Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane- l,2-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 487

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, reaction products with 1,2-propanediol

IUPAC Name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, reaction products with 1,2-propanediol

Constituent 2

Reference substance name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, polymer with 1,2-propanediol

EC Number:

613-407-9

Cas Number:

63948-88-9

IUPAC Name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, polymer with 1,2-propanediol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name:ZAN 573.
Synonym:ZAN 573/100 Both denominations refer to the same test item.
Batch number:130072591.
Description:
yellow to brown solid (coarse powder).
Container:one tube.
Storage condition:at room temperature.
Date of receipt:26 August 2014.
Re-test date:24 January 2016.
Molecular weight:UVCB-substance composed of constituents with varying molecular weight.
Purity:100%.
Correction factor applied:none

Method

Target gene:

Micronuclei formation was the chromosome aberration examined

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats induced with PB and BNF

Test concentrations with justification for top dose:

2.29, 6.86, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/mL.

Vehicle / solvent:

according to available solubility data, the vehicle was dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: DMBA,

Details on test system and experimental conditions:

Cell cultures were grown in 24-well plates at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. Each treatment was coupled to an assessment of cytotoxicity. In the preliminary toxicity test for the substance without S9 activation, the exposure was 3 hours, with a 24 hour recovery period, and also a 24 hour exposure with a 20 hour recovery period. The exposure period for the substance with S9 activation was 3 hours, with a 24 hour recovery period. For the main study without S9, the exposure period without S9 activation was of 3 hours duration, with a 24 hour recovery period, followed by a second study of 24 hour exposure duration with a 20 hour recovery period. For the main study of the substance with S9 activation, there were two experiments where the exposure duration was 3 hours, with a 24 hour recovery period.
Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.
After the final cell counting, the cells were washed twice and fixed. Cells from three dose-levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before a "blinded" analysis. For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).
Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
The test item was dissolved in dimethylsulfoxide (DMSO).

Evaluation criteria:

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
1. a dose-related increase in the frequency of micronucleated cells was observed,
2. for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
3. a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.
Evaluation of a negative response: a test item is considered negative if none of the criteria for a positive response were met.

Statistics:

Chi-square analysis was applied with a significance level of 0.05.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
WITHOUT S9 METABOLIC ACTIVATION:
Following the 3-hour treatment without S9 mix, a severe toxicity was observed at dose-levels ≥ 100 µg/mL, as shown by a 100% decrease in the PD.

Following the 24-hour treatment without S9 mix, a slight to severe toxicity was observed at dose-levels ≥ 100 µg/mL, as shown by a 39 to 100% decrease in the PD.

Following the 3-hour treatment with S9 mix, a moderate to severe toxicity was observed from the lowest tested dose-level (i.e. 10 µg/mL), as shown by a 58 to 100% decrease in the PD.

Based on toxicity data obtained from the preliminary experiment, the following dose-levels were tested using a treatment volume of 1% (v/v) in culture medium: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL. However, the highest dose-level of 100 µg/mL induced neither the recommended level of cytotoxicity nor a precipitation in the culture medium. No slides were prepared from this experiment. A new treatment was thus performed as the first experiment.

The following range of dose-levels was used in the first experiment (3 h treatment + 24 h recovery) and second experiment (24 h treatment + 20 h recovery): 2.29, 6.86, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/mL.

A slight to marked precipitate was observed in the culture medium exclusively at the beginning of the treatment periods at dose levels ≥ 555.6 µg/mL and ≥ 1666.7 µg/mL, in the first and second experiments, respectively.

Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels ≥ 61.7 µg/mL as shown by a 28 to 100% decrease in the PD.
Following the 24-hour treatment, a moderate to severe toxicity was induced at dose-levels ≥ 555.6 µg/mL as shown by a 50 to 100% decrease in the PD.

Based on the level of cytotoxicity, the dose-levels selected for micronucleus analysis were as follows:
6.86, 20.6 and 61.7 µg/mL for the 3-hour treatment, the latter inducing a 28% decrease in the PD, and the higher dose-level being too cytotoxic,
61.7, 185.2 and 555.6 µg/mL for the 24-hour treatment, the latter inducing the recommended level of toxicity (50% decrease in the PD).

WITH S9 METABOLIC ACTIVATION:
Based on toxicity data obtained from the preliminary experiment, the following dose-levels were tested using a treatment volume of 1% (v/v) in culture medium: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL. However, the highest dose-level of 100 µg/mL induced neither the recommended level of cytotoxicity nor a precipitation in the culture medium. No slides were prepared from this experiment. A new treatment was thus performed as first experiment.

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were as follows:
2.29, 6.86, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/mL for the first experiment,
0.25, 0.76, 2.29, 6.86, 20.6, 61.7, 185.2 and 555.6 µg/mL for the second experiment.

In the first experiment, a slight to severe toxicity was induced at dose-levels ≥ 6.86 µg/mL, as shown by a 25 to 100% decrease in the PD.

In the second experiment, a severe toxicity was induced at dose-levels ≥ 185.2 µg/mL, as shown by a 100% decrease in the PD.
Based on the level of cytotoxicity, the dose-levels selected for micronucleus analysis were as follows:
2.29, 6.86 and 20.6 µg/mL for the first experiment, the latter inducing the recommended level of cytotoxicity (50% decrease in the PD).
6.86, 20.6 and 61.7 µg/mL for the second experiment, higher dose-levels being too cytotoxic.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No increase in the frequency of micronucleated cells was observed in any of the 3 three consecutive doses selected for evaluation.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

ZAN 573 did not induce chromosome damage in cultured L5178Y TK+/-mouse lymphoma cells, in the absence or in the presence of metabolic activation. This study is informative for evaluation of the toxicity of members of the cyclic acid anhydride category, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.