Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
3-aminopropan-1-ol
EC Number:
205-864-4
EC Name:
3-aminopropan-1-ol
Cas Number:
156-87-6
Molecular formula:
C3H9NO
IUPAC Name:
3-aminopropan-1-ol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 111 v. 22.11.16
- Test substance No.: 15/0348-2

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital i.p. and β-naphthoflavone orally induced rat liver S9-mix
Test concentrations with justification for top dose:
Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.
1st experiment
without metabolic activation: 50, 100, 200, 400, and 800 µg/mL
with metabolic activation: 50, 100, 200, 400, and 800 µg/mL
2nd experiment
without metabolic activation: 75, 150, 300, 600, and 800 µg/mL
with metabolic activation: 75, 150, 300, 600, and 800 µg/mL
Vehicle / solvent:
culture medium (Ham's F12); due to the good solubility of the test substance in culutre medium
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
whithout metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Two independent experiments (replicates) were performed.

During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

For each test group, about 20x10^6 logarithmically growing cells per flask (300 cm²) were seeded into about 40 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours.

After the attachment period, the medium was removed from the flasks and the treatment medium (containing the test substance) with or without metabolic activation was added. The cultures were incubated for 4 h (exposure period) at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.

The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2x10^6 cells were seeded in 20 mL medium (in 175 cm2 flasks). The flasks were left to stand in the incubator for about 3 days at 37°C, relative humidity of ≥ 90% and 5% (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm2 flasks with 2x10^6 cells. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (3rd passage).

For selection of the mutants, two 175 cm2 flasks with 2x10^6 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 – 7 days. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

Relative survival after treatment (Cloning efficiency 1 [CE1] adjusted by cell loss)
For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.

Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.

In all cases, after seeding the flasks or petri dishes were incubated for 6 - 8 days to form colonies. These colonies were fixed, stained and counted.
The absolute and relative cloning efficiencies (%) were calculated for each test group.

Changes in pH were recorded by a change in the indicator color of the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured at least for the top concentrations and for the negative controls with and without S9 mix.
Osmolality was measured in at least the top concentrations and the negative controls with and without S9 mix.
Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.

Assessment 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/vehicle control 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:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
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
Key result
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:
not applicable
Positive controls validity:
valid
Additional information on results:
MUTANT FREQUENCY
No biologically relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (without S9 mix: MFcorr.: 0.85 – 5.48 per 10^6 cells; with S9 mix: MFcorr.: 0.28 – 6.86 per 10^6 cells) were close to or within the respective vehicle control values (MFcorr.: 1.98 – 5.87 per 10^6 cells) and clearly within the range of the 95% control limit of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 5.97 per 10^6 cells; with S9 mix: MFcorr.: 0.00 – 7.91 per 10^6 cells.
In both experiments, no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 133.70 – 148.39 per 10^6 cells; with S9 mix: MFcorr.: 131.35 – 134.98 per 10^6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 10^6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 10^6 cells.

CYTOTOXICITY
Cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values were not observed in both experiments in the absence and presence of S9 mix, up to the highest applied concentrations.

CELL MORPHOLOGY
After 4 hours treatment in both experiments in the absence of metabolic activation and in the 1st Experiment in the 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. However, in the presence of S9 mix, after 4 hours treatment in the 2nd Experiment the morphology and attachment of the cells was adversely influenced (grade > 2) at the highest applied concentration of 800 μg/mL.

TREATMENT CONDITIONS
The pH values of the test substance preparations (stock solutions) were adjusted to a physiological value by adding small amounts of HCl.
Then, osmolality and pH values were not influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest applied test substance concentrations.

Applicant's summary and conclusion