Bis(16-methylheptadecyl) malate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no data on test substance purity, limited documentation).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

(no data on colony sizing, very high concentrations tested, limited documentation; no data on analytical purity of the test substance)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Di(2-Ethylhexyl)adipate
- Analytical purity: no data

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Fischer´s medium (F0) was supplemented with 2 M L-glutamine, sodium pyruvate (110 µg/mL) 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (F10P).

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Experiment 1: 312.5, 625, 1250, 2500, and 5000 µg/mL
Experiment 2: 1800, 2600, 3400, 4200, and 5000 µg/mL
Experiment 3 and 4: 1000, 2000, 3000, 4000, and 5000 µg/mL

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (+S9): 3-Methylcholanthrene (MCA; 2.5 µg/mL); (-S9): mehtyl metahnesulphonate (MMS; 15 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
If no clear positive response was observed in two experiments in the absence of S9 mix, 2 experiments were performed in the presence of S9-mix. Insignificant responses were investigated by further tests involving RLN (uninduced S9) activation.

DURATION
- Preincubation period: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 11-14 days at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 14-17 days

SELECTION AGENT (mutation assays): trifluorothymidine (3 µg/mL)
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: duplicates per concentration per experiment (negative control: 3)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth

Evaluation criteria:

Increase in mutant frequency compared to negative control.
Mutant fraction (MF) was calculated as follows: 200 x (mutant clones per plate)/(total clones per plate) = mutants/10E+06 clonable cells

Quality control criteria:
- The cloning efficiency of the solvent control has to be between 50% and 60%.
- Average mutant fraction of the solvent controls has to be > 15 and 110 mutants per 10E+06 surviving cells.
- At least two solvent control cultures have to be accepted.
- A positive control culture is rejected if a chi-square test for consistency of the acceptable mutant fractions shows P<5%.
- A positive control culture is rejected if the relative total growth (RTG) is < 1%.

Statistics:

The statistical analysis was based upon the mathematical model proposed for this system and consisted of a dose-trend test and a variance anaysis of pair-wise comparisons of each dose against the vehicle control.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

only in experiment 4, an increase in mutant fraction was observed from 2000 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from approx. 1000 µg/mL in all experiments

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at 1000 µg/mL, but testing was continued up to 5000 µg/mL

RANGE-FINDING/SCREENING STUDIES: a screening test was performed to find the highest applicable concentration
The first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/mL .

ADDITIONAL INFORMATION ON CYTOTOXICITY: significant toxicity (RTG < 50%) was observed in all experiments (exp 1: from 1250 µg/mL, exp 2: from 1800 µg/mL, exp 3: from 1000 µg/mL, and exp 4: from 2000 µg/mL)

In experiment 4, the LOED (lowest observed effective dose) was 2000 µg/mL. However, the test substance was considered as negative in this chromosome aberration test, because:
- precipitation occurred from 1000 µg/mL
- no clear dose-relationship in mutant frequency increase was observed,
- cytotoxicity occurred from 1000 µg/mL
- the positive result was not reproducible in the other experiments, although the same conditions (with S9, same concentration) were applied and the relative total growth was comparable, no increase in the average mutation factor was observed in experiment 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. Results of the Mouse Lymphoma Cell Assay

Experiment 1 - without S9						Experiment 2 - without S9
Conc. (µg/mL)	CE (%)	RTG (%)	MC	MF	AVG MF	Conc. (µg/mL)	CE (%)	RTG (%)	MC	MF	AVG MF
Acetone	78	96	60	26		Acetone	92	100	75	27
0	84	85	49	19	24	0	63	97	31	16	22
	91	105	65	24			103	104	61	20
	85	114	67	26			84	98	63	25
312.5	75	74	64	28	26	1800	88	26	41	16	18
	80	80	57	24			74	25	47	21
625.0	88	75	35	13	20	2600	73	24	51	23	18
	80	70	64	27			88	23	36	14
1250	69	25	58	28	23	3400	97	20	48	17	16
	75	38	39	17			93	15	44	16
2500	79	13	50	21	27	4200	78	15	35	15	20
	70	16	68	32			69	13	50	24
5000	87	13	48	18		5000	74	12	37	17	17
	lethal						72	13	36	17
(MMS) 15	32	25	112	119	118*	(MMS) 15	44	29	110	83	83*
	44	24	156	118			39	34	97	84

 

Experiment 3 - with S9						Experiment 4 – with S9
Conc. (µg/mL)	CE (%)	RTG (%)	MC	MF	AVG	CE (%)	RTG (%)	MC	MF	AVG MF
Acetone	80	98	72	30		102	116	82	27
0	76	109	55	24	33	109	101	114	35	37
	70	98	88	42		100	101	111	37
	73	94	78	36		68	82	102	50
1000	65	39	47	24	32	93	70	116	42	40
	75	38	90	40		100	67	115	38
2000	50	27	53	36	33	111	28	294	88	73*
	48	22	43	30		101	32	176	58
3000	59	18	55	31	34	93	23	226	81	85*
	59	19	65	37		91	21	242	89
4000	56	16	61	37	39	78	20	187	80	80*
	53	22	66	41		100	26	238	79
5000	40	16	37	31	34	94	28	240	85	81*
	48	13	53	37		77	27	177	77
(MCA) 2.5	29	16	187	214	222*	55	11	603	364	363*
	32	21	223	231		55	11	592	361

CE = cloning efficiency (%)

RTG = relative total growth (%)

MC = mutant colony count

MF = mutant fraction (mutant colony per 10E+06 clonable cells)

AVG MF = group average mutant fraction

* Statistically significant different from control (P < 5%)

MMS = methyl metahnesulphonate

MCA = 3-methylcholanthrene

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative