Reaction mass of dimethyl adipate and dimethyl glutarate and dimethyl succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study similar to OECD 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only 4 of 5 recommended strains (TA1535, TA1537, TA98, TA100) were used in this study

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-dimethyl butanedioate; 1,5-dimethyl pentanedioate; 1,6-dimethyl hexanedioate

EC Number:

906-170-0

Molecular formula:

CH3CO2(CH2)nCO2CH3 Where n = 2, 3 and 4

IUPAC Name:

1,4-dimethyl butanedioate; 1,5-dimethyl pentanedioate; 1,6-dimethyl hexanedioate

Test material form:

other: liquid

Details on test material:

See information in the field "Confidential details on test material"

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

0, 100, 500, 1000, 5000, 10000 µg/plate for all bacterial strains with and without S9

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains with S9), 9-aminoacridine (TA1537 without S9), N-methyl-N'-nitrosoguanidine (TA1535 and TA100 without S9), 2-nitrofluorene (TA98 without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: as impregnation on paper disk (spot test in a closed system)

Experiments without S9 were performed by adding 10E8 bacteria (in 0.1 mL) to 2 mL of standard top agr, mixing immediately, and pouring on a Davis minimal agar plate. Experiments with S9 were performed in the same manner except that 0.5 mL of S9 mix was added before mixing.
A sterile disk saturated with test substance was placed in the center of one of two replicate plates. The plate with the disk was inverted and the other plate was stacked on top of it. Both plates were sealed in a plastic bag and incubated for 48 hours at 37°C.

Evaluation criteria:

A chemical was judged to be mutagenic or nonmutagenic in the assay after considering the various pieces of information provided by the statistical analysis

Statistics:

The data points (number of revertants/plate = x) were transformed prior to analysis using the power transformation y = xE0.2. Two analyses were performed. In the first analysis, the response observed at each concentration was compared to the control by a t-test of significance to determine which dose levels produced significant increases in mutation frequency. In the second analysis, the significance of the dose-response relationship was tested. Linear, quadratic, and higher order dose-response effects were tested in an F-test of significance. In addition, an analysis was conducted to determine whether the dose-response relationship was different in different trials.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to the top concentration of 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

This strain was tested in another assay described in Groot 2019 endpoint of this dossier

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In an intial cytotoxicity experiment on strain TA1535, DBE was tested at concentrations up to 10000 µg/plate and induced no toxicity.
No indication of mutagenic activity was observed in the spot test. The mutation frequencies were not significantly greater than te solvent control values and significant dose-response effects were not observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenicity testing in strain TA1535

 

Test concentration (µg/plate)	Number of revertants per plate (duplicate plates)
	Trial 1		Trial 2
Without metabolic activation
0 (DMSO)	21	32	24	34
100	37	32	31	33
500	35	38	29	32
1000	40	30	27	25
5000	31	36	32	37
10000	40	37	35	39
Positive control (MNNG, 4 µg/plate)	1521	1876	700	1070
Without metabolic activation
0 (DMSO)	9	22	15	21
100	15	23	20	18
500	12	20	16	18
1000	14	25	9	14
5000	13	28	25	19
10000	19	25	24	17
Positive control (2-AA, 10 µg/plate)	294	259	283	266

MNNG: N-methyl-N'-nitro-N-nistrosoguanidine2-AA: 2-aminoanthracene

Table 2: Mutagenicity testing in strain TA1537

 

Test concentration (µg/plate)	Number of revertants per plate (duplicate plates)
	Trial 1		Trial 2
Without metabolic activation
0 (DMSO)	11	15	11	8
100	13	15	10	13
500	14	8	18	9
1000	13	7	7	14
5000	6	13	9	14
10000	13	12	12	11
Positive control (9-AAC, 50 µg/plate)	580	411	383	357
Without metabolic activation
0 (DMSO)	12	11	10	9
100	8	12	10	13
500	11	9	11	9
1000	13	8	15	7
5000	9	10	12	8
10000	12	13	9	15
Positive control (2-AA, 10 µg/plate)	328	228	176	172

9-AAC: 9-aminoacridine          2-AA: 2-aminoanthracene

Table 3: Mutagenicity testing in strain TA98

 

Test concentration (µg/plate)	Number of revertants per plate (duplicate plates)
	Trial 1		Trial 2
Without metabolic activation
0 (DMSO)	31	34	37	32
100	26	22	28	30
500	20	27	30	26
1000	22	18	24	31
5000	26	23	30	31
10000	29	26	29	23
Positive control (2-NF, 25 µg/plate)	2772	2624	2619	2388
Without metabolic activation
0 (DMSO)	47	41	43	39
100	37	42	45	48
500	39	35	41	48
1000	43	47	58	54
5000	40	36	47	38
10000	39	37	34	54
Positive control (2-AA, 10 µg/plate)	1200	1363	1166	1114

2-NF: 2-nitrofluorene   2-AA: 2-aminoanthracene

Table 4: Mutagenicity testing in strain TA100

 

Test concentration (µg/plate)	Number of revertants per plate (duplicate plates)
	Trial 1		Trial 2
Without metabolic activation
0 (DMSO)	115	112	143	104
100	129	114	141	143
500	130	124	113	129
1000	105	115	112	115
5000	116	90	136	105
10000	100	121	130	98
Positive control (MNNG, 4 µg/plate)	1908	1989	1430	17995
Without metabolic activation
0 (DMSO)	146	127	143	172
100	120	115	139	114
500	146	117	130	138
1000	132	99	145	176
5000	117	114	126	136
10000	128	118	155	148
Positive control (2-AA, 5 µg/plate)	1102	820	863	1012

MNNG: N-methyl-N'-nitro-N-nistrosoguanidine2-AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

DBE is not mutagenic in the Ames reverse mutation assay (spot test format) with strains TA1535, TA1537, TA98 and TA100 up to the test concentration of 10000 µg/plate.

Executive summary:

The mutagenic potential of DBE has been assessed in the Salmonella typhimurium Ames test according to OECD guideline n° 471 and EC guideline n° B13/14.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were tested in presence and in absence of metabolic activation system from liver fraction of rats (S9 mix). The negative control was DMSO.

DBE was tested in 2 independent experiments performed according to the spot test method in which a sterile disk saturated with DBE was placed in the center of the plate.

Each strain of bacteria was exposed to dose-levels of 0, 100, 500, 1000, 5000 or 10000 µg/plate of the test item (two plates/dose-level). In each experiment, negative and positive controls were included using duplicate plates.

After 48 hours of incubation at 37°C in a sealed plastic bag, the number of revertant colonies per plate was scored in each experiment, for each strain and for each experimental point. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

No noteworthy toxicity was induced in an initial cytotoxicity experiment on TA1535.

Negative and positive controls responded adequately. The study was therefore considered valid.

The test substance DBE did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the 4 tester strains.

The test item did not demonstrate any in vitro mutagenic activity in this bacterial test system, therefore DBE is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).