in the frequency of revertants was noted in the presence or absence of metabolic activation inSalmonella typhimuriumstrains TA1535, TA1537, TA98 andEscherichia coliWP2 uvrA when tested up to cytotoxic concentrations. Similar results were obtained in two independent experiments using plate incorporation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key bacterial reverse mutation test (Shell, 1998), the test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics) was examined for mutagenic activity using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98 and 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver S9 fraction for metabolic activation. Two tests were performed: Test #1 (8, 40, 200, 1000, 5000 ug/plate), Test #2 (1000, 2000, 3000, 4000, 5000 ug/plate).  The material was not cytotoxic. In all cases, the test material did not induce any significant changes in the number of revertant colonies, with or without metabolic activation. It is concluded in this study that hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics is not a mutagenic agent. 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).

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a study conducted according to OECD TG 471 and in compliance with GLP (WIL Research, 2014a). No evidence of substance induced increase in the frequency of revertants was noted in the presence or absence of metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and Escherichia coli WP2 uvrA when tested up to limit concentrations. Similar results were obtained in two independent experiments using plate incorporation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a study conducted according to OECD TG 471 and in compliance with GLP (WIL Research BV, 2014b). No evidence of substance induced increase in the frequency of revertants was noted in the presence or absence of metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and Escherichia coli WP2 uvrA when tested up to limit concentrations. Similar results were obtained in two independent experiments using plate incorporation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a study conducted according to OECD TG 471 and in compliance with GLP (WIL Research, 2014b). No evidence of substance induced increase in the frequency of revertants was noted in the presence or absence of metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and Escherichia coli WP2 uvrA when tested up to limit concentrations. Similar results were obtained in two independent experiments using plate incorporation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Hydrocarbons, C14 -C16, n-alkanes, <2% aromatics

The mutagenic potential of the test material was assessed in the Salmonella typhimurium microsomal assay according to the Ames test in compliance with Good Laboratory Practice (Petroquimica, 1985). The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 mutants were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 72 hours of incubation at 37°C, the revertant colonies were scored. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The test substance was then tested in another experiment performed in the same way as the range-finding test. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test). Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Under the conditions of this study, the test material did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Isohexadecane

In a supporting reverse gene mutation assay in bacteria (EC Erdolchemie, 1990), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to isohexadecane at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate in the presence and absence of mammalian metabolic activation. No cytotoxicity was observed with all the dose tested. Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used (+/- S9). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, isohexadecane did not induce in vitro mutagenic activity in the bacterial test system in the presence and the absence of S9 activation system.

In Vitro Chromosome Aberration in Mammalian Cells

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

In a key in vitro Sister Chromatid Exchange Assay in Mammalian Cells (Chevron Phillips, 1983), 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 test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) mutagenic activity.

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics has been tested for potential for cytogenicity in a chromosome aberration study conducted according to OECD TG 473 and in compliance with GLP (Harlan, 2014). No evidence of a test substance induced increase in the number of cells with aberrations was observed when tested in peripheral human lymphocytes in the presence or absence of metabolic activation up to limit concentrations. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for clastogenicity under the conditions of the test.

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key 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 included 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. This finding does not warrant the classification of this test material as a genotoxin under Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics has been tested for potential for cytogenicity in a chromosome aberration study conducted according to OECD TG 473 and in compliance with GLP in two independent experiments (Harlan, 2014b). No evidence of a test substance induced increase in the number of cells with aberrations was observed when tested in peripheral human lymphocytes in the presence or absence of metabolic activation up to limit concentrations. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for clastogenicity under the conditions of the test.

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics

In an in vitro chromosome aberration test (ExxonMobil, 1991), Chinese Hamster Ovary cells were exposed to the test material at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation. Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, the test material is not classified according to the criteria of the CLP Regulation (1272/2008).

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics has been tested for potential for cytogenicity in a chromosome aberration study conducted according to OECD TG 473 and in compliance with GLP in two independent experiments (Harlan, 2014c). The study was carried out using human lymphocytes in the presence of metabolic activation (four hours exposure) and in the absence of metabolic activation (four and twenty two hours (continuous) exposure). After four hours exposure in the absence of metabolic activation in the first test, significant increases in chromosomal aberrations were observed at the lowest and median concentrations (out of five concentrations tested). The increase at the lowest concentration was within the range of historical control data, and therefore considered to be biologically irrelevant. The increase at the median concentration exceeded the range of historical control data. In the absence of dose dependency and as the findings were not observed when the experiment was repeated using the same exposure times, nor with continuous exposure in the absence of metabolic activation, it is concluded that this increase did not represent a potential for cytogenicity. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for clastogenicity under the conditions of the test.

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics has been tested for potential for cytogenicity in a chromosome aberration study conducted according to OECD TG 473 and in compliance with GLP in two independent experiments (Harlan, 2014d). No biologically relevant evidence of a test substance induced increase in the number of cells with aberrations was observed when tested in peripheral human lymphocytes in the presence or absence of metabolic activation up to cytotoxic or limit concentrations. A single statistically significant increase in the number of cells with aberrations was observed in the first experiment, which was within the range of historical controls so not considered biologically relevant. An increase above the level of historical controls was observed at one concentration in experiment 2, but this was neither statistically significant nor dose-dependent so not considered biologically relevant. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for clastogenicity under the conditions of the test.

In vitro Gene Mutation study in Mammalian Cells

C10-C13 isoalkanes

In a key mammalian cell gene mutation assay (Chevron Phillips, 1982), exposure to eight graded doses of the test material (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.

Isododecane

In a key mammalian cell gene mutation assay (INEOS, 1996), exposure to the test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane was not considered to be mutagenic in this test system.

In Vivo

Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key dominant lethal assay (ExxonMobil Corp, 1978), the test material 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, the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resorptions were unaffected by test material exposure. Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses. Based on these data, the test material when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test. This finding does not warrant the classification as a genotoxin under EU GHS guidelines.

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

In a key in vivo dominant lethal assay (ExxonMobil Corp., 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.  Based on these data, the test material when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test. 

Hydrocarbons, C10-C13, n-alkanes, <2% aromatics

In a key in vivo study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics) was tested in the mammalian bone marrow micronucleus assay using CD-1 mice. The test material was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes. Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner. These data indicate that Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/Kg.

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key in vivo study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics) was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/Kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively. Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing. The test material did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group. The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the classification of Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics as a genotoxin under EU GHS guidelines.

n-decane and n-dodecane

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 observed in in vitro and in vivo genotoxicity assays do not warrant the classification of Hydrocarbons, C11-C16 n-alkanes, isoalkanes, <2% aromatics as a genotoxin according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).