Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-23 - 2018-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted 21st July 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
2016-11-02
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctyl hydrogen phosphate
EC Number:
221-485-7
EC Name:
Dioctyl hydrogen phosphate
Cas Number:
3115-39-7
Molecular formula:
C16H35O4P
IUPAC Name:
Dioctyl hydrogen phosphate
Constituent 2
Chemical structure
Reference substance name:
Octyl dihydrogen phosphate
EC Number:
223-638-3
EC Name:
Octyl dihydrogen phosphate
Cas Number:
3991-73-9
Molecular formula:
C8H19O4P
IUPAC Name:
octyl dihydrogen phosphate
impurity 1
Chemical structure
Reference substance name:
Trioctyl phosphate
EC Number:
217-305-1
EC Name:
Trioctyl phosphate
Cas Number:
1806-54-8
Molecular formula:
C24H51O4P
IUPAC Name:
trioctyl phosphate
impurity 2
Chemical structure
Reference substance name:
Octanol
EC Number:
249-405-6
EC Name:
Octanol
Cas Number:
29063-28-3
Molecular formula:
C8H18O
IUPAC Name:
octan-1-ol
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Since the test item was found freely soluble but toxic in the preliminary test, the selection of the highest dose level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
The selected dose levels were:
* 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 1535 and TA 1537 strains in the first experiment,
* 62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain in both experiments, for the TA 102 strain in the first experiment and for the TA 1535 strain in the second experiment,
* 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 100 strain in both experiments and for the TA 102 strain in the second experiment,
* 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1537 strain in the second experiment

Experiments with S9 mix
The selected dose levels were:
* 125, 250, 500,1000, 2000 and 4000 µg/plate for all the strains in the first experiment and for the TA 1535 strain in the second experiment,
* 6.17, 18.5, 55.6, 166.7, 500, 1500 and 4000 µg/plate for the TA 98, TA 100 and TA 102 strains in the second experiment,
* 7.81, 15.6, 31.3, 62.5,125, 250 and 500 µg/plate for the TA 1537 strain in the second experiment.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Based on available solubility data
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar; Direct plate incorporation method for the preliminary test, both experiments without S9 mix and the first experiment with S9 mix; The second experiment with S9 mix was performed according to the pre-incubation method.

DURATION
- Exposure duration: 48-72h

NUMBER OF REPLICATIONS:
3 replicates per bacterial strain and concentration

The number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK).
Rationale for test conditions:
Range-finding study
Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 µL/plate, the highest recommended dose level of 5000 µg/plate was achievable. Thus, the dose levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

A moderate to strong toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at dose levels >= 500 µg/plate in the TA 100 strain without S9 mix, >= 1000 µg/plate in the TA 98 and TA 102 strains without S9 mix, and >= 2500 µg/plate in the three strains with S9 mix.
Microcolonies of toxicity, which prevented the scoring of revertants, were recorded without S9 mix in the TA 100 (2500 and 5000 µg/plate) and TA 102 strains (5000 µg/plate) without S9 mix, as well as in the TA 100 strain with S9 mix (5000 µg/plate).
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.
The test item is considered to have shown mutagenic activity in this study if:
* a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
* and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
* neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
* nor any evidence of a dose-response relationship is noted

Results and discussion

Test results
Species / strain:
other: All strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least six analysable dose levels for each strain and test condition. The study was therefore considered to be valid.

Experiments without S9 mix:
A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at the following dose levels:
* >= 1000 µg/plate for the TA 1535 and TA 98 strains in the first experiment,
* >= 500 µg/plate for the TA 100 and TA 102 strains in both experiments, as well as for the TA 1535 and TA 98 strains in the second experiment,
* >= 250 µg/plate for the TA 1537 strain in both experiments.
Microcolonies of toxicity, which prevented the scoring of revertants, were recorded in the TA 1537 strain at 2000 µg/plate

Experiments with S9 mix:
A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at the following dose levels:
* 4000 µg/plate for the TA 1535 strain in the first experiment,
* >= 2000 µg/plate for the TA 98, TA 100 and TA 102 strains in the first experiment,
* >= 1500 µg/plate for the TA 102 strain in the second experiment,
* >= 500 µg/plate for the TA 100 strain in the second experiment,
* >= 62.5 µg/plate for the TA 1537 and TA 98 strains in the second experiment.
Moreover, microcolonies of toxicity, which prevented the scoring of revertants, were recorded as follows:
* at 4000 µg/plate for the TA 100 strain in the first experiment, as well as in the TA 1535 and TA 102 strains in he second experiment;
* at dose levels >= 1500 µg/plate for the TA 98 and TA 100 strains in the second experiment.

Applicant's summary and conclusion

Conclusions:
The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, either with or without S9 mix. These results met thus the criteria of a negative response.
Executive summary:

Reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate has been tested according to OECD 471 and under GLP. No increase in the number of revertants was observed for the test substance tested up to cytotoxic or limit concentrations in any of the test strains either with or without metabolic activation. It is concluded that the test substance is not mutagenic to bacteria under the conditions of the test.

Categories Display