Dimethyl isophthalate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Evaluation of a three-exposure bone marrow micronucleus protocol: Results with 49 chemicals

Author:

Shelby MD, Erexson GL, Hook GL and Tice RR

Year:

1993

Bibliographic source:

Envir Molec Mutagen 21(2): 160-179

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: U.S. National Toxicology Program (NTP) Protocol for Bone Marrow Erythrocyte Micronucleus Assay

Version / remarks:

1993

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

older version, 1993

Deviations:

yes

Remarks:

Bone marrow smears from each animal were evaluated for the percentage of PCE among 200 erythrocytes in this 1993 study, rather than the current requirement (2017) of 500 erythrocytes.

Principles of method if other than guideline:

Repeated exposure of animals to test substance, after which animals are euthanised and bone marrow from femurs is extracted and erythrocytes examined for evidence of micronucleated polychromatic cells. Cytotoxicity measures indicate whether test material was absorbed and bone marrow exposed.

GLP compliance:

no

Remarks:

but performed in the spirit of GLP according to U.S. NTP quality standards

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl terephthalate

EC Number:

204-411-8

EC Name:

Dimethyl terephthalate

Cas Number:

120-61-6

Molecular formula:

C10H10O4

IUPAC Name:

dimethyl terephthalate

Test material form:

solid: crystalline

Details on test material:

dimethyl terephthalate. No information on purity

Specific details on test material used for the study:

Identical substance as used in NTP assays, obtained from NTP Chemical Repository.
Vendor was Radian Corporation, Austin, TX, USA
Chemicals (n = 49) were coded and sent to two laboratories for testing. Data was analyzed and results were called before the test chemicals were decoded, to avoid bias.

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

Mice were obtained from the NTP production facility at Taconic Farms.
Age was between 9 and 14 weeks.
Weight was 25-33 g, +/- 2 g.
Conditions used were those standard for in vivo NTP assays.
Idenification numbers were randomly assigned to mice prior to euthanasia.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Corn oil

Details on exposure:

The substance was prepated in corn oil and suspended using a Tek-Mar Tissumizer.
All test chemicals were administered within 30 minutes of preparation.
All IP treatments were at a volume of 0.4 ml per mouse.
Suspended DMBA (positive control) was stored at room temperature.

Duration of treatment / exposure:

IP injection daily on 3 consecutive days

Frequency of treatment:

IP injection daily on 3 consecutive days

Post exposure period:

Generally, animals were observed/monitored twice daily.
Mice were euthanized with CO2 24 hours after the the third treatment.
Animals were euthanised with CO2 asphyxiation. Bone marrow was extracted for analysis of erythrocyte chromosomal material (2 slides/tissue/mouse).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 750 mg/kg bw/day (actual dose received)

Remarks:

While the aim of the high dose was 2000 mg/kg (the limit dose), the dose of 1750 mg/kg was selected because the chemical could not be suspended in corn oil at concentrations that would permit higher doses.

Doses / concentrations 2

Dose / conc.:

875 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

438 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5-7

Control animals:

yes, concurrent vehicle

Positive control(s):

yes: 9,10-dimethylbenzanthracene;
- Justification for choice of positive control(s): demonstrated to result in appropriate increased incidence of micronucleated PCEs in mice
- Route of administration: IP
- Doses / concentrations: 12.5 mg/kg. Purchased from Eastman Kodak (Rochester, NY, USA).

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Duplicate slides were prepared fixed in absolute methanol and stained with acridine orange

Evaluation criteria:

Bone marrow smears were evaluated using fluorescence microscopy for determination of the percentage of polychromatic erythrocytes and micronucleated erythrocytes. Two hundred erythrocytes were determined per slide. The maximum administered dose determination experiments were conducted to more accurately estimate the maximum dose to be tested in the primary MN test. The number of MN-PCEs were evaluated among 2000 PCEs, and the number of PCEs were determined among 200 erythrocytes. The initial test utilized bone marrow tissue. In some cases peripheral blood smears (obtained 2 days after the last treatment in the dose determination experiment) were scored to verify a positive result in the initial bone marrow MN test. The frequency of MN-PCE among 2000 PCE and the percentage of PCE among 2000 erythrocytes were determined. If peripheral blood smears were unavailable or did not exhibit a significant increase in MN-PCE, a second in vivo BM test was conducted.

Statistics:

The data were analyzed using a specially designed software package, the MN Assay Data Management and Statistical Software Package (version 1.4, ILS, 1990). The level of significance was set at an alpha level of 0.05. The number of MN-PCE was pooled within the each dose group and analysed using a trend Test (1-tailed), which incorporated a variance inflation factor to account for excess animal variablity. If the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analysed for a significant positive trend after data at the highest dose only has been excluded. In this case, the alpha level is adjusted to 0.01 to protect against false positives. The % PCE data was analysed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each groups and the concurrent solvent control group was by an unadjusted 1-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Initial results were negative at all doses through 1750 mg/kg. No mortality was observed at any dose.

Applicant's summary and conclusion

Conclusions:

The test was negative (not genotoxic). Formation of micronuclei in newly-formed bone marrow erythrocytes was not elevated in male mice after 3 IP administrations of DMTP up to the high dose of 1750 mg/kg bw (actual dose). This is the highest dose technically possible for suspension in the corn oil vehicle, and can be considered the limit dose, just shy of 2000 mg/kg bw. Data can be read-across from DMTP to dimethyl isophthalate (DMIP), based on common functional groups. The substances are isomers. The similarities in structure are likely to apply to metabolites as well, with DMIP breaking down to isophthalic acid, just as DMTP is known to break down to terephthalic acid; these are isomers. Similarity in structures and break-down products for the two substances is the basis for the reading-across of data for this endpoint. This is adequate to fulfill the information requirements of Annex IX, to be the basis for classification and labelling decisions, and for risk assessment.