Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-03-20 to 2012-07-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Read-across from GLP guideline study with the source substance Hexadecyl dihydrogen phosphate. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
3539-43-3 (Hexadecyl dihydrogen phosphate)
IUPAC Name:
3539-43-3 (Hexadecyl dihydrogen phosphate)
Constituent 2
Reference substance name:
Hexadecyl dihydrogen phosphate
EC Number:
222-581-1
EC Name:
Hexadecyl dihydrogen phosphate
Cas Number:
3539-43-3
IUPAC Name:
hexadecyl dihydrogen phosphate

Method

Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (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
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (with and without metabolic activation):
50, 100, 250, 500, 750, 1000, 2500 µg/mL
Experiment I
without metabolic activation: 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 650, 1000 and 1750 µg/mL
and with metabolic activation: 0.010, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/mL
Experiment II
without metabolic activation: 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 650 µg/mL
and with metabolic activation: 0.75, 2.5, 7.5, 25, 75, 250, 750 and 1500 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was suspended in cell culture medium and processed by ultrasound for 20 min.
Controlsopen allclose all
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: (without metabolic activation)
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 316 μg/mL; Experiment II without S9: ≥ 100 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the HPRT test using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT test), V79 cells cultured in vitro were exposed to the test item suspended in (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 650, 1000 and 1750 µg/mL (without metabolic activation, Experiment I)

- 0.010, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/mL (with metabolic activation, Experiment I)

- 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 650 µg/mL (without metabolic activation, Experiment II)

- 0.75, 2.5, 7.5, 25, 75, 250, 750 and 1500 µg/mL (with metabolic activation, Experiment II).

The test item was tested up to cytotoxic concentrations.

A biologically relevant growth inhibition was observed in experiment I and II without metabolic activation. In experiment I without metabolic activation the relative growth was 17.5% for the highest concentration (1750 µg/mL) evaluated. In experiment II without metabolic activation the relative growth was 10.2% for the highest concentration (650 µg/mL) evaluated.

No biologically relevant growth inhibition was observed in experiment I and II with metabolic activation. In experiment I the highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 80.8%. In experiment II the highest concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 76.2%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.53 was found at a concentration of 3.16 µg/mL with a relative growth of 124.0%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.93 was found at a concentration of 3.16 µg/mL with a relative growth of 105.6%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.63 was found at a concentration of 0.0316 µg/mL with a relative growth of 104.3%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.00 was found at a concentration of 0.75 µg/mL with a relative growth of 98.7%.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitromutagenicity (mammalian forward gene mutation) data.