Pentane-1,5-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Pentane-1,5-diol

EC Number:

203-854-4

EC Name:

Pentane-1,5-diol

Cas Number:

111-29-5

Molecular formula:

C5H12O2

IUPAC Name:

pentane-1,5-diol

Specific details on test material used for the study:

Purity: 97.8 area-%
Water content: 0.03 g/100 g

Method

Target gene:

- Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster V79 cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix (phenobarbital and ß-naphthoflavone induced) from male Wistar rat livers

Test concentrations with justification for top dose:

1st Exp: 68.8; 137.5; 275.0; 550.0 and 1100.0 µg/mL (with and without S9 mix, 4-hour exposure)
2nd Exp.: 137.5; 275.0; 550.0; 750.0 and 1100.0 µg/ml (with and without S9 mix, 4-hour exposure)

Vehicle / solvent:

Due to the good solubility of the test substance in culture medium, the aqueous culture medium
(Ham's F12) was selected as vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: 2

Rationale for test conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative contro value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological
effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data .(95% control limit)

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity
Test procedures Workgroup Report. A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle
control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TREATMENT CONDITIONS:
Osmolality and pH values were not relevantly influenced by test substance treatment.

CELL MORPHOLOGY:
After 4 hours treatment neither in the absence nor presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test
group tested for gene mutations.

CYTOTOXICITY:
There was no relevant decrease in the number of colonies as described by the relative survival in the presence and absence of S9 mix up to the highest applied test substance concentration.

Any other information on results incl. tables

See "Attached background material" for additional information on results (tables)

Applicant's summary and conclusion

Conclusions:

Thus, in the absence and the presence of metabolic activation, 1,5-Pentanediol is not a
mutagenic substance in the HPRT locus assay using CHO cells under the experimental
conditions chosen.

Executive summary:

The substance 1,5-Pentanediol was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Four independent experiments were carried out, one Experiment with and without the addition of liver S9 mix from phenobarbital- and -naphthoflavone induced rats (exogenous metabolic activation) and three experimental parts in the presence of S9 mix.

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in all experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest applied test substance concentration.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies both without S9 mix and after the addition of a metabolizing system in all experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance 1,5-Pentanediol is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.