Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 2019 - 12 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Required by REACH

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide
EC Number:
619-409-6
Cas Number:
99208-50-1
Molecular formula:
C18H13O4P
IUPAC Name:
10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: DOPO-HQ
Batch: 108376001
Purity: 99.7%
Appearance: White powder
Expiry Date: 20 March 2020
Storage Conditions: At 2-8°C

Method

Target gene:
S. typhimurium: histidine
E. coli: tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: his C 3076; rfa-; uvrB- his D 3052; rfa-; uvrB-; R-factor his G 46; rfa-; uvrB- his G 46; rfa-; uvrB-; R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: trp-; uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9 were used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation.
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: strains: TA 1537, TA 98 4-nitro-o-phenylene-diamine (10 µg/plate in strain TA 98, 50 µg/plate in strain TA 1537) With S9: strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA 2-aminoanthracene ; 2.5 µg/plate ; 10.0 µg/plate in WP2 uvrA
Details on test system and experimental conditions:
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution
buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. ‎3.4.3 Precultures),
2000 µL Overlay agar
Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes.
100 µL Test solution at each dose level (solvent or reference mutagen solution
(positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution
buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. ‎3.4.3 Precultures),
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.
Rationale for test conditions:
The study was performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.
The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform and give reliable data on the ability of an agent to interact with DNA and produce mutations.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
plate incorporation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
NaN3
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
plate incorporation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
4-NOPD
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
plate incorporation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
4-NOPD
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
plate incorporation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
NaN3
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
plate incorporation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
MMS
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
plate incorporation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
plate incorporation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
a minor reduction in number of revertants below the indication factor of 0.5
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
plate incorporation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
plate incorporation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
plate incorporation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
pre incubation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
NaN3
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
pre incubation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
4-NOPD
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
pre incubation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
4-NOPD
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
pre incubation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
NaN3
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
pre incubation test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
a minor reduction in number of revertants below the indication factor of 0.5
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
MMS
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
pre incubation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
pre incubation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
pre incubation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
a minor reduction in number of revertants below the indication factor of 0.5
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
pre incubation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
pre incubation test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2-AA

Applicant's summary and conclusion

Conclusions:
It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, DOPO-HQ is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of DOPO-HQ to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate as well. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No relevant toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without metabolic activation. Only in a few strains a reduction below the indication factor of 0.5 was observed.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with DOPO-HQ at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies