Bis(hydroxyethyl) terephthalate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

The basis for this read-across approach is that the target substance is expected to undergo transformation into terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1). The toxicity of the metabolites will accurately predict the toxicity of the bis(2-hydroxyethyl)terephthalate (BHET; 959-26-2; 213-497-6). Refer to the JUSTIFICATION FOR READ-ACROSS OF TOXICOLOGICAL INFORMATION in Section 13 of this dossier for further details.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

SIDS Reliability (1): valid without restriction

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Terephthalic acid was supplied by the BP Amoco Chemicals Corporation. Purity was not noted but typically exceeds 99%

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were about 6-8 weeks of age at study initiation.

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

Animals (5/sex/group) were dosed ip with vehicle (negative control), 200, 400 or 800 mg/kg TPA, or 50 mg/kg cyclophosphamide (positive control) in corn oil in a volume of 20 ml/kg body weight and were sacrificed at 24 hours. Additional animals (5/sex/group) were treated with vehicle or 800 mg/kg TPA and sacrificed at 48 hours. An additional group of 5 males and 5 females were dosed with 800 mg/kg TPA as a replacement group in case of mortality.

Duration of treatment / exposure:

24 hours for 200, 400, 800 mg/kg groups, and negative and positive control groups. 48 hours for additional 800 mg/kg group. Animals were sacrificed at the end of the period.

Frequency of treatment:

once

Post exposure period:

24 hours for 200, 400, 800 mg/kg groups, and negative and positive control groups. 48 hours for additional 800 mg/kg group.

Dose / conc.:

200 mg/kg bw (total dose)

Dose / conc.:

400 mg/kg bw (total dose)

Dose / conc.:

800 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

intraperitoneal dosed with corn oil vehicle: 50 mg/kg cyclophosphamide

Tissues and cell types examined:

bone marrow, polychromatic erythrocytes

Details of tissue and slide preparation:

Bone marrow samples from animals sacrificed at 24 and 48 hours were scored under oil immersion (2000 polychromatic erythrocytes (PCE) per animal) for the presence of micronuclei. The number of micronucleated normocytes per 2000 PCE was also assessed. The proportion of PCE to total erythrocytes was also recorded on a per 1000 erythrocyte basis.

Evaluation criteria:

Sample were scored under oil immersion (2000 polychromatic erythrocytes (PCE) per animal) for the presence of micronuclei. The number of micronucleated normocytes per 2000 PCE was also assessed. The proportion of PCE to total erythrocytes was also recorded on a per 1000 erythrocyte basis.

Criteria for a valid test: The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the negative control vehicle. The incidence rate in the positive control group must be significantly increased relative to the negative control (p<0.05, Kastenbaum-Bowman Tables.

Statistics:

Statistical significance for the incidence of micronucleated polychromatic erythrocytes was determined using Kastenbaum-Bowman Tables.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mortality was observed in 1/15 male mice that had been treated with 800 mg/kg bw TPA. One mouse from the replacement group was used in place of the animal that died. Clinical signs following treatment with either dose of TPA included lethargy and piloerection. Negative and positive control animals appeared normal during the course of the study.

The total number of micronuclei observed per 20,000 PCE in animals treated with vehicle, 200, 400, or 800 mg/kg bw for 24 hours was 4, 8, 5, and 4, respectively. The incidence of micronuclei in animals treated with 800 mg/kg bw also did not differ from the vehicle control at 48 hours (2/20,000 vs. 8/20,000, respectively). There was no difference between males and females.

The test was valid, as the incidence of micronuclei in the vehicle control was not greater than 5/1,000 PCE and the number of micronucleated cells in the positive controls (382/20,000) was significantly different (p<0.05) from the vehicle control. Reductions (up to 9%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the treated groups relative to the vehicle control, suggesting that the test article did not inhibit erythropoiesis. Greater reductions in this ratio were observed in animals treated with cyclophosphamide (25-29%).

Conclusions:

Terephthalic acid (202-830-0; 100-21-0) was negative in the test.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Rodent Dominant Lethal Mutagenesis Assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

AUG 1976 to DEC 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Rodent Dominant Lethal Test)

Version / remarks:

pre-dates methodology: 1977 - Rodent Dominant Lethal Mutagenesis Assay

Principles of method if other than guideline:

Rodent Dominant Lethal Mutagenesis Assay

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

Polyester grade EG was received from Union Carbide Corporation, Hahnville, Louisiana, on January 22, 1975. The sample was 99.93% monoethylene glycol by analysis at the time of shipment. On March 27,1978, the sample was analyzed by silylation gas chromatography and found to be 99.82% monoethylene glycol plus 0.18% diethylene glycol.

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: 155 days
- Assigned to test groups randomly: Yes
- Housing: stainless-steel wire cages
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Photoperiod: controlled lighting (12 hr light)

Route of administration:

oral: feed

Details on exposure:

15 male F2 rats per dosage group from the reproduction study were removed from their dosing regimen at 155 days of age and bred to three separate sets (one set per week) of 15 untreated females.

- Premating exposure period for males (P and F1) as appropriate: gestation (F2 generation from a three-generation study) and life time through mating (age 155 days at start of first mating period)
- Premating exposure period for females (P and F1) as appropriate: 0 days

Duration of treatment / exposure:

3 consecutive weekly mating periods

Frequency of treatment:

daily

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Control animals:

yes, concurrent no treatment

Positive control(s):

15 diet control F2 males were given an intraperitoneal dose of 500 mg/kg bw of triethylenemelamine (TEM, Polysciences, Inc., Warrington, Pa.) on the day before mating (positive controls), and mated with three groups of females as previously described.

Tissues and cell types examined:

Each female was killed on Day 12 of gestation, and the ovaries and uteri were examined for the number of live and dead fetuses.

Statistics:

Continuous data such as body weights were compared by analysis of variance validated by Bartlett’s test for homogeneity of variance. Duncan’s multiple range test was used to identify individual mean differences when indicated by a significant F value.

Where Bartlett’s test indicated heterogeneous variances, t-tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the method of Wiel. Discontinuous data, such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the Chi-squared test and by Fisher's exact test.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Reproductive parameters, including the percentage of pregnant rats, total number of early fetal deaths per pregnant female, and median number of fetal deaths were unaffected by ethylene glycol treatment in all three mating periods. No significant dominant lethal effects were observed in females mated to ethylene glycol-treated males.

Conclusions:

Ethane-1,2-diol (203-473-3; 107-21-1) is negative for germ cell gene mutation according to a dominant lethal mutagenicity study.

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Bis(hydroxyethyl) terephthalate

EC Number:

213-497-6

EC Name:

Bis(hydroxyethyl) terephthalate

Cas Number:

959-26-2

Molecular formula:

C12H14O6

IUPAC Name:

bis(hydroxyethyl) terephthalate

Test material form:

solid

Specific details on test material used for the study:

Bis(2-hydroxyethyl) terephthalate value is read-across from supporting terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1) data.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Based on a mammalian erythrocyte micronucleus assay, terephthalic acid (202-830-0; 100-21-0) is not genotoxic.

Based on a rodent dominant lethal mutagenesis assay, ethane-1,2-diol (203-473-3; 107-21-1) is not genotoxic.

Information on the source substances is considered to be directly applicable to an equivalent molar amount of the target substance; therefore, BHET was predicted to be non-genotoxic.

Applicant's summary and conclusion

Conclusions:

BHET is predicted to be non-genotoxic.

Executive summary:

The cytogenicity of terephthalic acid was assessed in a GLP-compliant assay according to OECD 474. Male and female ICR mice were exposed by intraperitoneal injection to 200, 400, or 800 mg/kg terephthalic acid. The incidence of micronuclei did not differ from the vehicle control. The assay was considered valid. Terephthalic acid was reported as negative under the conditions of this mammalian erythrocyte micronucleus test. The mammalian germ cell gene mutation potential of ethylene glycol was assessed in a rodent dominant lethal mutagenesis assay. The F2 males from a three-generation reduction study were exposed in utero and up to 155 days of age prior to being mated with untreated females. The concentrations assessed were 40, 200 and 1 000 mg/kg bw/day. Reproductive parameters, including the percentage of pregnant rats, total number of early fetal deaths per pregnant female, and median number of fetal deaths were unaffected by ethylene glycol treatment. Treatment of male rats with ethylene glycol in the diet during gestation, up to mating and subsequent mating to untreated females over 3 weeks produced no indication of a dominant lethal mutagenic effect. Information on the source substances is considered to be directly applicable to an equivalent molar amount of the target substance; therefore, BHET was predicted to be non-genotoxic.