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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Older proprietary study commissioned by the US FDA

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1971
Report Date:
1974

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro cytogenetic study investigating anaphase chromosomes in human fibroblast cells.
GLP compliance:
no
Remarks:
: study pre-dates GLP
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test material, adipic acid (referred to as Compound FDA 71-50), of food processing quality, was supplied byt the Food and Drug Administration.
A read across is proposed based on structural similarities between the substances.

Method

Target gene:
Not applicable - chromosome aberration study
Species / strain
Species / strain / cell type:
other: human fibroblasts
Details on mammalian cell type (if applicable):
Human embryonic lung cultures, negative for adventitious agents (viruses, mycoplasma). The cells were at passage 19. The cells had been transferred using 0.025% trypsin and planted in 32 oz. prescription bottles containing 40 ml tissue culture medium. When growth was approximately 95% confluent the cells were removed from the glass using trypsin, centrifuged, and frozen in tisue culture medium containing DMSO. Cells were frozen in vials in the vapour phase of liquid nitrogen at a concentation of 2x10E6 cells/ml.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0 (saline), 2, 20 and 200 µg/ml
Vehicle / solvent:
Saline (0.85%).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
Remarks:
Migrated to IUCLID6: 0.1 µg/ml
Details on test system and experimental conditions:
When needed, the frozen cell vials were removed from liquid nitrogen, quick-thawed in a 37°C water bath, washed free of DMSO, suspended in tissue culture medium (MEM plus 1% glutamine, 200 units/ml penicillin and 200 µg/ml of streptomycin and 15% foetal calf serum) and planted in milk dilution bottles at a concentration of 5x10E5 cells/ml. The test material was added to the bottles, using three bottles per dose level, 24 hours after planting.

A preliminary toxicity test was conducted to determine the dose levels for the main study. Logarithmic doses of the test material in saline were added to a series of tubes containing 5x10E5 cells/ml which were almost confluent. The cells were examined at 24, 48 and 72 hours. Any cytopathic effect or inhibition of mitoses was scored as toxicity. Five more closely spaced dose levels were employed within the two logarithmic dosages, the higher of which showed toxicity and the lower no effect.

Cells were incubated at 37°C and examined twice daily to determine when an adequate number of mitoses were present. Cells were harvested by shaking when sufficient mitoses were observed (usually 24-48 hours after planting), centrifuged, and fixed in absolute methanol:glacial acetic acid (3:1) for 30 minutes. The cultures were centrifuged, decanted and suspended in acetic acid-orcein stain (2%) and a drop of suspension placed on a clean dry slide and covered with a coverglass. The coverglasses were sealed with clear nail polish and examined immediately.
Evaluation criteria:
The following was recorded: acentric fragments, bridges, multipolar cells, cells with greater than 10 aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. Mitotic indices were obtained by counting at least 200 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.
Statistics:
Formal statistical analysis was not carried out.

Results and discussion

Test results
Species / strain:
other: human fibroblasts
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The negative controls contained two cells with bridges, one of which contained an acentric fragment. The test compound was negative except for one cell which contained a bridge at the high dosage level. The positive controls were within normal limits.
Remarks on result:
other: strain/cell type: embryonic lung cultures
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary data

Treatment Group

Mitotic Index*

No. Cells

% Cells with Acentric Fragments

% Cells with Bridges

% Multipolar cells

% Cells with other Aberrations**

% Cells with Aberrations***

Adipic Acid 2 µg/ml

1

100

0

0

0

0

0

Adipic Acid 20 µg/ml

1

100

0

0

0

0

0

Adipic Acid 200 µg/ml

1

100

0

1

0

0

1

Saline Control

1

100

1

2

0

0

3

Positive Control (TEM)

1

100

3

12

0

0

15

* Percent of cells in mitosis: 200 cells observed/dose level

** Cells that have polyploidy, pulverisation, fragments or greater than 10 aberrations

*** Duplicate aberrations in a single cell will cause this value to be a % less than a summation of the % aberration seen.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No evidence of clastogenicity was seen under the conditions of this assay.
Executive summary:

Adipic acid was tested in an in vitro chromosome aberration assay, with human lung fibroblasts. The cells were cultured with the test material at concentrations of 0 (saline), 2, 20 and 200 µg/ml. Chromosome aberrations were evaluated when the cells were in anaphase. Only one cell (in the 200 µg/ml group) was found to contain a bridge, no other aberrations were seen. Therefore, adipic acid was negative for mutagenic activity in the in vitro mammalian cell chromosome aberration assay.