Bis(tridecyl) adipate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study (Draft)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(tridecyl) adipate

EC Number:

241-029-0

EC Name:

Bis(tridecyl) adipate

Cas Number:

16958-92-2

Molecular formula:

C28H54O4 - C36H70O4

IUPAC Name:

1,6-ditridecyl hexanedioate

Details on test material:

- Name of test material (as cited in study report): Bis(tridecyl)adipate
- Physical state: clear colourless liquid
- Analytical purity: 100% (UVCB)
- Lot/batch No.: J08120
- Expiration date of the lot/batch: 2014-06-07
- Storage condition of test material: room temperature in the dark

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats with induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4

Test concentrations with justification for top dose:

Preliminary toxicity test (in TA 100 and WP2 uvrA): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation
Main Assay (Experiment I and II): 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL, but was fully soluble in dimethyl formamide at the same concentration and in acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. Acetone was selected as the vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: ENNG (2-5 µg/mL for WP2 uvrA, TA 100, TA1535); 9-AA (80 μg/plate for TA 1537); 4NQO (0.2 μg/plate for TA98); +S9: 2-AA (1-10 µg/plate for TA 100, TA 1535, TA 1537 and WP2 uvrA); BP (5 μg/plate for TA98)

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-AA:9-Aminoacridine; 4-NQO: 4-nitroquinoline-1-oxide; 2-AA: 2-aminoanthracene; BP: benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (Preliminary toxicity test and Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 20 min (Experiment II)
- Exposure duration: 48 h (all experiments)

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method determination of the number of revertant colonies and inspection of the bacterial background lawn

Evaluation criteria:

There were several criteria for determining a positive result. Any, one, or all of the following were used to determine the overall result of the study:
-a dose-related increase in mutant frequency over the dose range tested.
- a reproducible increase at one or more concentrations.
- biological relevance against historical control ranges.
- statistical analysis of data as determined by UKEMS.
- fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Statistics:

Mean values and standard deviations of the mean number of revertants were calculated. Statistical analysis of data was performed according to guidelines from UKEMS.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: a test item precipitate (globular in appearance) was noted by eye at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: in the Preliminary toxicity test, the test item was non-toxic to strains TA 100 and WP2 uvrA up to the maximum concentration of 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: the mean number of revertants of vehicle and positive controls was within the normal range of the respective historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment I (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)
EXPERIMENT I (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 100	TA1535	WP2 uvrA	TA98	TA 1537
SC	108 ± 14	14 ± 2	19 ± 2	21 ± 4	10 ± 1
Test material
50 µg	91 ± 12	13 ± 2	18 ± 6	19 ± 2	7 ± 4
150 µg	95 ± 21	15 ± 2	22 ± 2	18 ± 5	8 ± 4
500 µg	91 ± 7	14 ± 4	21 ± 5	16 ± 1	8 ± 2
1500 µg	94 ± 6	13 ± 1	18 ± 10	22 ± 8	6 ± 2
5000 µg	93 ± 3	10 ± 2	20 ± 2	16 ± 3	8 ± 3
PC
ENNG	614 ± 28	442 ± 54	937 ± 42	-	-
4-NQO	-	-	-	239 ± 30	-
9-AA	-	-	-	-	912 ± 96
S9-Mix	With
Concentration (per plate)	TA 100	TA1535	WP2 uvrA	TA98	TA 1537
NC	91 ± 2	11 ± 3	26 ± 8	27 ± 3	11 ± 0
Test material
50 µg	101 ± 12	11 ± 2	25 ± 4	24 ± 4	9 ± 3
150 µg	96 ± 11	11 ± 1	28 ± 6	31 ± 2	10 ± 1
500 µg	104 ± 11	10 ± 1	29 ± 4	23 ± 8	10 ± 4
1500 µg	89 ± 3	11 ± 1	22 ± 7	23 ± 10	12 ± 5
5000 µg	99 ± 10	12 ± 1	24 ± 8	24 ± 6	10 ± 2
PC
2-AA	1238 ± 270	231 ± 11	286 ± 14	-	237 ± 12
BP	-	-	-	199 ± 11	-
SC = Solvent control; PC = Positive control substances; SD = standard deviation; ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-AA:9-Aminoacridine; 4-NQO: 4-nitroquinoline-1-oxide; 2-AA: 2-aminoanthracene; BP: benzo(a)pyrene

Table 2. Test results of experiment II (pre-incubation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)
EXPERIMENT II (pre-incubation)
S9-Mix	Without
Concentration (per plate)	TA 100	TA1535	WP2 uvrA	TA98	TA 1537
SC	79 ± 6	15 ± 5	26 ± 3	15 ± 3	10 ± 4
Test material
50 µg	72 ± 2	12 ± 2	24 ± 5	26 ± 12	9 ± 1
150 µg	82 ± 13	12 ± 2	27 ± 5	16 ± 3	11 ± 3
500 µg	66 ± 1	8 ± 2	18 ± 1	22 ± 5	11 ± 1
1500 µg	74 ± 3	13 ± 4	27 ± 5	16 ± 1	5 ± 1
5000 µg	79 ± 1	11 ± 4	23 ± 6	21 ± 7	11 ± 4
PC
ENNG	728 ± 131	892 ± 86	946 ± 35	-	-
4-NQO	-	-	-	118 ± 8	-
9-AA	-	-	-	-	534 ± 206
S9-Mix	With
Concentration (per plate)	TA 100	TA1535	WP2 uvrA	TA98	TA 1537
NC	75 ± 5	14 ± 6	34 ± 9	24 ± 4	9 ± 2
Test material
50 µg	75 ± 4	12 ± 3	25 ± 0	21 ± 1	7 ± 3
150 µg	83 ± 22	10 ± 2	22 ± 9	26 ± 2	12 ± 4
500 µg	70 ± 18	10 ± 7	29 ± 4	21 ± 2	8 ± 2
1500 µg	76 ± 12	11 ± 6	24 ± 2	21 ± 3	11 ± 2
5000 µg	76 ± 22	10 ± 2	29 ± 5	26 ± 3	10 ± 3
PC
2-AA	1506 ± 102	201 ± 30	246 ± 22	-	316 ± 32
BP	-	-	-	130 ± 7	-
SC = Solvent control; PC = Positive control substances; SD = standard deviation; ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-AA:9-Aminoacridine; 4-NQO: 4-nitroquinoline-1-oxide; 2-AA: 2-aminoanthracene; BP: benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative