Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May - 17 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD Guideline 476 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

EC Number:

939-579-8

Molecular formula:

Empirical Formula : C11H21N3O3Na C2nH4n+1 (n=4 – 9), C11H21N3O3NaC18H35, C11H21N3O3NaC18H33 Molecular formula of the two main constituents : C23H46N3O3Na

IUPAC Name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Details on test material:

- Name of test material (as cited in study report): Chimexane HB
- Physical state: Colourless to pale yellow, translucent to slightly opaque gel
- Analytical purity: 55.9 % active content
- Lot/batch No.: 0126344
- Date received: 22 May 2006
- Expiration date of the lot/batch: March 2007
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

hprt gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source of cells: Dr Donald Clive, Burroughs Wellcome Co, North Carolina, USA.
- Type and identity of media: Growth media, RPMI 1640
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for spontaneous mutant frequency: Yes
- Other details: For each experiment, one vial was thawed and the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. The cells were allowed to grow well and subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

5 % S9 mix; S9 fraction was prepared from liver homogenates of Sprague Dawley male rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Cytotoxicity study (range-finder experiment):
- Without S9 mix: 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/mL
- With S9 mix: 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL
Main study:
- Experiment 1 (-S9): 20, 40, 60, 80, 100, 110, 120, 130, 140 and 150 μg/mL
- Experiment 1 (+S9): 15, 30, 60, 80, 100, 120, 140, 150, 160 and 175 μg/mL
- Experiment 2 (-S9): 20, 40, 60, 80, 90, 100, 110, 120 and 140 μg/mL
- Experiment 2 (+S9): 20, 40, 60, 80, 100, 120, 130, 140, 150, 160 and 175 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified water
- Test item was dissolved in purified water with the aid of vortex mixing, warming at 37 or 60 °C and sonication as required to give the maximum required treatment solution concentration. This solution was filter-sterilised (Pall Acrodisc 32 filter, 0.2 μm pore size) and further dilutions were made using purified water. The test article solutions were protected from light and used within 1.25 h of initial formulation of the test article

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water diluted 10-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 2 and 3 µg/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water diluted 10-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.10 and 0.15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In RPMI 1640 medium

DURATION
- Exposure duration: 3 h, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Viability scoring: 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 11-12 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG) at 15 µg/mL (final concentration)

NUMBER OF REPLICATIONS:
- Vehicle and test substance groups (single cultures for cytotoxicity study and duplicate cultures for experiment 1 and 2).
- Single culture for positive control groups for experiments 1 and 2.

NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6-TG resistance respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival (%RS)

OTHER:
- Wells containing viable clones were identified by eye using background illumination and counted.
- Cell concentrations in the selected cultures were determined using a Coulter counter.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hypoxanthine phosphoribosyl transferase (hprt) locus in mouse lymphoma L5178Y cells if:
- the assay was valid
- the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
- there was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
- the effects described above were reproducible.

- Test article was considered positive in this assay if all of the above criteria were met.
- Test article was considered negative in this assay if none of the above criteria are met.
- Results that only partially satisfy the assessment criteria described above were to be considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity may require careful interpretation when assessing their biological significance.
- Extreme caution should be exercised with positive results obtained at levels of RS lower than 10 %.

Statistics:

- Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: Test item was soluble in purified water at concentrations up to at least 89.44 mg/mL (equivalent to 50 mg/mL, allowing for 55.9 % active content of test article).

- Effects of osmolality: No marked effect on osmolality (greater than a shift of 50 mOsm/kg) as compared to concurrent vehicle controls.

- Effects of pH: Increases of greater than 1 pH unit were observed in the absence of S9 at 2500 mg/mL and above. No further pH measurements were considered necessary as no increases of 1 pH unit were observed at 1250 mg/mL and below and the highest concentration tested in the main experiments was limited by toxicity to a maximum of 150 mg/mL. No increases of greater than 1 pH unit were observed in the presence of S9.

Precipitation:
- Cytotoxicity study (range-finder experiment): Upon addition of the test article to the cultures, no precipitate was observed. However, after the 3 h treatment incubation period, precipitate was observed at 312.5-2500 μg/mL in the presence of S9.
- Experiment 1: Upon addition of the test article to the cultures, no precipitate was observed. However, after the 3 h treatment incubation period, precipitate was observed at the highest two concentrations tested in the presence of S9 (160 and 175 μg/mL).
- Experiment 2: Upon addition of the test article to the cultures, no precipitate was observed. However, after the 3 h treatment incubation period, precipitate was observed at the highest five concentrations tested in the presence of S9 (130-175 μg/mL).

RANGE-FINDING/SCREENING STUDIES:
- Six concentrations were tested, in the absence and presence of S9, ranging from 4.688 to 150 μg/mL.
- Highest concentrations that survived treatment (75 μg/mL in the absence of S9 and 156.3 μg/mL in the presence of S9) yielded 84 and 16 % relative survival, respectively.
- See table 7.6.1/1 for more details.

MUTAGENICITY TESTS:
- When tested up to toxic concentrations, no statistically significant increases in mutant frequency were observed following treatment with Chimexane HB at any concentration tested in Experiments 1 and 2. A weak linear trend was observed in the presence of S9 in Experiment 2 but, as no significant increases in mutant frequency were observed and the effect was not reproduced between experiments, this was not considered biologically relevant.
- It may be noted that cultures treated at 130 μg/mL in the presence of S9 in Experiment 2 were highly toxic, yielding 5 % relative survival. These data were included in the statistical analysis as there was a steep concentration-related response between 100 and 130 μg/mL but there were no statistically significant increases in mutant frequency at the latter, highly toxic concentration.
- See table 7.6.1/2 and 7.6.1/3.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with the historical data of the laboratory.

Remarks on result:

other: other: TK+/- cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: Range-finder experiment 

Treatment (μg/mL)	-S9 % RS	+S9 % RS
0	100	100
4.688	98	NT
9.375	65	NT
18.75	80	NT
37.5	72	NT
75	84	NT
150	0	NT
156.3	NT	16

 

%RS: Percentage Relative Survival; NT: Not tested

Note: Concentrations of 312.5-5000 mg/mL treated in the presence of S9 were not plated, due to excessive toxicity

Table 7.6.1/2: Summary of mutation data -Experiment 1 (3 hour treatment in the absence and presence of S9) 

Treatment (µg/mL)	-S9		Treatment (µg/mL)	+S9
	%RS	MF§		%RS	MF§
0	100	5.26	0	100	4.41
20	92.87	5.19 NS	15	102.30	4.36 NS
40	83.20	5.23 NS	30	103.00	3.19 NS
60	88.23	3.96 NS	60	94.84	4.54 NS
80	74.26	4.12 NS	80	89.05	4.80 NS
100	27.70	4.33 NS	100	94.88	4.69 NS
110	17.82	2.30 NS	120	87.57	3.90 NS
-	-	-	140	53.95	6.46 NS
-	-	-	150	13.97	4.68 NS
Linear trend                                      NS			Linear trend                                      NS
NQO 0.1	60.64	24.25	B[a]P 2	63.59	42.40
NQO 0.15	65.39	45.77	B[a]P 3	51.63	102.29

 

Table 7.6.1/3: Summary of mutation data -Experiment 2 (3 hour treatment in the absence and presence of S9) 

Treatment (µg/mL)	-S9		Treatment (µg/mL)	+S9
	%RS	MF§		%RS	MF§
0	100	5.16	0	100	4.73
20	105.89	5.19 NS	20	96.17	4.25 NS
40	86.65	6.15 NS	40	95.32	3.41 NS
60	58.80	7.64 NS	60	97.73	4.86 NS
80	11.70	4.75 NS	80	100.67	4.26 NS
-	-	-	100	69.92	7.26 NS
-	-	-	120	24.50	7.36 NS
-	-	-	130 PP	5.46	7.13 NS
Linear trend                                      NS			Linear trend                                      *
NQO 0.1	78.79	35.61	B[a]P 2	83.80	80.00
NQO 0.15	62.72	95.81	B[a]P 3	47.65	124.46

 

§ : 6-TG resistant mutants/10^6 viable cells 7 days after treatment ; %RS : Percent relative survival adjusted by post treatment cell counts ; NS: Not significant ; *, **, *** Test for linear trend: x^2 (one-sided), significant at 5, 1 and 0.1 % level respectively ; PP : Precipitation observed by eye following the treatment incubation period

Applicant's summary and conclusion

Conclusions:

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

Under the test conditions, Chimexane HB did not induce any mutations in mammalian cells (strain: L5178Y mouse lymphoma cells) with and without metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, mouse lymphoma L5178Y tk+/- cells were exposed to Chimexane HB dissolved in purified water in RPMI 1640 medium for 3 h, with and without metabolic activation (5 % S9 fraction of male Sprague Dawley rats liver induced with Aroclor 1254), at the following concentrations:

Cytotoxicity study (range-finder experiment):

- Without S9 mix: 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/mL

- With S9 mix: 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL

Main study:

- Experiment 1 (-S9): 20, 40, 60, 80, 100, 110, 120, 130, 140 and 150 μg/mL

- Experiment 1 (+S9): 15, 30, 60, 80, 100, 120, 140, 150, 160 and 175 μg/mL

- Experiment 2 (-S9): 20, 40, 60, 80, 90, 100, 110, 120 and 140 μg/mL

- Experiment 2 (+S9): 20, 40, 60, 80, 100, 120, 130, 140, 150, 160 and 175 μg/mL

 

In the cytotoxicity study, the highest concentrations that survived treatment (75 μg/mL in the absence of S9 and 156.3 μg/mL in the presence of S9) yielded 84 and 16 % relative survival, respectively. In experiment 1, the highest concentrations analysed were 110 μg/mL in the absence of S9 and 150 μg/mL in the presence of S9, which yielded 18 and 14 % relative survival, respectively. In experiment 2, the highest concentrations analysed were 80 μg/mL in the absence of S9 and 130 μg/mL in the presence of S9, which yielded 12 and 5 % relative survival, respectively. When tested up to toxic concentrations, no statistically significant increases in mutant frequency were observed following treatment with Chimexane HB at any concentration tested in experiments 1 and 2. A weak linear trend was observed in the presence of S9 in experiment 2 but, as no significant increases in mutant frequency were observed and the effect was not reproduced between experiments, this was not considered biologically relevant. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [4-nitroquinoline 1-oxide at 0.10 or 0.15 µg/mL (without S9 mix) and benzo(a)pyrene at 2 or 3 µg/mL (with S9 mix)] indicating the validity of the study.

 

Under the test conditions, Chimexane HB is not considered as mutagenic in mammalian cells, as measured in the hprt locus of L5178Y mouse lymphoma cells.