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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key study: EU Method B.14 (GLP study). The substance was determined to be negative with and without metabolic activation in Salmonella strains.

Key study: EU Method B.10 (GLP study). The substance was determined to be negative with and without metabolic activation in human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

Concentration range in the first main test (with metabolic activation): 1000 - 5000 µg/ml
Concentration range in the second main test (with metabolic activation): 333 - 3330 µg/ml
Concentration range in the first main test (without metabolic activation): 1000 - 2400 µg/ml
Concentration range in the second main test (without metabolic activation): 100 - 1800 µg/ml
Concentration range in the second main test (without metabolic activation): 100 - 560 µg/ml

Vehicle / solvent:

Dimethylsulfoxide

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours
Fixation time:
First test with metabolic activation: 24 h fixation time.
Second test with metabolic activation: 48 h fixation time.
First test without metabolic activation: 24 h fixation time.
Second test without metabolic activation: 24 h fixation up to 560 ug/ml and 48 h fixation up to 1800 ug/ml.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity 1st experiment (3h exposure, 24h fixation): with and without S9 mix: 1000 µg/mL 2nd experiment (3h exposure, 48h fixation): with S9 mix: 1000 µg/mL; without S9 mix: 333 µg/mL 2nd experiment (3h exposure, 24h fixation): without S9 mix: 1000 µg

Additional information on results:

The test substance did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.
In the absence and presence of S9-mix the 48h fixation time in the second experiment an increase in the number of polypoploid cells was observed.
It is concluded that the test substance is not clastogenic in human lymphocytes under the experimental conditions.

Conclusions:

The substance was determined to be negative with and without metabolic activation system.

Executive summary:

A chromosome aberration test was conducted according to B.10 (GLP study).  The human lymphocytes cell line were treated with and without metabolic activation. The substance was solubilized in Dimethylsulphoxide and evaluated at a concentration ranging from 100 to 5000 µg/mL according to the test conditions. The substance was determined to be negative with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 100 - 5000 µg/plate
Concentration range in the main test (without metabolic activation): 100 - 5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide

Key result

Species / strain:

other: as specified above

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 µg/plate

Additional information on results:

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. No precipitation of the test substance was observed on the plates during the whole test period in all tester strains. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

Conclusions:

The substance was determined to be negative with and without metabolic activation system.

Executive summary:

An Ames test was conducted according to EU Method B.14 (GLP study). The Salmonella strains: TA98, TA100, TA1535 and TA1537 were used in the study and were treated with and without metabolic activation. The substance was solubilized in Dimethylsulphoxide and evaluated at a concentration ranging from 100 to 5000 µg/plate. The substance was determined to be negative with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key study: Read-across, test method similar to OECD 478. Based on read-across approach from experimental data on analogue hexanedioic acid, 1,6-diethyl ester (diethyl adipate), the test item AA-TMP was determined to be negative in a dominant lethal mutation assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance hexanedioic acid, 1,6-diethyl ester (diethyl adipate, DEA) which shares the same functional groups with the substance 1,1,1-Tri((1,3-dioxobutoxy)-methyl)-propane (AA-TMP) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: Read across from an analogue substance

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.

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

 

 

 

Conclusions:

Based on read-across approach from experimental data on the analogue diethyl adipate, the test item is considered as negative in a dominant lethal mutation assay.

Executive summary:

A dominant lethal mutation assay was carried out in Harlan/ICR albino Swiss male mice to investigate the germinal effects of diethyl adipate, DEA. 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.

Based on these results, the read-across approach was applied and AA-TMP can be considered as negative in a dominant lethal mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified for genetic toxicity according to the CLP Regulation (EC) no. 1272/2008.