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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
This IUCLID for dimethyl adipate is compiled using data from the substance itself, a structuraly related compound (dimethyl glutarate) and a mixture of dibasic esters (dimethyl adipate, succinate and glutarate). The toxicity of each of these susbtances is similar and the document attached justifies why data on the category members can be used to support the data gaps for dimethyl adipate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only 2 of 5 recommended strains (TA98, TA100) were used in this study
Principles of method if other than guideline:
As in sub-chronic studies DBE appeared to affect rat olfactory tissue (female more than male), a female rat olfactory S-9 was used to eliminate the possibility that olfactory tissue may be uniquely metabolizing DBE to mutagenic metabolites.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
See information in the field "Confidential details on test material"

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
other: TM677
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
0.0, 0.13, 0.21, 0.63, 1.07, 1.25, 2.13, 6.25 and 10.66 mmol/L in TA98 and TA100 assays with and without S9;
0, 0.28, 0.56, 2.82, 5.63 mM for TM677
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
additional positive controls substances: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
The negative control and positive indicators were assumed to be stable under the conditions of this study; no evidence of instability was observed.


S-9 ACTIVATION SYSTEMS

The 5-9 mix contained per mL: 0.3 mL of 5-9 diluted to 1.6 mg/mL with phosphate buffered saline (PBS) and 0.7 mL of a cofactor solution containing 8 micromoles MgCl2, 33 micromoles KCL, 5 micromoles glucose-6-phosphate, 4 micromoles NADP and 100 micromoles sodium phosphate
(pH 7.4). The rat liver S-9 (Sitek Research Laboratories. Rockville. MD Lot # 040187) was the 9,000 x g supernatant of liver homogenate (1 g wet liver: 3.0 mL PBS). The livers were obtained from 8 to 9 week old male Crl:CD@BR (Charles River, Kingston. NY) rats injected intraperitoneally
with Aroclor® 1254 (500 mg/kg) 5 days before sacrifice. The female rat olfactory S-9 was the 9.000 x g supernatant of olfactory homogenate (1 g wet olfactory tissue: 3 mL PBS). The olfactory tissue was obtained from 8 to 9 week old female Crl:CD@BR (Charles River, Kingston. NY) rats.
These rats were not pretreated with Arolcor.

REVERSE SUSPENSION ASSAY

The assay was performed in the presence and absence of a rat liver homogenate activation system (S-9 mix). Strains TA98 and TA100 were obtained from Dr. B. Ames. Berkeley. CA. Positive indicators and negative solvent controls were included in all assays. Overnight cultures of Salmonella typhimurium strains TA98 and TAI00 grown in Oxoid Nutrient Broth No 2 (NS) were diluted 1:4 with fresh NB. Treatments without activation(nonactivated) were conducted by adding
2.4 mL of diluted culture and 0.1 mL of the test sample in DMSO, control or positive indicator to 2.5 mL of NB. For activated treatments. 2.4 mL of diluted culture and 0.1 mL of the appropriate test sample was added to 2.5 mL S-9 mix (at a final protein concentration of 0.8 mg/5 mL). All
treatment tubes were glass and tightly sealed. The cultures were incubated for 2 hours in a 37°C shaking water bath (80-100 oscillations/min). The cultures were centrifuged at 3.000 rpm for 10 minutes and the cell pellet was resuspended in Davis Minimal Broth (DMB).

For mutagenesis evaluations. 1.0 ml of the cell suspension was added to 2 mL of molten top agar (0.6% Bacto-agar. 0.6% NaCl. 0.05 roM L-histidine. and 0.05 roM biotin). vortexed and poured on a Davis Minimal Base Agar (DMB containing 1.5% agar) plate. AlI exposures were plated in duplicate. The top agar was allowed to harden, plates inverted and incubated for approximately 36-48 hours at 37°C. Cytotoxicity experiments were run concomitantly by making dilutions (between 10^-4 and 10^-5) of the treatment suspensions using NB or phosphate buffered saline (PBS). Aliquots of 0.1 mL of the diluted cell sample were added to 2.0 mL of top agar, vortexed and poured on a Columbia Agar plate. The agar was allowed to harden. plates inverted and incubated for
approximately 18-36 hours at 37°C.

All colonies from individually labeled plates were counted with an Artek Automatic Colony counter. Mutant frequency was calculated by multiplying the average revertant count by the dilution factor for toxicity experiments then dividing by the average number of surviving cells for that dose group. In this report the frequency is expressed as the number of revertants per 10 cells. Only those trials meeting acceptability criteria for this assay are reported.


MICROFORWARD SUSPENSION ASSAY

Either a 0.1 mL aliquot-of quickly thawed or an overnight culture of TM677 was inoculated into 4.9 mL of DMB containing 0.05 mM biotin and 19 mg/mL dextrose. The culture was incubated at 37°C with shaking for 2 hours.
For cultures requiring activation 1 uL of test material and 10 uL of S-9 mix was added to 100 uL of bacterial culture. Test mixtures were incubated with gentle shaking at 37°C for 2 hours. PBS (0.4 mL) was then added to the reaction mixtures. An aliquot of 0.145 mL was removed and added to approximately 9.85 mL of 8 azaguanine (8-AG) supplemented top agar (0.6% purified agar. 100 mM NaCl, 120 mM sodium/potassium phosphate buffer pH 6.5. 0.05 mM biotin. and 0.5 mg/mL 8-AG). This was mixed and 2.5 mL was plated on purified agar (1.5% purified agar, 11 mM dextrose,
15 mM ammonium sulfate, 3 mM sodium citrate. 0.8 rmM magnesium sulfate. and 120 mM sodium/potassium phosphate buffer pH 6.5) plates in triplicate.

Plates were incubated for 48 hours at 37°C. Cytotoxicity was determined on the same samples. Aliquots of 0.145 mL were taken from the samples in the supplemented top agar and
added to approximately 4.85 mL PBS. Then 0.145 mL of this mixture was added to approximately 9.9 mL of top agar. This was vortexed and 2.5 mL was plated on Columbia agar plates in triplicate. All plates were allowed to harden, inverted and incubated for 24 hours at 37°C. Colonies from individually labeled plates were counted using an Artek
Automatic Colony Counter. Mutant frequencies were calculated by multiplying the average number of mutants in each dose group by the cytotoxicity dilution factor and then dividing by the number of surviving colonies. In this report mutant frequencies are expressed as mutants/l0 cells. Only those trials meeting acceptabililty criteria for this assay
are reported.

Evaluation criteria:
The guidelines below are used to aid in the evaluation and classification of a test sample along with sound scientific judgement and experience.
A test sample was classified as a POSITIVE when:
A) The number of induced revertants at one or more of the test sample concentrations studied are at least two times greater than the number of revertants in the solvent control. AND
B) There was a dose-response relationship.

A test sample is classified as a NEGATIVE when:
A) The criteria for a positive response is not met OR
B) There is no dose-response relationship.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TM677
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
DBE exhibited no toxicity to strain TA98 or TA100 without and with rat liver activation up to a maximum dose of 8.53 mg DBE/treatment (10.7 mM or 1.7 mg/mL). No mutagenic responses were observed in either strain with or without activation in two independent trials. Mutant frequencies are shown in Tables 1 and 2.
Microforward Suspension Assay: DBE exhibited no toxicity to strain TM677 i n the presence of olfactory activation up to a maximum dose of 100 µg
DBE/treatment (5.63 mM or 0.9 mg/ml). No mutagenic responses were observed in two independent trials . Mutant frequencies are shown i n Table 3.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenic activity of DBE in S. thyphimurium strainTA98with/without metabolic activation (rat liver S9 mix)

Concentration [mmol/L]

without S9

with S9

Trial 1a)

Trial 2a)

Trial 1a)

Trial 2a)

0.0 (control)

12.7

7.1

9.4

8.8

0.13

 

6.6

 

7.4

0.21

11.4

 

8.2

 

0.63

 

7.2

 

9.4

1.07

18.2

6.0

10.2

 

1.25

 

 

 

9.6

2.13

11.2

 

11.7

 

6.25

 

6.7

 

7.2

10.66

7.0

 

12.2

 

Positive control substances:

2-NF, 24 µMb)

518

84

 

 

2-AA, 21 µMb)

 

 

1052

1127

a)Average value from two plates per concentration and trial

b)2-NF: 2-Nitrofluorene, 2-AA: 2-Aminoanthracene

 

Table 2: Mutagenic activity of DBE in S. thyphimurium strain TM677 with metabolic activation (rat olfactory S9 mix)

Concentration [mmol/L]

without S9

with S9

Trial 1a)

Trial 2a)

Trial 1a)

Trial 2a)

0.0 (control)

58.2

77.4

49.1

88.1

0.13

 

71.4

 

73.5

0.21

62.0

 

38.0

 

0.63

 

64.0

 

75.1

1.07

63.0

 

43.3

 

1.25

 

57.3

 

74.6

2.13

55.3

 

44.2

 

6.25

 

55.2

 

76.9

10.66

58.8

 

40.2

 

Positive control substances:

MNNG, 54 µMb)

1441

1774

 

 

2-AA, 21 µMb)

 

 

1347

1900

a)Average value from two plates per concentration and trial

b)MNNG: N-methyl-N'-nitro-N-nitrosoguanidine, 2-AA: 2-Aminoanthracene

 

Table 3: Mutagenic activity of DBE in S. thyphimurium strainTA100with/without metabolic activation (rat liver S9 mix)

Concentration [mmol/L]

with S9

Trial 1a)

Trial 2a)

0.0 (control)

30

40

0.28

29

40

0.56

32

 

2.82

30

42

5.63

28

36

Positive control substances:

MNNG, 27 µMb)

1096

 

2-AA, 9.3 µMb)

300

348

a)Average value from two plates per concentration and trial

b)MNNG:N-methyl-N'-nitro-N-nitrosoguanidine, 2-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 suspension assay with strains TA98 and TA100 and in the microforward suspension assay with strain TM677.
Executive summary:

The mutagenic potential of DBE has been assessed in the Salmonella typhimurium/microsomal assay according to OECD guideline n° 471 and EC guideline n° B13/14, and in compliance with Good Laboratory Practice.

For the reverse suspension assay Salmonella typhimurium strains TA98 and TA100 were used in presence and in absence of metabolic activation system from liver fraction of Aroclor 1254-induced rats (S9 mix). The test article was suspended in DMSO. DBE was tested in 2 independent experiments performed according to the pre-incubation method in which bacteria were incubated with the test substance and S9 mix for 2 h at 37°C before plating.

Each strain of bacteria was exposed to dose-levels of 0.0, 0.13, 0.21, 0.63, 1.07, 1.25, 2.13, 6.25 and 10.66 mmol/L of the test item (two plates/dose-level). In each experiment, negative and positive controls were included using duplicate plates.

After 36 to 48 hours of incubation at 37°C, 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. In the microforward suspension assay Salmonella typhimurium strains TM677 was used in presence of metabolic activation system from olfactory tissue homogenate fractions of uninduced female Crl:CD BR rats (S9 mix). The test article was suspended in DMSO. DBE was tested in 2 independent experiments performed according to the pre-incubation method in which bacteria were incubated with the test substance and S9 mix for 2 h at 37°C before plating (0, 0.28, 0.56, 2.82, 5.63 mM test substance concentration, three plates/dose-level). In each experiment, negative and positive controls were included using triplicate plates. After 24 hours of incubation at 37°C, 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 any of the 3 tester strains. 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 3 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).