Condensation products of Dihydrofuran-2,5-dione, mono-alkenyl derivs. with polyethylene amine and acetic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

January 2008 - May 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Modern GLP study conducted in accordance with OECD test guideline. Restriction due to the fact that the study was conducted on read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): [EC number: 425-620-2 read cross material]
- Substance type: Viscous amber liquid
- Physical state: Liquid
- Analytical purity: Purity of test material received stated to be 100%
- Purity test date: No data
- Lot/batch No.: S#1C405-096/B#A32817/NB#30527-KG
- Expiration date of the lot/batch: 16 October 2008
- Stability under test conditions: No data
- Storage condition of test material: room temperature in the dark

Method

Target gene:

The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells.

Test concentrations with justification for top dose:

Concentration of Final
Experiment treatment solution concentration
(mg/mL) (ug/mL)
Range finder: 0.4688 4.688
0.9375 9.375
1.875 18.75
3.75 37.5
7.5 75
15 150
1 0.25 2.5
0.5 5
1 10
2 20
3 30
4 40
5 50
7.5 75
15 150
30 300

2 1 10
2 20
4 40
6 60
7.5 75
10 100
15 150

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was miscible with acetone at concentrations up to at least 500.2 mg/mL.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Controls 2

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: Final concentration in medium: 0.15 and 0.20 ug/mL

Controls 3

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: Final concentration in medium: 2.00 and 3.00 ug/mL

Details on test system and experimental conditions:

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: Two (except for positive control treatments, which only had single cultures).

NUMBER OF CELLS EVALUATED: 384 wells at 2,000 cells per well were plated for TFT resistance

Evaluation criteria:

PARAMETERS ASSESSED:
The following parameters were calculated:
- Suspension Growth (SG)
- Relative Suspension Growth (RSG)
- Relative Total Growth (RTG)
- Mutant Frequency (MF)

ACCEPTANCE CRITERIA
The assay was considered valid if all the following criteria were met:
1. The mean mutant frequencies in the negative (vehicle) control cultures fell within the normal range (50 to 170 mutants per 106 viable cells);
2. At least one positive control should show either an absolute increase in mean total MF of at least 300x10-6 (at least 40% of this should be in the
small colony MF), or an increase in small colony mutant frequency of at least 150x10-6 above the concurrent vehicle control;
3. The mean RTG for the positive controls should be greater than 10%;
4. The mean cloning efficiencies of the negative controls from the Mutation Experiments were between the range 65% to 120% on Day 2;
5. The mean suspension growth of the negative controls from the Mutation Experiments was between the range 8 to 32;
6. There should be no excessive heterogeneity between replicate cultures.

EVALUATION CRITERIA
For valid data, the test article was considered to be mutagenic in this assay if the MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF (Global Evaluation Factor) and the linear trend test was positive. This indicated a positive, biologically relevant response.

The test article was considered as negative in this assay if the MF of all test concentrations were less than the sum of the mean control mutant frequency plus GEF.

Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

All calculations were performed either manually or by cmoputer using validated software.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
In Experiment 1 (2.5-300 ug/mL), upon addition of the test article to the cultures, precipitation was observed at the six highest concentrations (30-300 ug/mL) in the absence and presence of S9. Following the 3-hour treatment incubation period, precipitate was observed at the highest two concentrations (150 and 300 ug/mL) in the absence and presence of S9. The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and the higher concentration was discarded.
In Experiment 2 (10-150 ug/mL), upon addition of the test article to the cultures, precipitation was observed at the five highest concentrations (40-150 ug/mL) in the absence and presence of S9. Following the 3-hour treatment incubation period, precipitate was observed at the highest two concentrations in the absence of S9 (100-150 ug/mL) and at the highest concentration in the presence of S9 (150 ug/mL). The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence of S9 was retained and the higher concentration was discarded.

RANGE-FINDING/SCREENING STUDIES:
In the rangefinding study, precipitation was observed at the three highest concentrations (37.5-150 ug/mL). Following the incubation period, precipitate was observed in the supernatant at the highest two concentrations (75 and 150 ug/mL) in the absence and presence of S9.
The highest concentration tested (150 ug/mL) gave 110% Relative Total Growth in the absence and presence of S9.
No significant changes in osmolality or pH were observed at the highest concentration tested (150 ug/mL).

Any other information on results incl. tables

The acceptance criteria were met and the study was therefore accepted as valid.

In Experiments 1 and 2, the MF of the concentrations plated were all less than the sum of the mean control MF plus the GEF, indicating a negative result. In addition, for the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the negative controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 25% to 28% in Experiment 1 and from 33% to 43% in Experiment 2. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals NQO and B[a]P.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to the solubility limit in culture medium in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

EC# 425-620-2 was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells in a GLP study conducted in accordance with OECD Test Guideline 476 using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S9). A 3-hour treatment incubation period was used for all experiments performed in the absence and presence of S9.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 4.688 to 150 µg/mL (limited by solubility in culture medium). The highest concentration tested (150 µg/mL) gave 110% relative total growth (RTG) in the absence and presence of S9.

Accordingly, for Experiment 1 ten concentrations, ranging from 2.5 to 300 µg/mL, were tested in the absence and presence of S9. Two days after treatment, the highest concentration selected to determine viability and TFT resistance was 150 µg/mL, which gave 114% and 82% RTG in the absence and presence of S9, respectively. In Experiment 2 seven concentrations, ranging from 10 to 150 µg/mL, were tested in the absence and presence of S9. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 100 µg/mL in the absence of S9 and 150 µg/mL in the presence of S9, which gave 76% and 88% RTG, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S9) and benzo[a]pyrene (with S9). Therefore the study was accepted as valid.

In Experiments 1 and 2, the mutant frequencies of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF, 126 mutants per 106 viable cells), indicating a negative result.

It is concluded that EC 425-620-2 did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to the solubility limit in culture medium in two

independent experiments in the absence and presence of a rat liver metabolic activation system (S9).