Diundecyl phthalate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Di(isononyl) Phthalate (DINP) and Di(isodecyl) Phthalate (DIDP) Are Not Mutagenic

Author:

Mckee R H

Year:

2000

Bibliographic source:

J. Appl. Toxicol. 20, 491–497 (2000)

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

(this information was not provided)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich

EC Number:

271-091-4

EC Name:

1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich

Cas Number:

68515-49-1

IUPAC Name:

bis(8-methylnonyl) phthalate

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 17-35 g
- Housing: husbandry was consistent with the recommendations of the National Institute of Health (NIH), 1985.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No information

Duration of treatment / exposure:

single dose 1 day

Frequency of treatment:

Once

Post exposure period:

24, 48 and 72 hours (killing times)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

(vehicle control)

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide. 5 males and 5 females. Killing time: 24 h post administration
- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes cells (from both femora)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range-finding study was used to select doses for the definitive test, but, because there was no evidence of toxicity, the highest dose was 5 g/kg based on TSCA (Toxic Substances Control Act) guidelines (US EPA 798.5395).

TREATMENT AND SAMPLING TIMES:
Both femurs were removed from each treated mouse and the proximal ends were cut to expose the bone marrow, which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifugation and slides were prepared.
DETAILS OF SLIDE PREPARATION:
After fixation in methanol, the slides were stained with acridine orange for ca. 1–2 min.

METHOD OF ANALYSIS:
Slides were evaluated at 400 X by fluorescence microscopy.
A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei.

Statistics:

Statistical analysis included means and standard deviations of the micronucleus test data, and a test of equality of means was performed by standard one-way analysis of variance (Snedecor G., 1989).
Because the only statistically significant differences were between the positive and negative control groups, no additional statistical analysis was carried out.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
There was no evidence of toxicity up to the highest dose tested.

RESULTS OF DEFINITIVE STUDY
- Mortality: All animals survived.
- Signs of toxicity: there was no evidence of toxicity.
- Induction of micronuclei (for Micronucleus assay): There were no significant differences between treatment groups and controls for either male or female mice at any dose or collection time.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values.
- Positive control: Treatment with cyclophosphamide, the positive control, induced a significant increase in micronuclei in both males and females, confirming the adequacy of the testing procedure.

Any other information on results incl. tables

Table 1. In vivo micronucleus evaluation of DIDP (68515–49–1) in male CD-1 mice a

Treatment	Dose	Harvest time (h)	% Micronucleated PCEs (1000 per animal)			Ratio PCE/NCE mean ± S.E.
			Males	Females	Total	Males	Females
Vehicle control (corn oil)	10 ml kg-1	24	0.06 ± 0.04	0.04 ± 0.02	0.05 ± 0.02	0.67 ± 0.31	0.58 ± 0.18
		48	0.00 ± 0.00	0.04 ± 0.02	0.02 ± 0.01	0.59 ± 0.08	1.00 ± 0.08
		72	0.08 ± 0.04	0.02 ± 0.02	0.05 ± 0.02	0.89 ± 0.17	1.09 ± 0.13
Positive control (cyclophosphamide)		24	2.04 ± 0.34*	1.76 ± 0.28*	1.90 ± 0.21*	0.50 ± 0.12	0.48 ± 0.08
DIDP dose	1250 mg kg-1	24	0.16 ± 0.04	0.10 ± 0.05	0.13 ± 0.03	0.35 ± 0.08	0.97 ± 0.09
		48	0.06 ± 0.02	0.06 ± 0.04	0.06 ± 0.02	0.69 ± 0.14	1.15 ± 0.10
		72	0.02 ± 0.02	0.10 ± 0.04	0.06 ± 0.03	0.79 ± 0.15	1.10 ± 0.14
	2500 mg kg-1	24	0.08 ± 0.04	0.08 ± 0.04	0.08 ± 0.02	0.49 ± 0.08	0.83 ± 0.19
		48	0.02 ± 0.02	0.06 ± 0.02	0.04 ± 0.02	0.91 ± 0.16	1.00 ± 0.15
		72	0.04 ± 0.02	0.00 ± 0.00	0.02 ± 0.01	0.62 ± 0.16	0.96 ± 0.11
	5000 mg kg-1	24	0.08 ± 0.04	0.06 ± 0.02	0.07 ± 0.02	0.38 ± 0.05	1.05 ± 0.17
		48	0.00 ± 0.00	0.02 ± 0.02	0.01 ± 0.01	0.90 ± 0.10	1.02 ± 0.17
		72	0.02 ± 0.02	0.06 ± 0.04	0.04 ± 0.02	0.89 ± 0.03	1.27 ± 0.19

a Values are means ± SEM; *significantly greater than the corresponding vehicle control (P < 0.05).

PCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte

Applicant's summary and conclusion

Conclusions:

DIDP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Executive summary:

An in-vivo micronucleus test was performed with Di(isodecyl) Phthalate (DIDP) according to OECD guideline 474 and EU method B.12. Five mice per sex and per group were exposed once (single dose) to test item diluted in corn oil at doses of 0 (vehicle control), 1250, 2500 and 5000 mg/kg bw, based on preliminary results. Animals were killed 24, 48 and 72 hours after administration of the test compound. Cyclophosphamide was used as a positive control (5 mice per sex at 20 mg/kg and killed 24 h post treatment). For each animal, bone marrow was extracted from both femora and the slides were prepared for erythrocyte micronuclei observation. There were no significant differences between treatment groups and controls for either male or female mice at any dose or collection time. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DIDP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.