Bis(1-methylheptyl) adipate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jul - 9 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Messungen und Naturschutz, Karlsruhe, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dihexyl adipate

EC Number:

203-757-7

EC Name:

Dihexyl adipate

Cas Number:

110-33-8

Molecular formula:

C18H34O4

IUPAC Name:

dihexyl adipate

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Chemical name: Dihexyl adipate
- Physical state: colourless liquid
- Analytical purity: ≥ 97.5% (97.7% active substance content)
- Purity test date: 2012-06-01
- Lot/batch No.: 12027013
- Expiration date of the lot/batch: 2013-05-30
- Storage condition of test material: normal conditions, keep container tightly closed, and store at dry and well ventilated place

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I: 16, 50, 158, 500, 1581 and 5000 µg/plate* (with and without metabolic activation)
Experiment II: 480, 860, 1540, 2780 and 5000 µg/plate* (with and without metabolic activation)
* corresponding to an active substance content of 97.7%

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.05 mL per plate)
- Justification for choice of solvent/vehicle: The test substance is poorly soluble in water (0.3 g/L).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

-S9: 2-NF (1.5 µg/plate, TA 98), SA (0.25-0.5 µg/plate, TA 100 and TA 1535), CHP (0.15 µL/plate, TA 102), 9-AA: 9-aminoacridine (49.5 µg/plate, TA 1537); +S9: BaP: (1.5 µg/plate, TA 98), 2-AA: (2 µg/plate, TA 98; 2.5-5 µL/plate, all other strains)

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

cumene hydroperoxide

other: 2-aminoanthracene

Remarks:

2-NF: 2-nitrofluorene; SA: sodium azide; CHP: cumene hydroperoxide; 9-AA: 9-aminoacridine; BaP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

Evaluation criteria:

According to OECD guideline 471, a sample was considered mutagenic if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without the metabolic activation system. Statistical methods were used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive result. For the evaluation of the results achieved, the increase of revertants in a sample was expressed as the induction rate [IR], (IR = number of revertants in the sample/number of revertants in the solvent control). In the case of a reproducible increase of the number of revertants and if additionally a statistically significant difference between the mean values was demonstrated the sample was evaluated as positive. The sample was evaluated as positive, if at least a doubling of revertant colonies with the sample compared to the control was observed (Induction rate [IR] ≥ 2).

Statistics:

For statistical evaluation the software SigmaStat Version 3.5 (Systat Software Inc., Point Richmond, USA) was applied. First, the data were checked for normality, then for homogeneity of variance. If variance was not equal for Ames test data, an ANOVA on the ranks applying the Kruskal-Wallis Test was performed followed by a pairwise multiple comparison (Dunn’s test) of the different treatments. In the case of variance homogeneity, one-way ANOVA was performed followed by multiple comparisons versus control group (Dunnett’s test). Statistics were only performed if the induction rates were ≥ 1.5. (Kim and Margolin, 1999).

REFERENCE:
Kim, B.S., Margolin, B.H. (1999): Statistical methods for the Ames Salmonella assay: A Review, Mutat. Res., 436, 113-122

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants in the negative control was within the historical ranges of each tester strain and the positive controls induced at least a doubling of the revertants in comparison to the negative controls. The number of mean revertants per plate after treatment with the positive control substance sodium azide in TA 1535 was significantly different in experiment I compared to experiment II (4644 in experiment I vs. 188 in experiment II). However, the positive control sodium azide fulfilled the criteria for the validity of the assay.

Any other information on results incl. tables

Table 1. Test results of first experiment

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± RSD)
EXPERIMENT I (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC (water)	26 ± 28	100 ± 16	824 ± 5	11 ± 13	8 ± 16
SC (DMSO)	19 ± 36	92 ± 7	756 ± 8	10 ± 29	6 ± 46
Test item
16 µg	26 ± 19	107 ± 15	957 ± 14	12 ± 40	6 ± 44
50 µg	26 ± 45	120 ± 8	748 ± 10	8 ± 15	5 ± 40
158 µg	19 ± 16	100 ± 6	691 ± 6	12 ± 46	5 ± 40
500 µg	21 ± 34	93 ± 5	817 ± 19	16 ± 26	4 ± 48
1581 µg	19 ± 11	96 ± 14	825 ± 16	8 ± 25	6 ± 29
5000 µg	19 ± 6	98 ± 4	797 ± 7	12 ± 25	7 ± 42
PC
2-NF (1.5 µg)	147 ± 3	-	-	-	-
SA (0.25 µg)	-	-	-	4644 ± 10	-
SA (0.5 µg)	-	6755 ± 19	-	-	-
CHP (0.15 µL)	-	-	1647 ± 10	-	-
9-AA (49.5 µg)	-	-	-	-	1201 ± 19
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC (water)	24 ± 10	113 ± 7	706 ± 7	13 ± 44	8 ± 26
SC (DMSO)	22 ± 34	110 ± 10	565 ± 8	14 ± 12	8 ± 22
Test item
16 µg	22 ± 23	112 ± 9	716 ± 2	15 ± 33	2 ± 50
50 µg	22 ± 10	108 ± 16	684 ± 20	21 ± 26	9 ± 22
158 µg	23 ± 18	122 ± 7	612 ± 3	21 ± 15	9 ± 24
500 µg	23 ± 22	110 ± 21	599 ± 12	14 ± 21	9 ± 29
1581 µg	20 ± 12	120 ± 12	643 ± 8	24 ± 17	7 ± 53
5000 µg	23 ± 16	103 ± 14	633 ± 2	18 ± 11	9 ± 53
PC
BP (1.5 µg)	100 ± 12	-	-	-	-
2-AA (2.0 µg)	280 ± 7	-	-	-	-
2-AA (2.5 µL)	-	944 ± 21	1776 ± 2	93 ± 30	-
2-AA (5.0 µL)	-	-	-	-	162 ± 17
NC = negative control; SC = Solvent control; PC = Positive control substances; RSD = relative standard deviation; 2-NF: 2-nitrofluorene; SA: sodium azide; CHP: cumene hydroperoxide; 9-AA: 9-aminoacridine; BaP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Table 2. Test results of second experiment

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± RSD)
EXPERIMENT II (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC (water)	20 ± 26	132 ± 5	810 ± 6	16 ± 3	7 ± 31
SC (DMSO)	21 ± 25	110 ± 9	618 ± 7	16 ± 15	7 ± 45
Test item
480 µg	20 ± 26	96 ± 13	911 ± 7	14 ± 12	3 ± 58
860 µg	26 ± 25	113 ± 4	885 ± 11	12 ± 9	7 ± 8
1540 µg	19 ± 5	119 ± 11	729 ± 12	9 ± 34	5 ± 20
2780 µg	22 ± 25	104 ± 5	876 ± 12	15 ± 28	4 ± 71
5000 µg	17 ± 12	106 ± 13	876 ± 5	12 ± 37	5 ± 60
PC
2-NF (1.5 µg)	119 ± 18	-	-	-	-
SA (0.25 µg)	-	-	-	188 ± 11	-
SA (0.5 µg)	-	476 ± 33	-	-	-
CHP (0.15 µL)	-	-	2745 ± 9	-	-
9-AA (49.5 µg)	-	-	-	-	1205 ± 20
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC (water)	24 ± 26	112 ± 15	878 ± 4	15 ± 25	8 ± 25
SC (DMSO)	22 ± 23	123 ± 7	620 ± 18	12 ± 16	7 ± 42
Test item
480 µg	22 ± 28	120 ± 6	576 ± 13	10 ± 40	7 ± 52
860 µg	21 ± 19	113 ± 12	591 ± 27	15 ± 14	5 ± 66
1540 µg	19 ± 21	129 ± 10	644 ± 16	16 ± 4	7 ± 25
2780 µg	18 ± 30	127 ± 1	683 ± 13	15 ± 37	6 ± 17
5000 µg	20 ± 27	120 ± 8	711 ± 14	12 ± 20	8 ± 7
PC
BP (1.5 µg)	239 ± 21	-	-	-	-
2-AA (2.0 µg)	281 ± 22	-	-	-	-
2-AA (2.5 µL)	-	760 ± 21	1733 ± 28	74 ± 5	-
2-AA (5.0 µL)	-	-	-	-	127 ± 4
NC = negative control; SC = Solvent control; PC = Positive control substances; RSD = relative standard deviation; 2-NF: 2-nitrofluorene; SA: sodium azide; CHP: cumene hydroperoxide; 9-AA: 9-aminoacridine; BaP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative