Butanoic acid, 3-oxo-, 1,1'-[2-[(1,3-dioxobutoxy)methyl]-2-ethyl-1,3-propanediyl] ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Dominant Lethal Mutations and Antifertility Effects of Di-2-Ethylhexyl Adipate and Diethyl Adipate in Male Mice

Author:

Singh A R

Year:

1975

Bibliographic source:

Toxicology and Applied Pharmacology 32, 566-576 (1975)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

not specified

Type of assay:

other: dominant lethal mutation assay (Cattanach et al., 1968)

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl adipate

EC Number:

205-477-0

EC Name:

Diethyl adipate

Cas Number:

141-28-6

Molecular formula:

C10H18O4

IUPAC Name:

diethyl adipate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Harlan Eastman Kodak Company, Rochester, New York, 14650

Test animals

Species:

mouse

Strain:

other: Harlan/ICR albino Swiss strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries, Inc., Cumberland, Indiana.
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: 25-30 g
- Fasting period before study: not specified.
- Housing:
- Diet (e.g. ad libitum): Purina Laboratory Chow, Ralston Purina Company, St. Louis, Missouri., provided ad libitum.
- Water (e.g. ad libitum): fresh, clean tap water provided ad libitum
- Acclimation period: not specified.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: None

Duration of treatment / exposure:

Single injection to males that were caged immediatley with two virgin females per week for 8 weeks.

Frequency of treatment:

Once

Post exposure period:

None

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mL/kg

Remarks:

Control, distilled water equivalent to the highest dose of test item administered.

Doses / concentrations 2

Dose / conc.:

0.44 other: mL/kg

Remarks:

1/5 of LD50 previously calculated.

Doses / concentrations 3

Dose / conc.:

0.73 other: mL/kg

Remarks:

1/3 of LD50 previously calculated.

Doses / concentrations 4

Dose / conc.:

1.1 other: mL/kg

Remarks:

1/2 of LD50 previously calculated.

Doses / concentrations 5

Dose / conc.:

1.46 other: mL/kg

Remarks:

2/3 of LD50 previously calculated.

No. of animals per sex per dose:

10 males per dose; 20 females weekly per dose.

Control animals:

yes, sham-exposed

Examinations

Tissues and cell types examined:

Uterine horns, ovaries (number of corpora lutea, number of implantations, pre-implantation loss, early and late fetal deaths, and viable fetuses)

Evaluation criteria:

Dominant lethal mutation was determined directly from the increased number of early fetal deaths in individual mice, and indirectly from the reduced number of total implantations (Epstein et al., 1970).

Statistics:

A two-factor (dose and time) analysis of variance was employed, using least-squares methods for unequal cell frequencies and matrix inversion techniques (Snedecor and Cochran, 1967). All data were tested for normality, and those yielding non-Gaussian distributions were appropriately corrected. To assure homogeneity of variance, the following transformations were used: (a) X2, rather than X, for number of implants/ pregnancy, and for number of live fetuses/pregnancy; (b) In(X + 0.1) for number of early fetal deaths; and (c) ln(100 — X), where X is the percentage of pregnancies, in evaluating incidences of pregnancies.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Late fetal deaths (i.e., nonviable, fully formed fetuses) were uncommon; their incidence tended to be equal to or less than that for controls. When present, these deaths apparently occurred randomly, without any dose- or time- relationship to treatment.
For rate of pregnancies, analyses indicated there was a significant (p < 0.05) effect from dose in the first 3 weeks, in which the higher doses produced fewer pregnancies than did the controls. However, no significant effect of dose or time (weeks) was revealed for the 4- to 8-week period.
There was no difference in the number of implants in the treated groups versus control animals.
For early fetal deaths, analyses of the data from all treated groups (with controls) indicated a significant effect of dosage. A similar analysis without controls did not reveal a dosage effect. The postmeiotic data showed a significant dose effect with controls. No dose-effect was apparent when the controls were deleted. For the prerneiotic stage, no significant effects were detected.
The treated groups showed a significant effect of time and dose- week interaction for the 8-wk period in the number of live fetuses/pregnant mouse when controls were included; when analyzed without controls, dose—week interaction was no longer significant. While the analyses did not reveal any significant effects during the first 3 wk after test item administration, significant effects (with and without controls) were noted for weeks and dose- week interaction during the premeiotic stage.

Any other information on results incl. tables

Table 1. Percentage of pregnancies in mice following administration of DEA a, b

Week no.	Control (c)	0.44	0.73	1.10	1.46
1	70	40	40	20	30
2	60	50	45	40	45
3	60	65	55	55	45
4	70	90	70	50	65
5	65	70	80	50	45
6	85	65	75	80	85
7	85	70	85	95	90
8	75	85	85	80	75
Overall mean and SE	71±3.5	67± 5.8	67± 6.3	59±8.7	60± 7.7

a Males were mated sequentially over a period of 8 wk after a single ip injection of DEA.

b Values are based on 20 females mated weekly to 10 males.

c In this group, animals were injected with distilled water equivalent to the highest dose of the test compound administered.

 

Table 2. Total number of implants per pregnancy in mice after ip administration of DEA to males (mean ± SE)

Week no.	Control	0.44	0.73	1.10	1.46
1	9.6± 0.34	10.4± 1.00	10.4± 0.50	9.5 ± 1.26	10.7 ± 0.71
2	9.8 ± 0.93	10.9 ± 0.38	8.7±1.32	9.5 ± 1.16	10.4± 0.80
3	10.7 ±0.47	9.9 ±0.42	9.7 ±0.63	10.0 ± 0.65	8.0 ± 1.22
4	11.3 ± 0.35	10.9 -r 0.25	10.7 ±0.50	10.6±0.40	9.2± 0.54
5	10.7 ± 0.46	10.2 ± 0.70	10.8 ± 0.31	11.3 ± 0.26	10.8 ± 0.98
6	10.5 ± 0.53	11.1± 0.54	11.1± 0.28	11.5 ± 0.35	11.6± 0.33
7	9.9 ± 0.79	10.2 ± 0.37	12.4± 0.45	11.5 ± 0.35	11.8 ± 0.42
8	11.1± 0.60	11.3 ± 0.24	11.2 ± 0.60	11.4 ± 0.31	10.5 ± 0.55
Overall mean ± SE	10.5 ± 0.56	10.6 ± 0.49	10.6 ± 0.57	10.7 ± 0.59	10.4 ± 0.69

 

Table 3. Early fetal deaths per pregnancy in mice after ip administration of DEA to males (mean ± SE)

Week no.	Control	0.44	0.73	1.10	1 .46
1	0.50 ±0.17	0.75 ± 0.49	1.25 ± 1.11	1.75 ± 1.44	2.17 ± 1.19
2	0.42 ± 0.26	0.70 ± 0.21	0.89 ± 0.31	1.50 ± 0.33	1.33 ±0.50
3	0.33 ± 0.19	0.46 -r 0.14	0.64± 0.36	1.00 ± 0.62	1.00 ± 0.50
4	0.21 ±0.11	0.44± 0.17	0.50 ± 0.20	0.80    0.25	1.08 ± 0.43
5	0.31± 0.17	0.50± 0.23	0.56 ± 0.20	0.80± 0.47	0.89 ± 0.39
6	0.41±0.15	0.69 ±0.31	0.67 ±0.25	0.56 ± 0.18	0.12 ± 0.08
7	0.41± 0.15	0.36J 0.17	0.71± 0.21	0.68 ± 0.32	0.44± 0.15
8	0.33 ± 0.16	0.41± 0.12	0.53 ± 0.21	0.69 ± 0.22	0.67 ± 0.32
Overall mean ± SE	0.37 ± 0.17	0.54 ± 0.23	0.72 ± 0.36	0.97 ± 0.49	0.96 ± 0.45

 

Table 4. Number of live fetuses per pregnancy in mice after ip administration of DEA to males (mean ± SE)

Week no.	Control	0.44	0.73	1.10	1.46
1	8.9 ± 0.38	9.6± 1.43	9.1± 1.38	7.8 ± 2.66	8.5 ± 1.77
2	9.3 ±0.82	10.1±0.43	7.8 ± 1.54	8.0± 1.25	9.1±1.20
3	10.3 ± 0.38	9.5 ± 0.37	9.1± 0.94	9.8 ± 0.63	8.1± 1.11
4	11.1± 0.37	10.3 ± 0.36	10.2± 0.48	9.8 ±0.39	7.5 ± 0.74
5	10.3 ±0.44	9.6± 0.68	10.3 ±0.31	10.0± 0.42	10.0± 0.90
6	9.8 ± 0.56	10.2 ± 0.79	10.3 ± 0.29	10.9 ± 0.40	11.0 ± 0.32
7	9.4 ± 0.77	9.9±0.39	11.6±0.51	10.7±0.57	11.2±0.51
8	10.7 ± 0.61	10.9 ± 0.26	10.5 ± 0.66	10.6± 0.35	9.9 ±0.64
Overall mean ± SE	10.0 ± 0.54	10.0 ± 0.59	9.9 ± 0.76	9.7 ± 0.83	9.4 ± 0.90

 

 

 

Applicant's summary and conclusion

Conclusions:

In a dominant lethal mutation assay with the test item in mice, no statistical significant difference was observed between treated groups and control, so the result of the test is negative.

Executive summary:

A dominant lethal mutation assay was carried out in Harlan/ICR albino Swiss male mice to investigate the germinal effects of the test item. The test item was administered intraperitoneally at dose levels of 1/5, 1 /3, l,'2, and 2/3 of the acute LD50 which correspond to 0.44, 0.73, 1.1 and 1.46 mL/kg. Ten males were injected at each dose level. Group of controls were injected ip with distilled water equivalent to the highest dose of test item administered. Immediately after injection, two virgin female mice were caged with each male mouse. During a period of 8 weeks the females were replaced weekly with two virgin females. Thus. each weekly group was composed of 20 female mice. Number of corpora lutea, total number of implantations, preimplantation losses, early and late fetal deaths, and viable fetuses were counted from the uterine horns and ovaries of sacrificed pregnant females. Dominant lethal mutation was determined directly from the increased number of early fetal deaths in individual mice, and indirectly from the reduced number of total implantations (Epstein et al., 1970). For rate of pregnancies, no significant effect of dose or time (weeks) was revealed during the study. There was no difference in the number of implants in the treated groups versus control animals. Late fetal deaths were lower in the treated animals but early fetal deaths were more common in treated groups. The authors claim that dosage had a significant effect on the number of live fetuses and that DEA thus increased the number of dominant lethals. Although there seems to be a dose response relationship for single weeks, no statistical significant difference exists between group and control data neither for any week nor for the overall data. Thus, the result of the test can be considered negative.