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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:
July, 19 2016 to January 31 2017
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:OXAQUIM S.A / 15000900
- Expiration date of the lot/batch: May 20, 2017

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Tissue: lung
Cell type: adherent

CELLS USED
- Source of cells: National Centre for Cell Science, Pune, INDIA
- Suitability of cells: Yes
- Cell cycle length, doubling time or proliferation index: 13-16 hours
- Methods for maintenance in cell culture if applicable:DMEM medium
- Modal number of chromosomes: 22 (±3)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:DMEM
- Properly maintained: [yes/no] YES
- Periodically checked for Mycoplasma contamination: [yes/no] YES
- Periodically checked for karyotype stability: [yes/no) YES
- Periodically 'cleansed' against high spontaneous background: [yes/no] HAT Treated
Metabolic activation:
with and without
Metabolic activation system:
Arochlor induced rat liver S9
Test concentrations with justification for top dose:
Concentrations selected:

62.5, 125, 250 and 500 ug/L

The highest dose was chosen for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item
Vehicle / solvent:
Vehicle, solvent used: DMSO
Justification: test item soluble in DMSO
Controls
Untreated negative controls:
yes
Remarks:
DMEM
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days


SELECTION AGENT (mutation assays):6-thioguanine


NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1.5x10^6 (single culture) and 5x10^2 (in duplicate) were seeded in DMEM10 for determination of mutation rate and toxicity



DETERMINATION OF CYTOTOXICITY
cloning efficiency; relative total growth;
Evaluation criteria:
A mutation assay is considered acceptable if it meets the following criteria:
- all the negative and positive control data values falls within the historical data
- Two experimental conditions (with and without metabolic activation) were tested

A test item ei classified as positive if:
- at least one of the test concentrations exhibits a statiscally significant increase compared with the concurrent negative control
- the increase in concentration-related
Any of the results are outside the distribution of the historical negative data

Statistics:
The number of mutant clolnies obtanined for the groups treated with the test item was compared to the solvent control groups using Mann-Whitney test. A trend is judged as significant whenever the p-value (probability value) is below 0.5. However, both, biological relevance and statictical significance was considered together



Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
valid
Remarks:
DMEM Medium
Positive controls validity:
valid
Remarks:
-S9 Ethyl methanesulfonate, +S9 7, 12 Dimethyl benz(a)anthracene

Any other information on results incl. tables

See the result tables attached in "attached background material" field

Applicant's summary and conclusion

Conclusions:
Conclusion
During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.
Therefore, OXALIC ACID is considered “ non-mutagenic” in this HPRT assay.
Executive summary:

This study was conducted to investigate the potential of OXALIC ACID to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The methods followed were as per OECD guideline No. 476, adopted on 28th July 2015.

The assay was performed in a independent experiment, using two parallel cultures each. The experiment I was performed with and without liver microsomal activation at a treatment period of 4 hours. Experiment II was performed for a treatment period of 4 hours with and 24 hours with out metabolic activation.

500 µg/mL concentration was chosen as the highest dose for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item.

The following concentrations were selected for both Phase - I and Phase - II based on cytotoxicity results.

500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL both in the presence and absence of metabolic activation

No relevant cytotoxic effect as indicated by the relative survival (RS) is cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count and as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%). The RS for the test item in the other tested concentrations were found to be more than 50% and hence the same doses were selected for the main experiment (Phase-I and Phase-II).

PHASE-I

In culture I, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 7.5, 8.7, 8.8, 12.9, 17.5, 23.8 and 152.1/106 cells respectively in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 4.3, 10.3, 11.7, 15.6, 24.7, 28.1 and 1181.2 per 106 cells in presence of metabolic activation.

In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 5.8, 9.5, 14.5, 15.7, 17.2, 26.9 and 169.9/106 cells respectively and in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 9.3, 12.8, 17.6, 17.8, 24.7, 28.7 and 1387.6 per 106 cells in presence of metabolic activation.

PHASE-II

In culture I, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 6.6, 6.9, 7.8, 9.9, 15.7, 20.9 and 170.6/106 cells respectively in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 5.2, 6.7, 10.2, 15.1, 19.0, 23.2 and 1022/106 cells in presence of metabolic activation.

In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 6.0, 7.5, 10.0, 13.3, 17.4, 23.7 and 155.2/106 cells respectively and in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 7.4, 10.7, 12.5, 17.3, 22.1, 25.2 and 1211.6 per 106 cells respectively in presence of metabolic activation.

In both the cultures, there was no distinct increase in the mutant frequency of OXALIC ACID when compared to respective vehicle control and the induction factor not exceeds more than three times the corresponding vehicle controls. No significant and reproducible dose dependent increase in mutant colony numbers was observed in either the Phase I or Phase II of the experiment.

The positive controls used, EMS in the absence of metabolic activation and DMBA  in the presence of metabolic activation, revealed significant increase in mutant colonies and induction factor is more than three times of vehicle control indicating that the test system were sensitive and the results are valid

Note: NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; T4: Test concentration 4; PC: Positive Control, EMS (ethyl methanesulfonate), DMBA (Dimethyl benz(a)anthracene)