1,2-Benzenedicarboxylic acid, isononyl ester, manuf. of, by-products from

Genetic toxicity: in vitro

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report, GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Alkenes, C7-9, hydroformylation products, distn. residues, heavy cracked fraction

EC Number:

308-482-7

EC Name:

Alkenes, C7-9, hydroformylation products, distn. residues, heavy cracked fraction

Cas Number:

98072-31-2

IUPAC Name:

98072-31-2

Details on test material:

Identity: MRD-08-196
Molecular weight: 294-350 (approx)
Batch number: 0304008
Expiry: April 2013
Appearance: Yellow liquid
Storage conditions: Room temperature
Purity/Assay: 100%
Date received: 6 April 2010

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

In this study, blood taken from healthy male non-smoking donors was pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test substance. Following treatment the cells were arrested at metaphase using the mitotic inhibitor, Colcemid®. Chromosomes in these metaphase cells were then examined for the presence of chromosome aberrations. The best estimate of the aberration frequency is at the first cell division after initiation of treatment since certain types of damage may be lost during subsequent cell divisions. In this laboratory the cell cycle time for human lymphocytes in whole blood culture is approximately 13-14 hours.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver

Test concentrations with justification for top dose:

First Test:
35.27, 58.79, 97.98, 163.30, 272.16, 453.60, 756, 1260, 2100 and 3500 µg/mL

Second Test:
In the absence of S9 mix: 21.16, 35.27, 58.79, 97.98, 163.30, 272.16, 453.60, 756, 1260, 2100 and 3500 µg/mL
In the presence of S9 mix (5% v/v): 35.27, 58.79, 97.98, 163.30, 272.16, 453.60, 756, 1260, 2100 and 3500 µg/mL

Vehicle / solvent:

Soluble in acetone at 350 mg/mL (1M)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Mitomycin C (-S9), Cyclophosphamide (+S9)

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Solvent, positive and treatment cultures were established approximately 48 hours after commencement of incubation of lymphocyte cultures. Duplicate cultures were prepared throughout for each 3 hour treatment in the absence and presence of S9 mix. All cultures were centrifuged and resuspended in fresh medium just before treatment.

For treatments in the presence of S9 mix, 1 mL of medium was removed from each culture and discarded. This was replaced with 1 mL of S9 mix (2% v/v final concentration), followed by 50 µL of each test substance dilution (giving the same series of final concentrations as above). Acetone was used as the solvent control and Cyclophosphamide at a final concentration of 5 µg/mL was the positive control.

Evaluation criteria:

The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the solvent control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected.

The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.

The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

Statistics:

Yes

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of CAS# 98072-31-2 to induce chromosomal aberrations in human lymphocytes cultured in vitro.

 

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

 

In order to determine the toxicity of CAS# 98072-31-2 to cultured human lymphocytes, the mitotic index was assessed for all cultures treated with the test substance and the solvent control, acetone. Justification for concentration selection was based on cytotoxicity. On the basis of these data, the following concentrations were selected for metaphase analysis:

 

First test

In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 35.27, 163.30 and
453.60 µg/mL.

 

In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 58.79, 272.16 and 453.60 µg/mL.

 

Second test

In the absence of S9 mix - 21 hour continuous treatment: 30, 45 and 55 µg/mL.

In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 272.16, 453.60 and 1260 µg/mL.

 

In both the absence and presence of S9 mix, CAS# 98072-31-2 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.

 

No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis, in either test. 

 

All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

It is concluded that CAS# 98072-31-2 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.