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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep-2018 to Feb-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted on 29 th July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
CAS No: 99-76-3
Melting point: 127 °C
Purity: 99.8 % (=<0.1 % p-HBA and =< 0.1 % unspecified impurity)
Date of Expiry: 29.11.2020

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and ß-Naphthoflavone induced rat liver homogenate
Test concentrations with justification for top dose:
The test item was found soluble in DMSO at 200 mg/mL. Since the test item showed slight precipitation and no changes in pH up to 2 mg /mL, same concentration was considered as the highest concentration in the initial cytotoxicity test.
There was no excessive cytotoxicity up to 2 mg/mL, therefore 2 mg/mL was selcted as the highest concentration for testing in the gene mutation test.
For concentrations i.e. 0.25, 0.50, 1 and 2 mg/ml were selcted for gene mutation test based on initial cytotoxicity test.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
The derivative of the CHO-K1, CHO AA8 cells were used as the test system as recommended in teh OECD TG 476. CHO AA8 cells, Batch No. 5000062 procured fromAmerican Type Culture Collection (ATCC) was used for the test.

Benozo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test.

Cytotoxicity was assessed by determining the adjusted cloning efficiency and relative survival in the test.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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:
Based on the results obtained, the test item is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells, according to OECD TG 476 " In vitro Mammalian cell genen mutation test using hprt and xprt genes" adopted on 29th july 2016.

The test item was found soluble in DMSO at 200 mg/mL. Slight precipitation was observed at 2 mg/ml. No change in pH and precipitate at and up to 2 mg/mL in culure medium. Based on the result of solubility, pH and precipitation test an initial cytotoxicity test was conducted at concentrations of 0.0625, 0.125, 0.25, 0.5 1 and 2 mg/mL using DMSO as vehicle in the presence and absence of metabolic activation (3 to 6 hours). The result of the initial cytotoxicity test indicated that the test item was not cytotoxic to CHO AA8 cells, as the relative survival of the treated CHO AA8 cellsat the test concentrations up to 2 mg/mL was in a range of 71.91 to 86.21 % when compared with the respective vehicle control, both in the presence and absence of metabolic activation. The gene mutation test was conducted at concentrations of 0.25, 0.5, 1 nd 2 mg/mL in for plates/groups in the presence and absence of metabolic activation (3 to 6 hours) Benozo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test. Cytotoxicity was assessed by determining the adjusted cloning efficiency and relative survival in the test.

There was no statistically significant increase in number of mutant colonies at any of concentrations tested when compared with the vehicle control. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control.

Based on the results obtained, the test item is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.