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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:
FROM 22 JUN 2010 TO 30 SEP 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
(according to German Chemical Law and OECD Principles of GLP)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Urea, reaction products with formaldehyde, glyoxal and methanol
EC Number:
296-665-1
EC Name:
Urea, reaction products with formaldehyde, glyoxal and methanol
Cas Number:
92908-36-6
Molecular formula:
C2 H2 O2 .C H4 N2 O .C H4 O .C H2 O
IUPAC Name:
Urea, reaction products with formaldehyde, glyoxal and methanol
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: October 16, 2014
- Certificate of analysis: N° 1873, 15/04/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of solvent: Stable in water
- Storage: At room temperature

Method

Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: supplemented minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: checked for spontaneous mutant frequency
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
All test item concentrations were adjusted to solvent content (8 % water))
Range finding pre-experiment: 15.8 - 2022.0 µg/mL (ca. 10 mM),
Experiment 1: 101.1, 202.2, 404.4, 808.8, 1213.2, 1617.6 and 2022.0 µg/mL (4 h, with and without metabolic activation)
Experiment 1A: 505.0, 1011.0, 1263.8, 1444.3, 1685.0, 1838.2 and 2022.0 µg/mL (4 h, without metabolic activation)
Experiment 2: 3.8, 7.5, 15.0, 30.0, 60.0, 80.0 and 100.0 µg/mL (24 h, without metabolic activation)
200.0, 400.0, 800.0, 1200.0, 1600.0 and 2000.0 µg/mL (4 h, with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (local tap water deionisised at Harlan CCR)
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 476
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: Experiment 1: 4 h (with and without metabolic activation); Experiment 2: 24 h (without metabolic activation), 4 h (with metabolic activation)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER: Two cultures were tested in each experiment
Evaluation criteria:
A test item is classified as positive if it induces either a reproducible concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent con-trols within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, bio-logical and statistical significance were 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. To clarify the results obtained a confirmatory experiment IA was performed in the absence of S9 mix. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The cell cultures were evaluated at the following concentrations:

exposure
period S9
mix concentrations in µg/mL
Experiment I
4 hours - 404.4 808.8 1213.2 1617.6 2022.0
4 hours + 202.2 404.4 808.8 1213.2 1617.6
Experiment IA
4 hours - 1263.8 1444.3 1685.0 1838.2 2022.0
Experiment II
24 hours - 15.0 30.0 60.0 80.0 100.0
4 hours + 200.0 400.0 800.0 1200.0 1600.0 2000.0*

* mutagenicity evaluation was performed only in culture I

The maximum concentration of the test item in the first experiment equals a molar concentration of about 10 mM adjusted to solvent content (8 % water) according to the substance definition of REACH (REGULATION (EC) No 1907/2006). In experiment II the evaluation of dose groups was limited by test item induced cytotoxic effects. No precipitation of the test item was observed up to the maximum concentration in all experiments.
Cytotoxic effects occurred in the first experiment in the highest guideline required dose group (2022.0 µg/mL). In the presence and absence of S9 mix the cloning efficiency and/or the cell density were reduced to 10-20 % of the solvent controls. In the second experiment in the absence of S9 mix following 24 hours treatment the cloning efficiency I was reduced to approximately 10 % at the highest evaluated test item concentration of 100.0 µg/mL. In the presence of S9 relevant cytotoxic effects occurred at 1200.0 µg/mL and above. At 1200.0 µg/mL the relative cloning efficiency I was reduced to 17.9 and 14.2 % in culture I and II.
In experiment I following 4 hours treatment in the absence of S9 mix with 1617.6 and 2022.0 µg/mL three mutation frequencies (36.6, 39.5 and 56.6 mutant colonies/106 cells) exceeded the laboratory's historical data range of 0.6-32.4 mutant colonies/106 cells. The induction factor exceeded the threshold of three times the corresponding solvent control in the highest dose group in culture I and in the two highest dose groups in culture II. To verify this results a confirmatory experiment IA was performed in the absence of S9 mix under identical conditions but using narrower spacing of concentrations. In the highest dose group required by the OECD guideline relevant cytotoxicity was observed. The cloning efficiency I was reduced to 21.4 and 17.7 % of the solvent control in culture I and II. Following treatment with 1263.8 µg/mL the mutation frequency (35.6 mutant colonies/106 cells) exceeded the historical data range. The induction factor remained well within the threshold of three times the corresponding solvent control in all evaluated dose groups. Therefore, the effect observed in experiment I was not reproducible and is judged as not being biologically relevant.
In the second experiment three mutation frequencies (35.3, 43.5 and 50.1 mutant colo-nies/106 cells) exceeding the laboratory's historical data range (0.8 ¿ 33.2 mutant colo-nies/106 cells) were observed following treatment in the presence of S9 mix. The induction factor remained well within the threshold of three times the corresponding solvent control in all evaluated dose groups.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. In experiment I culture II (absence of S9 mix) and in experiment II in both cultures (presence of S9 mix) significant dose dependent trends of the mutation frequency indicated by a probability value of <0.05 were deter-mined. However, the trends were judged as biologically irrelevant since the trend was either not reproducible (experiment I) or the mutation frequency did not exceed the threshold described above (experiment II).
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 7.1 up to 28.3 mutants per 106 cells; the range of the groups treated with the test item was from 7.4 up to 56.6 mutants per 106 cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells with or without metabolic activation.

Applicant's summary and conclusion

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay with and without metabolic activation.
Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in three independent experiments, using two parallel cultures each. Experiment I was performed with and without S9 liver microsomal activation and a treatment period of 4 hours. A confirmatory experiment IA was performed with a treatment time of 4 hours without metabolic activation. Experiment II was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The following concentrations were tested:

Experiment 1: 101.1, 202.2, 404.4, 808.8, 1213.2, 1617.6 and 2022.0 µg/mL (4 h, with and without metabolic activation)

Experiment 1A: 505.0, 1011.0, 1263.8, 1444.3, 1685.0, 1838.2 and 2022.0 µg/mL (4 h, without metabolic activation)

Experiment 2: 3.8, 7.5, 15.0, 30.0, 60.0, 80.0 and 100.0 µg/mL (24 h, without metabolic activation); 200.0, 400.0, 800.0, 1200.0, 1600.0 and 2000.0 µg/mL (4 h, with metabolic activation)

The highest concentration in the main experiments (2022.0 µg/mL) was equal to a molar concentration of 10 mM adjusted to solvent content (8 % water) according to the substance definition of REACH (REGULATION (EC) No 1907/2006). In Experiment II the highest evaluated concentration was limited by strong test item induced cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells with or without metabolic activation.

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