Paraffin waxes (Fischer-Tropsch), isomerization

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Paraffin waxes (Fischer-Tropsch), isomerization

Cas Number:

2658498-20-3

Molecular formula:

C25H52 - C150H302

IUPAC Name:

Paraffin waxes (Fischer-Tropsch), isomerization

Details on test material:

- Purity: 100%
- Molecular weight: Not applicable (UVCB)
- Physical state / Appearance: Solid / white
- Expiry Date / Retest Date: Not indicated
- Storage Conditions: Room temperature
- Stability in Solvent: Not indicated

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

Method

Target gene:

HPRT

Species / strain

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

 

 

 

 

Applicant's summary and conclusion

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.