2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministery of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines

Version / remarks:

March 2011

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

EC Number:

206-406-6

EC Name:

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Cas Number:

335-99-9

Molecular formula:

C7H4F12O

IUPAC Name:

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Test material form:

liquid

Method

Target gene:

mutant histidine gene and mutant tryptophan gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
The protein concentration of the S9 preparation was 32.0 mg/mL (Lot. No.: 140520B) in all experiments. The S9 mix comprised 10% S9 fraction.

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Depending on the toxic effects, observed in experiment I, seven, respectively six concentrations were tested in experiment II. 5000 μg/plate were chosen as maximal concentration in experiment II. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
Strains TA 1535, TA 98 and TA 100: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Strains TA 1537and WP2 uvrA: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Since the acceptance criterium of at least five analysable concentration was only just reached in strain TA 98 in the presence of S9 mix, this part of experiment II was repeated in this strain. It is reported as experiment IIa. The maximum concentration in experiment IIa was 1000 μg/plate. The following concentrations were tested in experiment IIa:
1; 3; 10; 33; 100; 333; and 1000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
On the day of the experiment, the test item 1H,1H,7H-perfluoroheptane-1-ol was dissolved in DMSO (purity > 99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981).

All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was proven to be stable for this period in a separate GLP study (Currenta File No.: 2018/0047/08) conducted at CURRENTA GmbH & Co. OHG.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene for TA 100, TA 1535, TA 1537, TA 98, and WP2 uvrA (+ S9) (2.5 µg/plate) 4-nitro-o-phenylene-diamine for TA1537 (50 µg/plate, -S9) and TA 98 (10 µg/plate, -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and pre-incubation

DURATION
- Preincubation period: 60 min at 37 °C ± 1.5 °C
- Exposure duration: at least 48 hours at 37 °C ± 1.5 °C in the dark

NUMBER OF REPLICATIONS: all plates were prepared in triplicate

DETERMINATION OF BACTERIOTOXICITY
- Method: gross appraisal of background growth

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation criteria:

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. Evaluation based on criteria mentioned above

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate in experiment II only

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >= 2500 µg/plate without S9 and at >=1000 µg/plate with S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >= 1000 µg/mL with and without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >= 2500 µg/mL without S9 and >= 1000 µg/mL with S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix in Exp. II only

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

Applicant's summary and conclusion

Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and Escherichia coli WP2 uvrA, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate were tested in experiment I and II. Based on the observed toxic effects, evident as a redution in the number of revertants (below the indication factor of 0.5), 1000 µg/plate were chosen as maximum concentration in experiment IIa.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose 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 and showed a distinct increase of induced revertant colonies.

The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.