Reaction mass of hexadecyl dihydrogen phosphate and dihexadecyl hydrogen phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic activity of Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate was investigated in one bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 102), in an in vitro gene mutation study in mammalian cells (Chinese Hamster V79 cells) and in one in vitro chromosome aberration study in human lymphocytes. Negative results were obtained in all studies with and without metabolic activation.

Link to relevant study records

Reference

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:

2012-03-20 to 2012-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

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

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

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.

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 300 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL

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.

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'.

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 locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin 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 controlsdidinduce the appropriate response. 

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

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphateshowed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA102. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate also yielded negative results in an in vitro gene mutation study in mammalian cells in concentration up to 2500 µg/mL.

Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate was assessed for its potential to induce chromosome aberrations in human lymphocytes in vitro. The test item did not induce chromosome aberrations. Therefore, Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate considered to be non-mutagenic in this in vitro chromosome aberration test when tested up to 1000 µg/mL.

In conclusion, Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate is not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study in human lymphocytes, and the in vitro chromosome aberration in the presence and absence of metabolic activation.

Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalian cells and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 476 and GLP.

Justification for classification or non-classification

Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate does not have to be not classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assay (up to 2500 µg/mL) and in the in vitro chromosome aberration study in concentrations up 1000 µg/mL.