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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Due to the toxicity of the test item a supplementary dose was studied.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Batch : 20180328 (28 March 2018)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch n°20180328
- Expiration date of the lot/batch: March 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable under the storage condition



Method

Target gene:
his-operon (S. typhimurium)
trp-operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains of Salmonella typhimurium are purchased from MOLTOXTM.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
Strains of Salmonella typhimurium are purchased from MOLTOXTM.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix. S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOXTM
Test concentrations with justification for top dose:
500, 250, 150, 50 , 15 and 5 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no
- Vehicle controls tested: medium with DMSO alone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: cis-Platinum (II) Diammine Dichloride and 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48-72 hour incubation period at 37° C

NUMBER OF REPLICATIONS:
- 3 culture plates per strain per dose or per control were prepared.
- If the first assay is positive, the second one is performed in the same manner.
- If the first assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay.repli

NUMBER OF CELLS EVALUATED: Not applicable, as the number of revertant colonies per plate was calculated.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:
Ensure that the criteria of validity of the study are well respected namely :
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
- Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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:
S. typhimurium TA 1537
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:
S. typhimurium TA 98
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:
S. typhimurium TA 100
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:
E. coli WP2 uvr A pKM 101
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

Applicant's summary and conclusion

Conclusions:
Doses (500, 250, 150, 50, 15 and 5 μg/plate) prepared from solutions of the test item Para-phenoxyphenol do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines 471.
Executive summary:

A test in accordance of OECD guideline n°471 was performed to test the capacity of 4 -phenoxyphenol to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A¯)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out

For assay n° 1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory (Table 11).

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (500, 250, 150, 50 , 15 and 5 μg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101).