Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of Ethoxylated phenol phosphate was investigated in a bacterial reverse gene mutation assay conducted according to OECD 471 with and without metabolic activation. There was no increase of mutant colonies compared to the negative control observed in all strains. Consequently, the test item is considered to be non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-02 to 2018-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- CAS number: 39464-70-5
- Appearance: Clear, yellow-amber liquid
- Purity: 100%
- Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH))


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All dilutions in the main tests of test item were made in the testing laboratory using Distilled water. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 2 hours after preparation.
Target gene:
Salmonella typhimurium: Histidine locus
E. coli: Tryptophane locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: 21 April 2015, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

MEDIA USED
- Type and identity of media:
- Minimal Glucose Agar
- Nutrient Broth No. 2
- Nutrient Agar
- Top Agar
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in Distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, distilled water
- Justification for choice of solvent/vehicle: In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO & Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
TA98 (4 µg/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO & Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535 (2 µg/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO & Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 (50 µg/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
DMSO & Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA (2 µL/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO & Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all Salmonella strains (2 µg/plate), with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO & Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
E.coli WP2 uvrA (50 µg/plate), with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation, pre-incubation

EXPERIMENTAL PERFORMANCE
Experiment 1 (plate incorporation):
A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No .2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45 °C. The equivalent number of minimal glucose agar plates (three plates per test item concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45 °C). This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.


Experiment II (pre-incubation):
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
For the pre-incubation method, bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
Before the overlaying, 50 µL of the test item formulation or its vehicle (or 50 µL of positive reference controls or their solvents), 100 µL of the overnight culture of bacterial cells and 0.5 mL of the S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into appropriate tubes to provide direct contact between bacteria and the test item. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37 ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 tubes per control and 3 tubes for each concentration level

EVALUATION OF MUTAGENICITY
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in all tester strains of the main tests
- at least five analysable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occured and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occured in at least one strain with or without metabolic activation.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
Key result
Species / strain:
other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Concentration Range Finding Test:
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation. No inhibitory or toxic effects of the test item were detected in the preliminary experiment.

Initial and Confirmatory Mutation Tests:
In the Initial Mutation Test (using plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 500 and 15.81 μg/plate concentrations without metabolic activation (the observed mutation factor value was: MF: 1.26). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 50 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.88). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test. Precipitate/slight precipitate was observed on the plates in the Confirmatory Mutation Test in all examined bacterial strains with metabolic activation at 5000 μg/plate concentration. No inhibitory or toxic effects of the test item were detected in the main tests.

ACCEPTANCE OF RESULTS:
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.


Remarks on result:
other: Experiment I
Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Ethoxylated phenol phosphate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, conducted according to OECD guideline 471, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and E. coli WP2 uvrA were exposed to Ethoxylated phenol phosphate (100% purity) in distilled water at concentrations of 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The mutagenic potential of Ethoxylated phenol phosphate was investigated in a bacterial reverse gene mutation assay conducted according to OECD 471 with and without metabolic activation. There was no increase of mutant colonies compared to the negative control observed in all strains. Consequently, the test item is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available data, the target substance Ethoxylated phenol phosphate does not warrant classification for mutagenicity.