Hydrocarbons, C18-C24, iso-alkanes <2% aromatics

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains (TA 98, 100, 1535, 1537) and Escherichia coli WP2uvrA (OECD 471).
Cytogenicity in mammalian cells: negative with and without activation in human lymphocytes (OECD Draft Guideline 487). Negative with and without activation in human lymphocytes (OECD 473).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-21 to 2015-01-12

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: 19.2, 33.7, 58.9, 103.1, 180.3, 315.6, 552.3, 966.5, 1691.4, 2960.0 µg/mL
Experiment IIA: 19.2, 33.7, 58.9, 103.1, 180.3, 315.6, 552.3, 966.5, 1691.4, 2960.0 µg/mL

Without metabolic activation:
Experiment I: 19.2, 33.7, 58.9, 103.1, 180.3, 315.6, 552.3, 966.5, 1691.4, 2960.0 µg/mL
Experiment IIA: 19.2, 33.7, 58.9, 103.1, 180.3, 315.6, 552.3, 966.5, 1691.4, 2960.0 µg/mL
Experiment IIB: 422.5, 845.0, 1267.5, 1690.0, 2535.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- 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 I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB the exposure period was 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 IIA, 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).

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 applicable

Positive controls validity:

valid

Additional information on results:

The test item Hydrocarbons, C18-C24, isoalkanes, <2% aromatics, dissolved in THF, 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 I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB the exposure period was 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 IIA, 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, 2960.0 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 in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix at 103.1 µg/mL and above, in Experiment IIA in the absence of S9 mix at 58.9 µg/mL and above and in Experiment IIB in the absence of S9 mix at 422.5 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 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 (1.0 – 2.5 % aberrant cells, excluding gaps) were within the range of the solvent control values (0.5 – 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data (see Appendix 2).
In Experiment I in the absence of S9 mix, one single increase in chromosomal aberrations, above the laboratory historical control data range (0.0 – 3.0 % aberrant cells, excluding gaps), was observed after treatment with 1691.4 µg/mL (4.5 % aberrant cells, excluding gaps). The value is not statistically significant and no dose-dependency was observed (Table 7). In Experiment IIA in the absence of S9 mix after treatment with 1691.4 µg/mL one single statistically significant increase in chromosomal aberrations (3.8 % aberrant cells, excluding gaps), above the laboratory historical control data range (0.0 – 2.5 % aberrant cells, excluding gaps), was observed. No dose-dependency was observed in this experimental part (Table 10). In Experiment IIB in the absence of S9 mix this finding could not be confirmed (Table 13).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Either EMS (550.0, 660.0 or 770.0 µg/mL) or CPA (7.5 or 15.0 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, C18-C24, 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	2.0	0.5
		Positive control^2	101.3	11.0	10.5^S	1.5
		58.9	93.0	3.0	2.0	0.5
		966.5^PS	89.9	1.5	1.5	0.0
		1691.4^PS#	88.0	4.8	4.5	0.0
		2960.0^PS	92.4	2.5	2.0	0.0
Exposure period 22 hrs without S9 mix
IIA	22 hrs	Solvent control^1	100.0	1.5	1.0	0.0
		Positive control^3##	35.7	40.0	39.0^S	12.0
		33.7	106.1	2.0	2.0	0.0
		966.5^PS	103.1	1.0	1.0	0.0
		1691.4^PS#	95.2	5.0	3.8^S	0.5
		2960.0^PS	116.0	0.0	0.0	0.0
IIB	22 hrs	Solvent control^1	100.0	1.0	1.0	0.0
		Positive control^4	32.3	18.5	18.5^S	2.5
		1267.5^PS	91.5	2.0	2.0	0.0
		1690.0^PS	94.9	2.5	2.5	0.0
		2535.0^PS	100.9	2.5	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    THF             0.5 % (v/v)

²     EMS       770.0 µg/mL

³     EMS       660.0 µg/mL

⁴     EMS       550.0 µg/mL

Table 2, cont.  Summary of results of the chromosomal aberration study with
Hydrocarbons, C18-C24, 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	3.5	2.5	0.0
		Positive control^2	44.3	19.0	15.5^S	1.5
		58.9	83.2	2.5	2.5	0.0
		966.5^PS	85.5	2.0	2.0	0.5
		1691.4^PS	90.8	1.0	1.0	0.0
		2960.0^PS	71.8	3.0	2.5	0.0
IIA	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^3	38.2	18.0	18.0^S	3.0
		58.9	93.5	2.0	2.0	0.5
		966.5^PS	86.9	2.0	1.0	0.0
		1691.4^PS	93.5	1.5	1.5	0.0
		2960.0^PS	106.5	1.0	1.0	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    THF             0.5 % (v/v)

²    CPA          15.0 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, C18-C24, 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, C18-C24, isoalkanes, <2% aromatics, dissolved in THF, 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. I	Exp. IIA & IIB	Exp. I & IIA
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 IIA, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2960.0 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 section 3.5.1. The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.

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

In the presence of S9 mix no relevant increase in chromosomal aberrations was observed. In Experiment I in the absence of S9 mix, one single increase in chromosomal aberrations, above the laboratory historical control data range (0.0 – 3.0 % aberrant cells, excluding gaps), was observed after treatment with 1691.4 µg/mL (4.5 % aberrant cells, excluding gaps). The value is not statistically significant and no dose-dependency was observed. In Experiment IIA in the absence of S9 mix after treatment with 1691.4 µg/mL one single statistically significant increase in chromosomal aberrations (3.8 % aberrant cells, excluding gaps), above the laboratory historical control data range (0.0 – 2.5 % aberrant cells, excluding gaps), was observed. No dose-dependency was observed in this experimental part. In Experiment IIB in the absence of S9 mix this finding could not be confirmed.

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

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-Aug-2014 to 28-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.

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9:10 µg/plate in DMSO for WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9:5 µg/plate in saline for TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative 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.

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed (observed as droplets).
Dose range finding test: at concentrations of 164 µg/plate and upwards.
Mutation experiment: at concentrations of 512 µg/plate and upwards.

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 with and without metabolic activation

An Ames test was performed with Hydrocarbons, C18-C24, 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, C18-C24, 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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo:
Cytogenicity: negative in Mammalian Bone Marrow Chromosome Aberration Test (OECD 474/EU B.11)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The experimental phases of the study were performed between 26 January 2011 and 14 March 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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. Read-across is considered to be reliability 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient Wistar HanTM (RCCHanTMWIST) rats were supplied by a reputable supplier. For full details please see full study report. At the start of the test the rats weighed 186 to 229 g and were approximately seven to twelve weeks old. After a minimum acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding (Supplied by a reputable supplier. For full details please see full study report). Free access to mains drinking water and food, Harlan Teklad 2014 Rodent Pelleted Diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK, was allowed throughout the study. The animals were also provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Supplied by a reputable supplier. For full details please seefull study report).

The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

The vehicle control was supplied by a reputable supplier as follows:
Supplier's identification: Arachis oil
Harlan serial number: V-4855
Date received: 14 June 2010
Description: Straw-coloured slightly viscous liquid
Expiry date: 31 January 2012
Storage conditions: Room temperature

Details on exposure:

Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours. In addition, two further groups of rats were included in the study; one group of seven rats was dosed via the oral route with the vehicle alone (arachis oil) and a second group of five rats was dosed orally with Cyclophosphamide.
Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test. The vehicle control group and positive control group were killed approximately 24 hours following treatment.
All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later. The experimental design is summarised as follows:

Treatment Group Dose Level(mg/kg) Concentration (mg/ml) Dose Volume (ml/kg) Kill Time (Hours After Dosing) Animal Numbers
1. Vehicle Control (Arachis oil) 0 0 10 24 1 – 7
2. Positive Control (Cyclophosphamide) 25 2.5 10 24 8 – 12
3. Test item 2000 200 10 48 13 – 19
4. Test item 2000 200 10 24 20 – 26
5. Test item 1000 100 10 24 27 – 33
6. Test item 500 50 10 24 34 – 40

Frequency of treatment:

Groups of rats were dosed once only via the oral route

Post exposure period:

All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.
Treatment with Mitotic Inhibitor
Animals were injected via the intraperitoneal route with a solution of Colchicine at 4 mg/kg 2 to 4 hours prior to bone marrow harvest. At thescheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue.

Remarks:

Doses / Concentrations:
Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg.
Basis:
other: The maximum recommended dose of 2000 mg/kg was used as the maximum dose, substance was formulated in Arachis oil at 50, 100 and 200 mg/ml and dosed at 10 ml/Kg to achieve actual dose levels.

No. of animals per sex per dose:

Groups, each of seven male rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of male rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours.

Control animals:

yes, concurrent vehicle

Positive control(s):

Five rats were dosed orally with Cyclophosphamide at a dose of 25 mg/kg. Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test.
The positive control item was supplied by Acros Organics, as follows:
Supplier's identification : Cyclophosphamide
Supplier’s lot number : A0277203
Harlan serial number : R-4723
Date received: 04 December 2009
Expiry date : 04 December 2011
Storage conditions: Approximately 4ºC, in the dark

Tissues and cell types examined:

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in
rat bone marrow cells. In addition any increases in polyploidy may indicate the induction of numerical aberrations

Details of tissue and slide preparation:

At the scheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue. The bone marrow was aspirated into 5 ml of warm Hanks buffered salt solution (HBSS) supplemented with demecolcine (Colcemid 0.1 µg/ml) and incubated at approximately 37°C for 30 minutes before being spun down in a centrifuge. The supernatant was removed and the cell pellet re-suspended in 0.075 M potassium chloride (KCl) at 37°C for approximately 15 minutes including centrifugation. The cells were re centrifuged and all but 1 ml of the supernatant removed. After re-suspension of the cell pellet, the cells were fixed by the addition of freshly prepared fixative (methanol:glacial acetic acid, 3:1). The fixative was changed several times and the cells stored at approximately 4ºC for at least 4 hours.
Slide Preparation and Staining
After storage the cell suspensions were centrifuged and the fixative removed to leave a sufficient amount to give a milky suspension on re-suspension of the cell pellet. A few drops of each cell suspension were dropped onto clean, wet slides and air-dried. When completely dry the slides were stained in 5% Giemsa for 10 minutes and rinsed in tap water and distilled water. When the slides were dry a cover slip was applied using a mounting medium.

Evaluation criteria:

Slide Evaluation
The stained slides were coded and examined ‘blind’ using light microscopy at x100 and x1000 magnifications. 100 metaphase cells of adequate quality were scored, if possible, from the slides prepared from each animal for both numerical and structural chromosome aberrations. Except where there were approximately 30 to 50% of cells with aberrations, then slide evaluation was terminated at 50 cells. A mitotic index (MI) value was also obtained for each animal by recording the number of metaphase cells that were associated with 1000 cells.
If the cell had 40 to 44 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS Guidelines for Mutagenicity Testing. The details of the classification of chromosome aberrations and the evaluation criteria applied to test data are given in Appendix 1 (see in attached section). A Senior Cytogeneticist checked aberrations recorded by the slide scorers.

Statistics:

Statistical Analysis
Comparisons were made between the vehicle control group and each treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations. Analysis of mitotic index data was performed using a Students T-Test following a √(x+1) transformation.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mortality Data and Clinical Observations
There were no premature deaths seen in any of the test item dose groups and no clinical signs were observed in animals dosed with the test item at any of the dose levels.

Evaluation of Bone Marrow Slides
A summary of the results of the chromosome aberration test is given in the attached Table 1. Individual and group mean data are presented in he attached Tables 2 to 7. (See in attached section).

No marked decreases in the mitotic index mean value were observed with any of the test item groups or the positive control group when compared to the vehicle control group.

All of the vehicle control animals gave values of chromosome aberrations within the expected range (see table 2 in the attached section).

The positive control group animals showed highly significant increases in the frequency of aberrations (see table 3 in the attached section) indicating that the test method itself was operating as expected. It should be noted that due to the toxic response seen with cyclophosphamide in the bone marrow the quality and morphology of the metaphases was less than perfect. Therefore it was necessary to score more than 100 metaphase cells for three of the five animals to demonstrate the sensitivity of the rats following exposure with cyclophosphamide.
There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group.

The test item did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups.

Due to the nature and format of the tables, please see the attached results tables.

Table 1: Group Mean Results of Chromosome Aberration Test

Table 2: Individual Results of Chromosome Aberration Test - 24-Hour Vehicle Control Group

Table 3: Individual Results of Chromosome Aberration Test - 24-Hour Positive Control Group: Cyclophosphamide 25 mg/kg

Table 4: Individual Results of Chromosome Aberration Test - 48-Hour Test Item Group: 2000 mg/kg

Table 5: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 2000 mg/kg

Table 6: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 1000 mg/kg

Table 7: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 500 mg/kg

Conclusions:

Interpretation of results: negative
The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non clastogenic to rat bone marrow cells in vivo.

Executive summary:

Introduction. 

The study was performed to assess the potential of the test item to produce damage to chromosomes or the mitotic apparatus when administered to rats.   The method used is compatible with that described in the revised OECD Guidelines for Testing of Chemicals No. 475 “Mammalian Bone MarrowChromosome Aberration Test”, Method B11 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 andUS, EPA, TSCA and FIFRA guidelines.

Methods. 

A range-finding test was not performed as the test item had been previously investigated at Safepharm Laboratories Ltd*(Project No 2041/0045) at a dose of 5000 mg/kg with no ill effects. Therefore, the maximum recommended dose of 2000 mg/kg was used as the maximum dose and at the request of the Sponsor only male animals were investigated via the oral route.

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, processed and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.

Further group of rats for the 24-hour time point were given a single oral dose of Arachis oil (seven rats) or dosed orally with Cyclophosphamide (five rats) to serve as vehicle and positive controls respectively.

Results. 

There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any dose level.

No marked decreases in the mitotic index mean value were observed in any of the test item groups when compared to the vehicle control group.

There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group. 

The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in any of the exposure groups.

The positive control item produced a marked increase in the frequency of chromosome aberrations.

Conclusion. 

The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non‑clastogenic to rat bone marrow cells in vivo.

*On the 9^thNovember 2008 Safepharm Laboratories Ltd changed its name to Harlan Laboratories Ltd.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The experimental phases of the study were performed between 26 January 2011 and 14 March 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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. Read-across is considered to be reliability 2.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

At the start of the test the rats weighed 184 to 231 g and were approximately seven to twelve weeks old. After a minimum acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
The animals were housed in groups of up to five in solid-floor polypropylene cages with woodflake bedding (Datesand Ltd, Cheshire, UK.). Free access to mains drinking water and food, Harlan Teklad 2014 Rodent Pelleted Diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK, was allowed throughout the study. The animals were also provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) which were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

Supplier's identification: Arachis oil
Serial number (laboratory): V-4855
Date received: 14 June 2010
Description: Straw-coloured slightly viscous liquid
Expiry date: 31 January 2012
Storage conditions: Room temperature

Details on exposure:

Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours. In addition, two further groups of rats were included in the study; one group of seven rats was dosed via the oral route with the vehicle alone (arachis oil) and a second group of five rats was dosed orally with Cyclophosphamide.
Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test. The vehicle control group and positive control group were killed approximately 24 hours following treatment.
All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later. The experimental design is summarised as follows:

Treatment Group Dose Level(mg/kg) Concentration (mg/ml) Dose Volume (ml/kg) Kill Time (Hours After Dosing) Animal Numbers
1. Vehicle Control (Arachis oil) 0 0 10 24 1 – 7
2. Positive Control (Cyclophosphamide) 25 2.5 10 24 8 – 12
3. Test item 2000 200 10 48 13 – 19
4. Test item 2000 200 10 24 20 – 26
5. Test item 1000 100 10 24 27 – 33
6. Test item 500 50 10 24 34 – 40

Frequency of treatment:

Groups of rats were dosed once only via the oral route

Post exposure period:

All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.
Treatment with Mitotic Inhibitor
Animals were injected via the intraperitoneal route with a solution of Colchicine at 4 mg/kg 2 to 4 hours prior to bone marrow harvest. At thescheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue.

Remarks:

Doses / Concentrations:
Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg.
Basis:
other: The maximum recommended dose of 2000 mg/kg was used as the maximum dose, substance was formulated in Arachis oil at 50, 100 and 200 mg/ml and dosed at 10 ml/Kg to achieve actual dose levels.

No. of animals per sex per dose:

Groups, each of seven male rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of male rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours.

Control animals:

yes, concurrent vehicle

Positive control(s):

Five rats were dosed orally with Cyclophosphamide at a dose of 25 mg/kg. Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test.
The positive control item was supplied by Acros Organics, as follows:
Supplier's identification : Cyclophosphamide
Supplier’s lot number : A0277203
Harlan serial number : R-4723
Date received: 04 December 2009
Expiry date : 04 December 2011
Storage conditions: Approximately 4ºC, in the dark

Tissues and cell types examined:

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in rat bone marrow cells. In addition any increases in polyploidy may indicate the induction of numerical aberrations

Details of tissue and slide preparation:

At the scheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue. The bone marrow was aspirated into 5 ml of warm Hanks buffered salt solution (HBSS) supplemented with demecolcine (Colcemid 0.1 µg/ml) and incubated at approximately 37°C for 30 minutes before being spun down in a centrifuge. The supernatant was removed and the cell pellet re-suspended in 0.075 M potassium chloride (KCl) at 37°C for approximately 15 minutes including centrifugation. The cells were re centrifuged and all but 1 ml of the supernatant removed. After re-suspension of the cell pellet, the cells were fixed by the addition of freshly prepared fixative (methanol:glacial acetic acid, 3:1). The fixative was changed several times and the cells stored at approximately 4ºC for at least 4 hours.
Slide Preparation and Staining
After storage the cell suspensions were centrifuged and the fixative removed to leave a sufficient amount to give a milky suspension on re-suspension of the cell pellet. A few drops of each cell suspension were dropped onto clean, wet slides and air-dried. When completely dry the slides were stained in 5% Giemsa for 10 minutes and rinsed in tap water and distilled water. When the slides were dry a cover slip was applied using a mounting medium.

Evaluation criteria:

Slide Evaluation
The stained slides were coded and examined ‘blind’ using light microscopy at x100 and x1000 magnifications. 100 metaphase cells of adequate quality were scored, if possible, from the slides prepared from each animal for both numerical and structural chromosome aberrations. Except where there were approximately 30 to 50% of cells with aberrations, then slide evaluation was terminated at 50 cells. A mitotic index (MI) value was also obtained for each animal by recording the number of metaphase cells that were associated with 1000 cells.
If the cell had 40 to 44 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS Guidelines for Mutagenicity Testing. The details of the classification of chromosome aberrations and the evaluation criteria applied to test data are given in Appendix 1. A Senior Cytogeneticist checked aberrations recorded by the slide scorers.

Statistics:

Statistical Analysis
Comparisons were made between the vehicle control group and each treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations. Analysis of mitotic index data was performed using a Students T-Test following a √(x+1) transformation.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A summary of the results of the chromosome aberration test is given in the attached Table 1. Individual and group mean data are presented in the attached Tables 2 to 7.
No marked decreases in the mitotic index mean value were observed with any of the test item groups or the positive control group when compared to the vehicle control group.
All of the vehicle control animals gave values of chromosome aberrations within the expected range (attached Table 2).
The positive control group animals showed highly significant increases in the frequency of aberrations (attached Table 3) indicating that the test method itself was operating as expected. It should be noted that due to the toxic response seen with cyclophosphamide in the bone marrow the quality and morphology of the metaphases was less than perfect. Therefore it was necessary to score more than 100 metaphase cells for three of the five animals to demonstrate the sensitivity of the rats following exposure with cyclophosphamide.
There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group.
The test item did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups.

Due to the nature and format of the tables, please see the attached results tables.

Conclusions:

Interpretation of results: negative
The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non clastogenic to rat bone marrow cells in vivo.

Executive summary:

Introduction. 

The study was performed to assess the potential of the test item ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ to produce damage to chromosomes or the mitotic apparatus when administered to rats. The method used is designed to be compatible with that described in the revised OECD Guidelines for Testing of Chemicals No. 475 “Mammalian Bone Marrow Chromosome Aberration Test”, Method B11 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 andUS, EPA, TSCA and FIFRA guidelines.

 

Methods. 

A range-finding test was not performed as the test item had been previously investigated at a dose of 5000 mg/kg with no ill effects. Therefore, the maximum recommended dose of 2000 mg/kg was used as the maximum dose and at the request of the Sponsor only male animals were investigated via the oral route.

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, processed and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.

Further group of rats for the 24-hour time point were given a single oral dose of Arachis oil (seven rats) or dosed orally with Cyclophosphamide (five rats) to serve as vehicle and positive controls respectively.

Results. 

There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any dose level.

No marked decreases in the mitotic index mean value were observed in any of the test item dose groups when compared to the vehicle control group.

There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group. 

The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in any of the exposure groups.

The positive control item produced a marked increase in the frequency of chromosome aberrations.

Conclusion. 

The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non-clastogenic to rat bone marrow cells in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data are available for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics from bacterial mutagenicity studies and an in vitro cytogenicity study. Supporting studies are also available for GTL Gasoil (C8-C26) and GTL Base Oil Distillates (C18-C50), including in vitro and in vivo micronucleus tests. There are no in vitro or in vivo mammalian mutagenicity toxicity studies for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics. Representative constituents of GTL substances obtained by the Fisher-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 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 vitro

A bacterial mutagenicity test was performed with Hydrocarbons, C18-C24, isoalkanes, <2% aromatics according to OECD 471 and GLP (WIL Research, 2014c). 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.

An in vitro chromosome aberration study was performed for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics according to OECD 473 and GLP (Harlan, 2015). In the absence and presence of metabolic activation, no clear cytotoxicity was observed up to the highest applied concentration, (2960.0 μg/ml). In the presence of metabolic activation no relevant increase in chromosomal aberrations was observed. In Experiment I in the absence of metabolic activation, one single increase in chromosomal aberrations, above the laboratory historical control data range (0.0 – 3.0% aberrant cells, excluding gaps), was observed after treatment with 1691.4 μg/ml (4.5% aberrant cells, excluding gaps). The value is not statistically significant and no dose-dependency was observed. In Experiment IIA in the absence of metabolic activation after treatment with 1691.4 μg/ml one single statistically significant increase in chromosomal aberrations (3.8% aberrant cells, excluding gaps), above the laboratory historical control data range (0.0 – 2.5% aberrant cells, excluding gaps), was observed. No dose-dependency was observed in this experimental part. In Experiment IIB in the absence of S9 mix this finding could not be confirmed. No evidence of an increase in polyploid metaphases was noticed after treatment. Appropriate mutagens were used as positive controls. They induced statistically significant increases in the number of cells with structural chromosome aberrations. It was concluded that the test substance was negative for cytogenicity up to limit concentrations under the conditions of the test.

Supporting data are available for GTL Gasoil (C8-C26) and GTL Base Oil Distillates (C18-C50).

GTL Gasoil (C8 - C26, branched and linear).

An Ames test was performed for GTL Gasoil according to Annex V guidelines and GLP (SafePharm, 2006a). Strains tested were Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2uvrA, both with and without metabolic activation (rat liver S9). A micronucleus test in human lymphocytes in vitro was carried out according to the draft OECD Test Guideline 487. The test results show that GTL Gasoil is not clastogenic or aneugenic (SafePharm, 2006c).

An in vitro chromosome aberration test in human lymphocytes with GTL Gasoil, carried out in accordance with OECD 473 was negative both with and without metabolic activation (Harlan, 2010a). The test substances was therefore considered to be non-clastogenic to human lymphocytes in vitro.

GTL Base Oil Distillates (C18-C50, branched, cyclic and linear)

GTL Base Oil Distillates has been tested in a bacterial mutagenicity study according to OECD 471 and under GLP using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA (SafePharm, 2006b). The test material was dissolved in tetrahydrofuran at concentrations up to 5000 µg/plate. Appropriate solvent and positive controls were included and gave expected results. No toxicity to bacterial cells was observed. No significant increase in the number of revertants was observed at any concentration with and without metabolic activation in any of the strains tested. The results were confirmed in a repeat experiment; both experiments used the direct plate incorporation method.

An in vitro micronucleus study has been conducted using GTL Base Oil Distillates following OECD draft guideline 487 and conducted under GLP conditions (SafePharm, 2006d). No increase in the incidence of micronuclei was observed in duplicate cultures of human lymphocytes at any concentration in either the initial experiment (4 hour exposure, 16 hour expression, with and without metabolic activation) or the repeat experiment (20 hour exposure without metabolic activation; 4 hour exposure, 16 hour expression, with metabolic activation). No test material induced toxicity was observed. The test material was dissolved in acetone, and the maximum concentration tested was 2500 µg/plate; higher concentrations could not be tested due to difficulties in formulating the test material in the vehicle. The vehicle controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes, and appropriate positive controls were concluded and induced significant increases in the number of cells with micronuclei. It was concluded that the test material is non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Further evidence of the lack of effects on chromosomes in vitro was obtained when GTL Base Oil Distillates was tested according to OECD 473 and under GLP (Harlan, 2010b). No statistically significant increase in the frequency of cells with chromosome aberrations was observed in either the initial or the repeat experiment when tested with and without metabolic activation up to a dose level that was limited by the onset of precipitate. Appropriate solvent and positive controls were included and gave expected results.

Evidence from the in vitro studies is supported by data from two in vivo studies. An in vivo chromosome aberration study was conducted according to OECD 475 and under GLP using GTL Gasoil (Harlan, 2011a). Male Wistar rats were dosed orally at 0, 500, 1000, and 2000 mg/kg bw in arachis oil. No increase in the incidence of cells with chromosome aberrations excluding gaps or of polyploid cells was observed in bone marrow up to the highest dose tested. No premature deaths or clinical signs were observed at any dose level. The positive control item produced a marked increase in the frequency of chromosome aberration. An in vivo chromosome aberration study on GTL Base Oil Distillates was conducted according to OECD 475 and under GLP (Harlan, 2011b). Male Wistar rats were dosed orally at 0, 500, 1000 and 2000 mg/kg bw in arachis oil. No increase in the incidence of cells with chromosome aberrations excluding gaps or of polyploid cells was observed in bone marrow up to the highest dose tested. No premature deaths or clinical signs were observed at any dose level. The positive control item produced a marked increase in the frequency of chromosome aberration.

Based on the weight of evidence from the available studies on the registered and the related substances it can be concluded that Hydrocarbons, C18-C24, isoalkanes, <2% aromatics is not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo data for the registered substance and relevant read-across substances, Hydrocarbons, C18-C24, isoalkanes, <2% aromatics is not genotoxic and does not require classification according to Regulation (EC) No. 1272/2008.