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Toxicological information

Genetic toxicity: in vitro

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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:
23 may 2016 - 25 aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 76582139K0
- Expiration date of the lot/batch: 17 Jan 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed by the sponsor

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (phenobarbital- and beta-naphthoflavone induced rats)
Test concentrations with justification for top dose:
1st Experiment
without S9 mix
0; 34.4; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL
with S9 mix
0; 34.4; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL
2nd Experiment
without S9 mix
0; 43.8; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
with S9 mix
0; 43.8; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL
Vehicle / solvent:
Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as most suitable vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Time schedule
Day 1: Seeding of the cells pretreated with "HAT" medium: in 300 cm² flasks
Day 2: Test substance incubation (approx. 20 – 24 hours after seeding); exposure
period (4 hours); removal of test substance by intense washing;
1st passage of the treated cells in 175 cm2 flasks and seeding of the cloning efficiency 1 (survival) in
60 mm petri dishes.
Day 5: 2nd passage of the treated cells
Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1
3rd passage of the treated cells; addition of selection medium ("TG" medium)
and seeding of the cloning efficiency 2 (viability) in 60 mm petri dishes
From day 16: Drying, fixation, staining and counting of the selected colonies and the cloning efficiency 2
In this study, all incubations were performed at 37°C with a relative humidity of ≥ 90% in a
5% (v/v) CO2 atmosphere.


SELECTION AGENT : 6-thioguanine

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: about 200 cells per concentration

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- OTHER: pH, Osmolality; Solubility; Cell morphology
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/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
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 examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Thus, in the absence and the presence of metabolic activation, Neopentylglycol is not a mutagenic substance
in the HPRT locus assay using CHO cells under the experimental conditions chosen.