Hydrocarbons, C15-C19, n-alkanes, isoalkanes,

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in bacterial mutagenicity assay (OECD TG 471).

Negative in chromosome aberration assay (OECD TG 473).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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.

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 control^1	100.0	2.5	2.5	0.5
		Positive control^2	120.8	10.0	10.0^S	2.0
		83.6	102.9	2.0	2.0	0.0
		783.7^PS	88.2	2.5	2.5	0.0
		1371.4^PS	91.8	3.0	3.0	0.0
		2400.0^PS	124.5	2.5	2.0	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^3#	31.9	40.0	39.0^S	12.0
		783.7^PS	106.1	2.5	1.5	0.0
		1371.4^PS	105.2	1.0	1.0	0.0
		2400.0^PS	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 control^1	100.0	0.5	0.5	0.0
		Positive control^2	31.6	9.5	9.5^S	1.5
		47.7	72.6	1.0	1.0	0.0
		783.7^PS	77.7	3.0	3.0^S	0.0
		1371.4^PS	88.9	0.0	0.0	0.0
		2400.0^PS	95.2	1.5	1.5	0.0
II	22 hrs	Solvent control^1	100.0	1.5	1.5	0.0
		Positive control^3	35.5	18.0	18.0^S	3.0
		27.3	107.6	1.0	0.5	0.0
		47.7^##	112.7	4.3	3.8	0.3
		83.6^PS	104.2	3.0	2.5	0.0
		783.7^PS	110.9	1.0	1.0	0.0
		1371.4^PS	116.4	1.5	1.5	0.0
		2400.0^PS	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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-Aug-2014 to 25-Aug-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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:

S. typhimurium TA 1537

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:

S. typhimurium TA 98

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:

S. typhimurium 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

1. SOFTWARE including version number
a) OECD QSAR Toolbox v4.0
c) VEGA 1.1.4
d) Danish QSAR Database, including the SAR models SciQSAR, LeadScope and CASE Ultra.

2. MODEL (incl. version number) - see above

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODELS
OECD QSAR Toolbox - smiles
VEGA - smiles
Danish QSAR database - CAS n umber, therefore substances without CAS numbers could not be used as input in the Danish QSAR toolbox.

Identifiers
2,6,10,10-tetramethylpentadecane C19, CCCCCC(C)(C)CCCC(C)CCCC(C)C, 1921-70-6
Eicosane C20, CCCCCCCCCCCCCCCCCCCC, 112-95-8
3,8,13-trimethylheptadecane C20, C(C)(CCCCC(C)CCCCC(C)CCCC)CC, No CAS number
Pentacosane C25, CCCCCCCCCCCCCCCCCCCCCCCCC, 629-99-2
3,10,18-trimethylpentacosane C28, C(C)(CCCCCCC(C)CCCCCCCC(C)CCCCCCC)CC, No CAS number
Triacontane C30, CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC, 638-68-6
3,13,23-trimethyltriacontane C33, C(C)(CCCCCCCCCC(C)CCCCCCCCCC(C)CCCCCCC)CC, No CAS number
n-pentatriacontane C35, CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC, 630-07-9
3,14,26-trimethylpentatriacontane C38, C(C)(CCCCCCCCCCC(C)CCCCCCCCCCCC(C)CCCCCCCCC)CC, No CAS number

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint:
Read-across using OECD QSAR Toolbox:
• Bacterial Reverse Mutation Assay (e.g. AMES test)
• In vitro Mammalian chromosome Aberration Test
• Mammalian Cell Gene Mutation Assay
• In vivo Micronucleus assay
VEGA:
• CAESAR model
• ISS model
• SarPy model
• KNN model
• Mutagenicity (AMES test) CONSENSUS model 1.0.2
Danish QSAR database:
In vitro Genotoxicity Endpoints
• Ashby Structural Alerts for DNA Reactivity
• Bacterial Reverse Mutation Test (Ames test) in S. typhimurium (in vitro): Direct Acting Mutagens (without S9); Base-Pair Ames Mutagens; Frameshift Ames Mutagens; Potent Ames Mutagens, Reversions ≥ 10 Times Controls
• Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells
• Chromosome Aberrations in Chinese Hamster Lung (CHL) Cells
• Mutations in Thymidine Kinase Locus in Mouse Lymphoma Cells
• Mutations in HGPRT Locus in Chinese Hamster Ovary (CHO) Cells
• Unscheduled DNA Synthesis (UDS) in Rat Hepatocytes
• Syrian Hamster Embryo (SHE) Cell Transformation
In vivo Genotoxicity Endpoints
• Sex-Linked Recessive Lethal (SLRL) Test in Drosophila m.
• Micronucleus Test in Mouse Erythrocytes

• Dominant Lethal Mutations in Rodents
• Sister Chromatid Exchange in Mouse Bone Marrow Cells• Comet Assay in Mouse
Structural alerts OECD QSAR Toolbox
• DNA Binding by OASIS 1.4
• DNA Binding by OECD
• Protein binding by OASIS v1.4
• Protein binding by OECD
• DNA alerts for AMES by OASIS v1.4
• DNA alerts for CA and MNT by OASIS v1.4
• In vitro mutagenicity (AMES test) alerts by ISS
• In vivo mutagenicity (Micronucleus test) alerts by ISS
• Protein binding alerts for chromosomal aberration by OASIS v1.2

- Unambiguous algorithm: The Tools used are accepted and validated for genetic toxicity prediction
- Defined domain of applicability:
OECD QSAR Toolbox: not applicable
Danish QSAR database: only predictions within the applicability domain are searched and displayed
VEGA: defined domain of applicability is not available to the reviewer, but the predictions are rated as good if the substance falls within the domain of applicability.
- Appropriate measures of goodness-of-fit and robustness and predictivity:
The results of the predictions were in good agreement
- Mechanistic interpretation:
The structural alerts on which the predictions are based are known to be associated with genetic toxicity

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain:
OECD QSAR Toolbox: read-across predictions were based on substances with similar structures.
Danish QSAR toolbox: All of the structures are within the applicability domains of all endpoints for at least one of the tools used, and within the domains of all tools for at least one endpoint.
VEGA: All the substances fall within the applicability domain of the tool.
- Structural and mechanistic domains: The structures assessed fall within the structural domain of the tools used. The mechanisms covered by the tools provide a wide coverage of the
- Similarity with analogues in the training set: The substances are in the same homologous series as analogues identified in the tools used.

6. ADEQUACY OF THE RESULT
The results of the QSAR tools are in agreement with the conclusion from existing measurements on the substance or close analogues that the substance is not genotoxic.

Reason / purpose for cross-reference:

read-across: supporting information

Principles of method if other than guideline:

Read-across was performed implementing a category approach using the OECD QSAR Toolbox v4.0.
QSAR analyses for mutagenicity were performed using two tools – VEGA 1.1.4 and Danish QSAR Database.
Mutagenicity-relevant profilers were applied using OECD QSAR Toolbox v4.0 to identify any structural alerts for DNA binding, protein binding and genotoxicity (including chromosomal aberration and mutagenicity).

GLP compliance:

no

Type of assay:

other: Genetic toxicity QSAR

Specific details on test material used for the study:

2,6,10,10-tetramethylpentadecane C19, CAS 1921-70-6
Eicosane C20, CAS 112-95-8
3,8,13-trimethylheptadecane C20
Pentacosane C25, CSA 629-99-2
3,10,18-trimethylpentacosane C28
Triacontane C30, CAS 638-68-6
3,13,23-trimethyltriacontane C33
n-pentatriacontane C35, CAS 630-07-9
3,14,26-trimethylpentatriacontane C38

Details on test system and experimental conditions:

a) Read-Across predictions were performed implementing a category approach using the OECD QSAR Toolbox v4.0. The category formation was based on structural similarity and chemical functional groups to identify the most relevant structural analogues and assign a final prediction to the target chemical for the following four models:
• Bacterial Reverse Mutation Assay (e.g. AMES test)
• In vitro Mammalian chromosome Aberration Test
• Mammalian Cell Gene Mutation Assay
• In vivo Micronucleus assay
b) QSAR predictions for mutagenicity were performed using two tools – VEGA 1.1.4 and Danish QSAR Database.
VEGA 1.1.4 provides mutagenicity prediction from the following four different models and also provides a final consensus prediction. For each of the above-listed models, VEGA hints the reliability in prediction to inform the user if the predicted substance falls within the model’s applicability domain or not.
• CAESAR model
• ISS model
• SarPy model
• KNN model
• Mutagenicity (AMES test) CONSENSUS model 1.0.2
The Danish QSAR database is able to predict several in vitro and in vivo genotoxicity endpoints for a chemical substance with a valid CAS number. For each endpoint, the predictions are made using a battery of three QSAR prediction models – SciQSAR, LeadScope and CASE Ultra. For each prediction coming from the three models as well as the battery model, reliability in prediction is hinted by informing the user if the substance was inside or outside the model’s applicability domain, otherwise the prediction is considered as inconclusive. 3 QSAR models (SciQSAR, LeadScope and CASE Ultra) for the following 17 genotoxicity endpoints were applied on each alkane:

In vitro Genotoxicity Endpoints
• Ashby Structural Alerts for DNA Reactivity
• Bacterial Reverse Mutation Test (Ames test) in S. typhimurium (in vitro): Direct Acting Mutagens (without S9); Base-Pair Ames Mutagens; Frameshift Ames Mutagens; Potent Ames Mutagens, Reversions ≥ 10 Times Controls
• Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells
• Chromosome Aberrations in Chinese Hamster Lung (CHL) Cells
• Mutations in Thymidine Kinase Locus in Mouse Lymphoma Cells
• Mutations in HGPRT Locus in Chinese Hamster Ovary (CHO) Cells
• Unscheduled DNA Synthesis (UDS) in Rat Hepatocytes
• Syrian Hamster Embryo (SHE) Cell Transformation

In vivo Genotoxicity Endpoints
• Sex-Linked Recessive Lethal (SLRL) Test in Drosophila m.
• Micronucleus Test in Mouse Erythrocytes
• Dominant Lethal Mutations in Rodents
• Sister Chromatid Exchange in Mouse Bone Marrow Cells
• Comet Assay in Mouse
c) Structural alerts:
Following mutagenicity-relevant profilers were applied using OECD QSAR Toolbox v4.0 to identify if any structural alerts for DNA binding, protein binding and genotoxicity (including chromosomal aberration and mutagenicity) were triggered for any of the nine alkanes.
• DNA Binding by OASIS 1.4
• DNA Binding by OECD
• Protein binding by OASIS v1.4
• Protein binding by OECD
• DNA alerts for AMES by OASIS v1.4
• DNA alerts for CA and MNT by OASIS v1.4
• In vitro mutagenicity (AMES test) alerts by ISS
• In vivo mutagenicity (Micronucleus test) alerts by ISS
• Protein binding alerts for chromosomal aberration by OASIS v1.2

Key result

Species / strain:

mammalian cell line, other: CHO, CHL, L5178Y, rat hepatocytes, SHE cells

Remarks:

Predictions were made for a number of mammalian cell lines

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Profiling results:

All of the alkanes were screened through nine genotoxicity-relevant profilers (listed in Figure 2) incorporated into the OECD QSAR Toolbox v4.0 to identify the DNA and protein binding potential of the alkanes and if any structural alerts for genotoxicity were triggered. This profiling exercise was the very first step in this work before any read-across or QSAR predictions were derived for all the nine alkanes. In the author's opinion, profiling can be considered as an initial screening approach to get a general idea about the genotoxicity potential of the alkanes on the basis of the triggered structural alerts.

None of the nine alkanes were associated with DNA and protein binding. Moreover, none of them were associated with any structural alerts for genotoxicity (including chromosomal aberration, in vitro/in vivo mutagenicity).

Read-across results using OECD QSAR Toolbox v4.0

Read-across predictions for the four models (Bacterial Reverse Mutation Assay (e.g. AMES test); In vitro Mammalian chromosome Aberration Test; Mammalian Cell Gene Mutation Assay; In vivo Micronucleus assay) were assigned for all the nine alkanes using a category approach. The approach was repeated in the same manner for all the alkanes and involved the following key steps:

a) All the chemicals from all databases and inventories incorporated into the OECD QSAR Toolbox v4.0 showing a reasonable level of structural similarity to the target alkane.

b) The retained dataset was then sub-categorised based on the chemical elements and functional groups.

c) Substances with no existing experimental data for genotoxicity endpoints were also excluded.

d) The remaining substances in the category were finally used to assign a read-across prediction.

Results for Mutagenicity predictions using VEGA 1.1.4

Four QSAR models incorporated into VEGA 1.1.4 to predict mutagenicity potential of the substances were applied on the nine alkanes. The battery prediction from these four models was also included and was considered as the key prediction from VEGA. None of the alkanes were predicted as mutagens.

Results for Mutagenicity predictions using Danish QSAR Database

3 QSAR models (SciQSAR, LeadScope and CASE Ultra) corresponding to 17 genotoxicity endpoints were applied on all the alkanes with a valid CAS number (5 alkanes in this case). Of these 17 endpoints, 1 corresponds to the Ashby structural alerts, 5 endpoints corresponds to the AMES test results, 6 endpoints covers other in vitro genotoxicity models and finally the last 5 endpoints covers the in vivo genotoxicity endpoints. None of the final predictions derived using the battery approach were positive for any of the genotoxic endpoints for any of the five alkanes.

Conclusions:

Representative constituents of GTL substances obtained by the Fisher-Tropsch process have been assessed for genetic toxicity using QSAR tools. The constituents assessed were C20, C25, C30 and C35 linear alkanes, and C19, C20, C33 and C38 methyl-branched alkanes with three branches (C20, C33 and C38) or four branches (C19). Based on the read-across analysis from OECD QSAR Toolbox v4.0, QSAR analyses from VEGA 1.1.4 and Danish QSAR Database and the OECD QSAR Toolbox profiling for structural alerts, it can be concluded that all the nine alkanes representative of GTL constituents in the C19-C38 range assessed are non-genotoxic and non-mutagenic.

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, C15-C19, 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

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

In Vitro

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, 2014c). 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, TA100 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 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 Cytotest, 2014). 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.

There are no in vitro or in vivo mammalian mutagenicity toxicity studies for Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics. Representative constituents of GTL substances obtained by the Fischer-Tropsch process have been assessed for genetic toxicity, including mammalian genotoxicity, using QSAR tools (KREATiS, 2017). The constituents assessed were C20, C25, C30 and C35 linear alkanes, and C19, C20, C33 and C38 methyl-branched alkanes with three branches (C20, C33 and C38) or four branches (C19). Based on the read-across analysis from OECD QSAR Toolbox v4.0, QSAR analyses from VEGA 1.1.4 and Danish QSAR Database and the OECD QSAR Toolbox profiling for structural alerts, it can be concluded that all the nine alkanes representative of GTL constituents in the C19-C38 range assessed are non-genotoxic and non-mutagenic.

In Vivo

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.

Justification for classification or non-classification

All in vitro and in vivo tests on the registration substance and analogues were negative; Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics is considered not to be mutagenic and should not be classified for mutagenicity according to the criteria of Annex VI of Regulation (EC) No 1272/2008.