Paraffin waxes (Fischer-Tropsch), isomerization

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1) Bacterial reverse mutation assay / Ames test

registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', OECD 471 and GLP, Salmonella typhimurium strains (TA 98, 100, 1535, 1537) and Escherichia coli WP2uvrA, result: negative with and without activation.

2) Mammalian cell micronucleus test

- registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', OECD 487 and GLP, human lymphocytes, result: negative with and without activation;

- supporting substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0), OECD 487 and GLP, human lymphocytes, result: negative with and without activation;

- supporting substance GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130), OECD 487 and GLP, human lymphocytes, result: negative with and without activation.

3) Mammalian cell gene mutation test using the hprt gene 

registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', OECD 476 and GLP, Chinese hamster lung cells (V79), result: negative with and without activation.

4) Mammalian chromosome aberration test

supporting substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0), OECD 473 and GLP, human lymphocytes, result: negative with and without activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2022 to 24 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

On the day of the experiment (immediately before treatment), the test item was suspended in THF by using a ultrasonic water bath and stirring by vortex. The final concentration of THF in the culture medium was 0.5% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures (1).
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period.

The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation (2):
Solvent control test item
100 µg/mL
Osmolarity [mOsm] 381 410
pH-value 7.37 7.38

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Table 1 Doses applied in the gene mutation assay with the test item
(concentrations given in bold letters were chosen for the mutation rate analysis)

exposure period S9mix concentrations in µg/mL
Main experiment
4 hours - 0.09 0.27 0.8 1.6P 3.1 P 6.3 P
4 hours + 0.09 0.27 0.8 1.6P 3.1 P 6.3 P

P = Precipitation visible at the end of treatment

The cultures at the two highest concentration with and without metabolic activation were not continued to avoid analysis of too many concentrations showing precipitation.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled .

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiment.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. A t-test is judged as significant if the p-value (probability value) is below 0.05.
A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data
However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment (solvent control: 381 mOsm, pH 7.37 versus 410 mOsm and pH 7.38 at 100 µg/mL).
- Evaporation from medium: Not examined
- Precipitation: Precipitation occurred at the concentration of 1.6 µg/mL and above after four hours treatment with and without S9 mix in the main experiment
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment
Test item concentrations between 0.8 µg/mL and 100 µg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The highest concentration was limited by the solubility properties of the test item.
The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 12.5 µg/mL and above with and without metabolic activation.
There was no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below, neither with nor without metabolic activation
Based on the results of the pre-experiment the following concentrations were applied in the main experiment:
0.09; 0.27; 0.8; 1.6; 3.1; and 6.3 µg/mL





COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:

No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted up to the highest concentration evaluated, which showed precipitation.

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

Summary Table

				relative	relative	rel. adjusted	(MF)	95%	statistical analysis
	conc.	P	S9	cloning	cell	cloning	mutant/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	colonies/	interval	t-test	linear
				%	%	%	10^6 cells			regression
Column	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 h treatment				mean values of culture I and II
Solvent control with THF			-	100.0	100.0	100.0	12.0	3.3 - 21.3
Positive control (EMS)	300.0		-	99.5	94.3	93.7	183.0	ꟷ	0.000^S
Test item	0.09	-	-	110.9	92.0	100.9	8.2	3.3 - 21.3	n.c.	0.156
Test item	0.27	-	-	106.5	88.0	94.0	11.5	3.3 - 21.3	n.c.
Test item	0.8	-	-	103.8	87.2	90.6	5.5	3.3 - 21.3	n.c.
Test item	1.6	P	-	97.0	91.4	89.0	6.1	3.3 - 21.3	n.c.
Test item	3.1	P	-	culture not continued#
Test item	6.3	P	-	culture not continued#
Solvent control with THF			+	100.0	100.0	100.0	11.2	3.4 - 22.5
Positive control (DMBA)	2.3		+	96.2	118.5	114.7	86.4	ꟷ	0.000^S
Test item	0.09	-	+	91.3	110.1	100.5	13.1	3.4 - 22.5	n.c.	0.027 ^I
Test item	0.27	-	+	92.1	97.6	89.6	10.6	3.4 - 22.5	n.c.
Test item	0.8	-	+	94.0	117.3	108.6	9.8	3.4 - 22.5	n.c.
Test item	1.6	P	+	95.5	98.4	93.7	7.3	3.4 - 22.5	n.c.
Test item	3.1	P	+	culture not continued#
Test item	6.3	P	+	culture not continued#

MF      Mutant frequency

P          Precipitation at the end of treatment

S          Significant trend (p < 0.05)
n.c.      not calculated (mean MF below the upper limit of the 95% control limit)

I           Inverse trend without biological relevance

#             cultures not continued to avoid to many concentrations showing precipitation

 

 

 

 

Conclusions:

- test item did not induce gene mutations at the HPRT locus in V79 cells;
- test item is considered to be non-mutagenic in this HPRT assay

Executive summary:

A study was performed to investigate the potential of the substance Paraffin waxes (Fischer-Tropsch), isomerization, suspended in THF, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (OECD 476; GLP).

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (100 µg/mL) was limited by the solubility of the test item. The maximum test item concentration of the main experiment was 6.3 µg/mL, limited by precipitation of the test item as well.

Based on the results of the pre-experiment the following concentrations were applied in the main experiment:

0.09; 0.27; 0.8; 1.6; 3.1; and 6.3 µg/mL where precipitation of the test material occurred at the concentration of 1.6 µg/mL and above after four hours treatment.

In the main experiment in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration (1.6 µg/mL), which showed precipitation.

Consequently, the concentrations of 0.09 to 1.6 µg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-04-27 till 2022-06-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Physical state / Appearance: Solid, white
Storage Conditions: Room temperature
Stability in Solvent: Not indicated

Species / strain / cell type:

lymphocytes:

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I: 0.13; 0.22; 0.39; 0.68; 1.2; 3.6; 6.3; 11.1; 33.3; 100 µg/mL,
experiment II: 0.10; 0.17; 0.30; 0.52; 0.91; 1.6; 2.8; 4.9; 8.6; 15.0 µg/mL.
With regard to the solubility properties of the test item and it being a UVCB, 100 μg/mL
were applied as top concentration for treatment of the cultures in experiment I.

Vehicle / solvent:

Tetrahydrofuran (THF)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

CELL CULTURES:
Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a male donor (32 years old) for Experiment I and from a male donor (22 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The cell cycle time for lymphocytes from each donor has been determined by BrdU (bromodeoxyuridine) incorporation to assess the average generation time (AGT) for the donor pool.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 – 2.0 x AGT (average generation time) (Whitwell et al, 2019). Any specific cell cycle time delay induced by the test item was not accounted for directly.

CULTURE CONDITIONS:
Blood cultures were established by preparing a 13% mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract (Exp. I) and 3 µg/mL as solvent lyophilizate (Exp. II), 10% FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5% CO2 in humidified air.

DOSE SELECTION:
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration should be 10 mM, 2 mg/mL, or 2 µL/mL, whichever is the lowest. When the test item is not of defined composition (e.g. UVCBs or environmental extracts) the top concentration may need to be higher (e.g. 5 mg/mL) in the absence of sufficient cytotoxicity.
At least three test item concentrations should be evaluated for cytogenetic damage. In case of test item induced cytotoxicity, measured by a reduced cytokinesis-block proliferation index (CBPI) and expressed as cytostasis, or precipitation (observed at the end of test item exposure by the unaided eye) the dose selection should reflect these properties of the test item. Where cytotoxicity occurs, applied concentrations should cover a range from no to approximately 55 ± 5% cytostasis. For poorly soluble test items, which are not cytotoxic at concentrations lower than the lowest insoluble concentration, the highest concentration analysed should produce turbidity or visible precipitation (phase separation for liquid test items).
With regard to the solubility properties of the test item and it being a UVCB, 100 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 0.13 to 100 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 2.1 µg/mL and above in the absence of S9 mix and at 11.1 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Considering the precipitation, 15.0 µg/mL (without S9 mix) were chosen as top treatment concentration for Experiment II.

PRE-EXPERIMENT:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 11 concentrations of the test item separated by no more than a factor of 1.7 to 3 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 3 hrs (with and without S9 mix). The preparation interval was 28 hrs after start of the exposure.
This preliminary test was designated Experiment I since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

CYTOGENETIC EXPERIMENT - PULSE EXPOSURE:
About 48 h after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test substance concentration. The culture medium was replaced with serum-free medium containing the test substance or control. For the treatment with metabolic activation S9 mix (50 µL/mL culture medium) was added. After 3 h the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10% FBS (v/v) in the presence of Cytochalasin B (4 – 6 µg/mL) and cultured for 25 hours until preparation (Clare et al, 2006, Lorge et al, 2006).

CYTOGENETIC EXPERIMENT - CONTINUOUS EXPOSURE:
About 48 h after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10% FBS) containing the test item and in the presence of Cytochalasin B (4 – 6 µg/mL). The cells were exposed for 28 hours until preparation (Whitwell et al, 2019).

PREPARATION OF CELLS:
The cultures were harvested by centrifugation 28 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded, and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.

Rationale for test conditions:

Human lymphocytes are the most common cells used in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level (Countryman and Heddle, 1976; Evans and O’Riordan, 1975).

Evaluation criteria:

Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.
1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOP DOSE:
The highest treatment concentration in this study, 100 µg/mL was chosen with regard to the solubility properties of the test item, the test item being a UVCB, and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

PRECIPITATION:
In Experiment I, precipitation of the test item in the culture medium was observed at 2.1 µg/mL and above in the absence of S9 mix and at 11.1 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 2.8 µg/mL and above at the end of treatment.

EFFECTS ON PH / OSMOLARITY:
No relevant influence on pH was observed.
The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect, however, is based on a final concentration of 0.5% THF in medium. As the osmolarity is measured by freezing point reduction, 0.5% of THF has a substantial impact on the determination of osmolarity (Hughes et al., 2008).

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**	95% Ctrl limit in %
Exposure period 3 h without S9 mix
I	28 h	Solvent control^1	1.85		0.4	0.01 – 0.92
		Positive control^2	1.63	25.8	10.30^S
		0.68	1.85	0.4	0.4
		1.2	1.91	n.c.	0.6
		2.1^P	1.83	2.5	0.75
Trend test: p-value 0.054
Exposure period 28 h without S9 mix
II	28 h	Solvent control^1	1.5		0.5	0.00 – 1.19
		Positive control^3	1.45	10	2.90^S
		0.91	1.59	n.c.	0.4
		1.6	1.46	7.6	0.5
		2.8^P	1.54	n.c.	0.7
Trend test: p-value 0.243
Exposure period 3 h with S9 mix
I	28 h	Solvent control^1	1.89		0.55	0.00 – 0.97
		Positive control^4	1.4	54.7	2.35^S
		3.6	1.84	5.4	0.6
		6.3	1.86	3.1	0.55
		11.1^P	1.86	3.9	0.5
Trend test: p-value 0.345
*	For the positive control groups and the test item treatment groups the values are related to the solvent controls
**	The number of micronucleated cells was determined in a sample of 2000 binucleated cells
^P	Precipitation occurred at the end of treatment
^S	The number of micronucleated cells is statistically significantly higher than corresponding control values
n.c.	Not calculated as the CBPI is equal or higher than the solvent control value
^1	THF	0.5 % (v/v)
^2	MMC	1.0 µg/mL
^3	Vinblastine	10 ng/mL
^4	CPA	17.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item Paraffin waxes (Fischer-Tropsch), isomerization is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Executive summary:

The test item Paraffin waxes (Fischer-Tropsch), isomerization, suspended in THF, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Stimulation period (hrs)	48	48	48
Exposure period (hrs)	3	28*	3
Cytochalasin B exposure (hrs)	25	28*	25
Total culture period (hrs)	76	76	76

* Co-treatment of test item and Cytochalasin B

In each experimental group, two parallel cultures were analysed. Per culture, 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (100 µg/mL of the test item) was chosen with regard to the solubility properties of the test item, the test item being a UVCB, and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with Guideline 487. The rationale for the dose selection is reported in section "Dose Selection". The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.

In Experiment I and II in the absence and presence of S9 mix, no cytotoxicity was observed at the highest evaluated concentration, which showed precipitation.

In the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The mean percentage of the micronuclei in all treated conditions was within the 95% control limit and none of the values were statistically significantly increased, when compared with the solvent control. There was also no concentration related increase in micronucleus formation, as judged by an appropriate trend test.  The outcome of the study is clearly negative.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Reference 3

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:

2022-02-08 - 2022-03-07

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physical state / Appearance: Solid, white
Storage Conditions: Room temperature
Stability in Solvent: Not indicated

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent used: THF
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 30 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: None
Water solubility: Not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at the same concentrations.
Other confounding effects: None

COMPARISON WIT HISTORICAL CONTROL DATA: In Experiment II in the absence of S9 mix, the data of the solvent control of strain WP2 uvrA were slightly above our historical control range (64 ± 8 vs. 63 colonies). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and therefore acceptable.
ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: reverse mutation assay migrated from the field Test System

Summary of Experiment I

 

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)TA 1535	Revertant Colony Counts (Mean ±SD)TA 1537	Revertant Colony Counts (Mean ±SD)TA 98	Revertant Colony Counts (Mean ±SD)TA 100	Revertant Colony Counts (Mean ±SD)WP2 uvrA
Activation	THF		16 ± 4	9 ± 3	32 ± 7	136 ± 11	53 ± 10
	Untreated		11 ± 5	11 ± 4	31 ± 10	128 ± 10	53 ± 7
	test item	3 µg	14 ± 2	10 ± 2	30 ± 9	123 ± 5	53 ± 4
	test item	10 µg	15 ± 1	9 ± 3	25 ± 7	128 ± 20	50 ± 12
	test item	33 µg	12 ± 3	9 ± 2	25 ± 7	132 ± 8	48 ± 13
	test item	100 µg	15 ± 1	9 ± 3	27 ± 4	122 ± 10	59 ± 7
	test item	333 µg	11 ± 1	10 ± 5	31 ± 1	131 ± 9	61 ± 3
	test item	1000 µg	10 ± 1^P	10 ± 4^P M	40 ± 7^P	124 ± 9^P	58 ± 5^P
	test item	2500 µg	13 ± 3^P M	9 ± 3^P M	32 ± 9^P	133 ± 14^P	73 ± 10^P
	test item	5000 µg	15 ± 1^P M	7 ± 3^P M	35 ± 3^P	125 ± 14^P	64 ± 6^P
	NaN3	10 µg	1107 ± 12			2032 ± 71
	4-NOPD	10 µg			522 ± 12
	4-NOPD	50 µg		129 ± 23
	MMS	2.0 µL					888 ± 59
With Activation	THF		14 ± 3	12 ± 2	37 ± 5	128 ± 7	65 ± 8
	Untreated		11 ± 1	12 ± 3	40 ± 8	134 ± 6	56 ± 11
	test item	3 µg	11 ± 4	13 ± 3	44 ± 5	128 ± 9	57 ± 8
	test item	10 µg	13 ± 2	9 ± 3	41 ± 5	125 ± 2	65 ± 7
	test item	33 µg	14 ± 2	11 ± 3	42 ± 7	131 ± 17	64 ± 15
	test item	100 µg	11 ± 4	11 ± 2	43 ± 7	133 ± 13	59 ± 6
	test item	333 µg	14 ± 5	13 ± 2	47 ± 9	118 ± 17	58 ± 6
	test item	1000 µg	16 ± 2^P M	13 ± 2^P M	31 ± 6^P M	137 ± 9^P	68 ± 6^P
	test item	2500 µg	11 ± 5^P M	10 ± 2^P M	30 ± 4^P M	101 ± 3^P M	57 ± 6^P M
	test item	5000 µg	15 ± 1^P M	7 ± 2^P M	32 ± 2^P M	93 ± 10^P M	47 ± 7^P M
	2-AA	2.5 µg	333 ± 40	445 ± 48	2638 ± 238	4091 ± 279
	2-AA	10.0 µg					271 ± 6

 

 

 

Summary of Experiment II

 

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)TA 1535	Revertant Colony Counts (Mean ±SD)TA 1537	Revertant Colony Counts (Mean ±SD)TA 98	Revertant Colony Counts (Mean ±SD)TA 100	Revertant Colony Counts (Mean ±SD)WP2 uvrA
Without Activation	THF		12 ± 6	12 ± 5	33 ± 7	128 ± 10	64 ± 8
	Untreated		17 ± 3	13 ± 3	30 ± 4	127 ± 5	59 ± 7
	test item	10 µg	11 ± 4	10 ± 4	32 ± 3	112 ± 9	62 ± 13
	test item	33 µg	12 ± 2	9 ± 4	26 ± 6	123 ± 14	64 ± 9
	test item	100 µg	12 ± 2	9 ± 2	21 ± 2	110 ± 9	63 ± 5
	test item	333 µg	11 ± 2	10 ± 1	31 ± 5	118 ± 20	73 ± 3
	test item	1000 µg	17 ± 3^P	10 ± 2^P M	35 ± 3^P	123 ± 18^P	54 ± 3^P
	test item	2500 µg	16 ± 5^P	10 ± 2^P M	33 ± 3^P	136 ± 6^P	60 ± 6^P M
	test item	5000 µg	10 ± 3^P M	9 ± 2^P M	26 ± 3^P M	109 ± 12^P	52 ± 6^P M
	NaN3	10 µg	1123 ± 21			1812 ± 22
	4-NOPD	10 µg			565 ± 18
	4-NOPD	50 µg		145 ± 14
	MMS	2.0 µL					713 ± 51
With Activation	THF		10 ± 4	16 ± 1	45 ± 8	119 ± 19	63 ± 2
	Untreated		8 ± 3	13 ± 3	45 ± 10	148 ± 6	68 ± 5
	test item	10 µg	11 ± 4	13 ± 3	42 ± 13	126 ± 17	71 ± 8
	test item	33 µg	15 ± 3	13 ± 4	47 ± 10	114 ± 8	77 ± 8
	test item	100 µg	9 ± 2	11 ± 5	37 ± 6	116 ± 16	73 ± 5
	test item	333 µg	11 ± 1	12 ± 3	37 ± 8	121 ± 3	63 ± 7
	test item	1000 µg	12 ± 3^P M	11 ± 3^P M	35 ± 3^P M	114 ± 7^P M	85 ± 7^P
	test item	2500 µg	10 ± 2^P M	12 ± 3^P M	31 ± 2^P M	108 ± 9^P M	54 ± 3^P M
	test item	5000 µg	9 ± 2^P M	10 ± 2^P M	30 ± 3^P M	105 ± 6^P M	44 ± 7^P M
	2-AA	2.5 µg	280 ± 15	441 ± 14	3097 ± 338	4389 ± 251
	2-AA	10.0 µg					326 ± 21

 

^{Key to Plate Postfix Codes:}              

^{P: Precipitate}

^{M: Manuel Count}

^{R:  Reduced Background growth}

^{D:  Densely Colored Plate}

 

^{Key to positive controls:}

^{NaN3: sodium azide}

^{4 -NOPD: 4 -nitro-o-phenylene-diamine}

^{MMS: methyl methane sulfonate}

^{2-AA: 2 -aminoanthracene}

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item Paraffin waxes (Fischer-Tropsch), isomerization is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item 'Paraffin waxes (Fischer-Tropsch), isomerization' was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item dissolved in THF was tested at the following concentrations:

• Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate


• Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at the same concentrations.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Paraffin waxes (Fischer-Tropsch), isomerization at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies. In Experiment II in the absence of S9 mix, the data of the solvent control of strain WP2 uvrA were slightly above our historical control range (64 ± 8 vs. 63 colonies). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and therefore acceptable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian bone marrow chromosome aberration test

supporting substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0), OECD 475 and GLP: negative 

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key 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

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:

- test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations
- 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 GTL base oil [‘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 GTL base oil (CAS 848301-69-9, EC 482-220-0) 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

TEST RESULTS 

1) Registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization'

The registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' 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. 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 test material 'Paraffin waxes (Fischer-Tropsch), isomerization' was considered to be non-mutagenic under the conditions of this test.

 

An OECD 487 guidline study was performed in order to evaluate the mutagenic potential of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' to induce formation of micronuclei in human lymphocytes. The test item, suspended in THF, was assessed in two independent experiments.

In each experimental group, two parallel cultures were analysed. Per culture, 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (100 µg/mL of the test item) was chosen with regard to the solubility properties of the test item, the test item being a UVCB, and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with Guideline 487. Appropriate mutagens were used as positive controls; they induced statistically significant increases in cells with micronuclei.

In Experiment I and II in the absence and presence of S9 mix, no cytotoxicity was observed at the highest evaluated concentration, which showed precipitation. In the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.

The mean percentage of the micronuclei in all treated conditions was within the 95% control limit and none of the values were statistically significantly increased, when compared with the solvent control. There was also no concentration related increase in micronucleus formation, as judged by an appropriate trend test.  The outcome of the study was clearly negative.

 

An OECD 476 GLP guideline study was performed to investigate the potential of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', suspended in THF, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (100 µg/mL) was limited by the solubility of the test item. The maximum test item concentration of the main experiment was 6.3 µg/mL, limited by precipitation of the test item as well. Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 0.09, 0.27, 0.8, 1.6, 3.1, and 6.3 µg/mL where precipitation of the test material occurred at the concentration of 1.6 µg/mL and above after four hours treatment. In the main experiment in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration (1.6 µg/mL), which showed precipitation. Consequently, the concentrations of 0.09 to 1.6 µg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation. No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells. The test material 'Paraffin waxes (Fischer-Tropsch), isomerization' was considered to be non-mutagenic in this HPRT assay.

 

2) Supporting substances [covering the low molecular weight fraction of the registration substance up to C50]

An in vitro micronucleus study has also been conducted using the closely related substances GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) and GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130), both following OECD draft guideline 487 and conducted under GLP conditions. No increase in the incidence of micronuclei was observed in duplicate cultures of human lymphocytes at any concentration in either the initial experiments or the repeat experiments. No test material induced toxicity was observed. The test materials were 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 both test materials are non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Further evidence of the lack of effects on chromosomes in vitro can be obtained from an OECD 473 guideline study of the related substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0). 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.

 

Moreover, in vivo data is available from a mammalian bone marrow chromosome aberration test on the related substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0), conducted according to OECD 475:

The 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.

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 and it 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.

 

Based on the available studies on the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' and closely related substances, it can be concluded that the registration substance is not genotoxic.

Justification for classification or non-classification

Based on the available in vitro and in vivo data, the substance 'Paraffin waxes (Fischer-Tropsch), isomerization' is not genotoxic and does not require classification according to Regulation 1272/2008/EC.