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 vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May - 08 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 474 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:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus 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 or slightly opaque gel
- Analytical purity: Aqueous solution at 55.9 % of active material
- Lot/batch No.: 0126344
- Expiration date of the lot/batch: March 2007
- Storage condition of test material: Stored at room temperature and protected from light
- Stability: Stable at T<70 °C

Test animals

Species:

rat

Strain:

other: OFA Sprague Dawley rat (toxicity assay) and CD Sprague Dawley rat (genotoxicity assay)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles RIVER origin, Saint-Germain-sur-l'Arbresle, France
- Weight at study initiation: 157-190 g (males); 140-176 g (females)
- Age at study initiation: 6-10 weeks
- Assigned to test groups randomly: Yes, animals were placed in groups of 3 or 2 by random-distribution.
- Housing: Animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which they were placed in groups of 3 or 2 by random-distribution.
- Diet: A04C10 irradiated rat/mouse feed (SAFE, Augy, France), ad libitum
- Water: Drinking water softened, treated by osmosis and filtered on 0.2 μm membrane, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 55 ± 15 %
- Ventilation: 20 times an hour
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Distilled water
- Lot/batch no.: UCV 161 (Fresenius)
- Concentration of test material in vehicle: 100, 150 and 200 mg/mL
- Stability in vehicle: Stable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item, Chimexane HB was dissolved at the maximum concentration of 200 mg/mL in distilled water.

DOSE VOLUME: 10 mL/kg bw

Duration of treatment / exposure:

- Vehicle control and treated groups: Two consecutive days treatment at 24 h interval
- Treated group for systemic exposure: One day
- Positive control: One day

Frequency of treatment:

- Vehicle control and treated groups: Once daily for two days
- Treated group for systemic exposure: Single treatment
- Positive control: Single treatment

Post exposure period:

- Vehicle control and treated groups: 24 h after the second treatment
- Positive control: 24 h after the single treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
Assay 1: 2000 mg/kg bw/day; Assay 2: 1000 and 1500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Preliminary toxicity assay: 3/sex/dose

Main assay:
Assay 1:
- Negative and positive control group: 10/sex/dose
- Treated group-2000 mg/kg bw/day: 5/sex/dose
- Treated group-2000 mg/kg bw for systemic exposure: 3/sex/dose (for 2 time points – 1 and 4 h)
- Negative control group for systemic exposure: 3/sex/dose

Assay 2:
- Negative and positive control group: 5/sex/dose
- Treated group-1000 and 1500 mg/kg bw/day: 5/sex/dose
- Treated group-1000 and 1500 mg/kg bw for systemic exposure: 3/sex/dose (for 2 time points – 1 and 4 h)

Note: 2 additional animals of each sex for the group treated at 1000 and 1500 mg/kg bw/day (assay 2) and 2000 mg/kg bw/day (assay 1), in parallel, but were sacrificed and examined only in case of mortality observed in the first five treated animals.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Intraperitoneal route (single injection)
- Dose: 25 mg/kg bw
- Vehicle: 0.9% (w/v) NaCl solution
- Concentration: 2.5 mg/mL
- Dose volume: 10 mL/kg bw

Examinations

Tissues and cell types examined:

- For each animal, the micronuclei were counted in 2000 polychromatic erythrocytes.
- Polychromatic (PCE) and normochromatic (NCE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes per animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Dose levels were selected on the basis of results of preliminary toxicity assay in which the test item was administered orally to Sprague-Dawley rats at the dose of 2000 mg/kg bw/day for two consecutive days at 24 h interval.

TREATMENT AND SAMPLING TIMES:
- Two consecutive treatments at 24 h interval followed by sacrificing the animals 24 h after second treatment (vehicle control and treatment groups)
- One day treatment followed by sacrificing the animals 24 h after administration (positive control group)

DETAILS OF SLIDE PREPARATION:
- At the sampling time, all the animals were sacrificed by CO2 asphyxiation.
- Femurs of the rats were removed and the bone marrow was extracted with foetal calf serum.
- After centrifugation, the supernatant was removed and a drop of the cell suspension was placed and spread on a slide.
- Slides were air-dried and stained with May Grunwald Giemsa.

Evaluation criteria:

- For a substance to be considered negative in the micronucleus test, there must be no statistically significant increase in the number of micronuclei observed compared with negative control animals.
- A test substance is considered positive in the micronucleus test if it induces either a dose related increase in the number of micronucleated PCE or a statistically significant positive response for at least one of the test points. However, both biological and statistical significance should be considered together.

Statistics:

- Statistical comparison for the polychromatic/normochromatic erythrocyte ratio and for the weight homogeneity within the sex of each group was performed using the Student's t test.
- Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test.
- Statistical analysis for micronucleus number was conducted, males and females separately and two sexes combined (if using the same doses for the two sexes) in case of homogeneity in the results within the sex of each group.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRELIMINARY TOXICITY ASSAY
- Dose: 2000 mg/kg bw/day for two consecutive days at 24 h interval.
- Solubility: Test item was dissolved at the maximum concentration of 200 mg/mL.
- Mortality: No mortality was observed.
- Clinical signs: Clinical signs limited to soft faeces were observed in all the 6 animals treated at 2000 mg/kg bw/day, between 6 and 24 h after the first treatment and up to the end of the observation period, i.e. 24 h after the second treatment.

RESULTS OF MAIN ASSAY

Induction of micronuclei (for Micronucleus assay):
- Assay 1: No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in the animals treated at 2000 mg/kg bw/day with Chimexane HB, in males and females separately, or in both sexes combined when compared with the respective control groups.
- Assay 2: No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in the animals treated at 1000 and 1500 mg/kg bw/day with Chimexane HB, in males and females separately. As heterogeneity in the spontaneous micronuclei frequency was observed between males and females, no statistical assessment of both sexes combined could be performed.
- See table 7.6.2/1

Ratio of PCE/NCE (for Micronucleus assay):
- Statistically significant decrease in the ratio PCE to NCE was noted in the males treated at 2000 (assay 1) and 1500 mg/kg bw/day (assay 2) and in animals (both sexes combined) treated at 1500 mg/kg bw/day (assay 2) when compared to the concurrent negative control groups. Slight decrease (non statistically significant) in the PCE/NCE ratio were observed animals treated at 1000 mg/kg bw/day (assay 2).

- Body weight means of the treated animals were not significantly different from the controls.

HISTORICAL DATA
- Results were compared with the historical data (January 2003 - November 2005) of the laboratory.

OTHERS:
Determination of plasma levels:
- Results shown that animals treated orally at 2000-1500 and 1000 mg/kg bw were clearly exposed to Chimexane HB. Indeed, 1 h after treatment, the mean plasma concentrations were of 2644 – 2197 and 3315 ng/mL in the males, respectively and of 3347 – 2656 and 2145 ng/mL in the females, respectively. 4 h after treatment, they were of 2291 – 1853 and 2579 ng/mL in the males at the doses of 2000-1500 and 1000 mg/kg bw, respectively and of 2819 – 2640 and 2070 ng/mL, respectively, in the females.
- These data demonstrate a satisfactory systemic exposure at the doses actually tested in the current study.
- All plasmas from animals of control groups were below the limit of quantification (LOQ = 5 mg/mL) as expected.

Any other information on results incl. tables

Table 7.6.2/1: Results of micronucleus assay

Dose	Sex	PCE/NCE Ratio		Micronuclei for 1000 PCE
		Mean ± SD	Student's t Test (p)	Mean ± SD	Mann-Whitney U rank Test
					UA	UB	p
Assay 1
Vehicle	M	1.66 ± 0.44	-	0.75 ± 0.89	-	-	-
	F	1.14 ± 0.15	-	0.75 ± 0.54	-	-	-
	M + F	1.40 ± 0.42	-	0.75 ± 0.72	-	-	-
Test item 2000 mg/kg bw/day	M	1.08 ± 0.16	<0.01	0.40 ± 0.42	20	30	NS
	F	1.30 ± 0.21	NS	1.40 ± 1.08	16.5	33.5	NS
	M + F	1.19 ± 0.21	NS	0.90 ± 0.94	94.5	105.5	NS
Cyclophosphamide 25 mg/kg bw	M	0.80 ± 0.15	<0.001	6.80 ± 2.00	0	100	<0.001
	F	0.73 ± 0.12	<0.001	8.95 ± 3.15	0	100	<0.001
	M + F	0.76 ± 0.14	<0.001	7.88 ± 2.80	0	400	<0.001
Assay 2
Vehicle	M	2.15 ± 0.49	-	0.20 ± 0.27	-	-	-
	F	1.10 ± 0.12	-	0.70 ± 0.27	-	-	-
	M + F	1.62 ± 0.65	-	-	-	-	-
Test item 1000 mg/kg bw/day	M	1.52 ± 0.42	NS	0.70 ± 0.57	5.5	19.5	NS
	F	1.00 ± 0.31	NS	0.80 ± 0.57	10.5	14.5	NS
	M + F	1.26 ± 0.44	NS	-	-	-	-
Test item 1500 mg/kg bw/day	M	1.24 ± 0.19	<0.01	0.50 ± 0.50	8	17	NS
	F	0.96 ± 0.11	NS	0.70 ± 0.84	14.5	10.5	NS
	M + F	1.10 ± 0.20	<0.05	-	-	-	-
Cyclophosphamide 25 mg/kg bw	M	1.00 ± 0.10	<0.001	5.20 ± 1.57	0	25	<0.01
	F	0.87 ± 0.20	NS	5.10 ± 1.29	0	25	<0.01
	M + F	0.94 ± 0.16	<0.01	-	-	-	-

 

NS: Non-significant at the threshold of p=0.05; As heterogeneity in the spontaneous micronuclei frequency was observed between males and females, no statistical assessment of both sexes combined could be performed in assay 2.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, Chimexane HB was not clastogenic in bone marrow cells of rats.

Executive summary:

In an in vivo bone marrow micronucleus assay performed according to OECD Guideline 474 and in compliance with GLP, Sprague-Dawley rats were orally administered with Chimexane HB in distilled water at the following dose levels:

 

Assay 1:

- Treatment group (5/sex/dose): 2000 mg/kg bw/day, 2 consecutive treatments at 24 h interval

- Treatment group for systemic exposure (3/sex/dose): 2000 mg/kg bw, single treatment, blood sample collection 1 and 4 h after treatment.

- Negative control group for systemic exposure (3/sex/dose): Vehicle (distilled water)

 

Assay 2:

- Treatment group (5/sex/dose): 1000 and 1500 mg/kg bw/day, 2 consecutive treatments at 24 h interval

- Treatment group for systemic exposure (3/sex/dose): 1000 and 1500 mg/kg bw, single treatment, blood sample collection 1 and 4 h after treatment.

 

The vehicle and positive control groups (10/sex and 5/sex in assay 1 and 2, respectively) received distilled water and cyclophosphamide at 25 mg/kg bw, respectively. Bone marrow smears were obtained from treatment, vehicle and positive control groups 24 h after last administration. Polychromatic (PCE) and normochromatic (NCE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes. For each animal, the micronuclei were counted in 2000 polychromatic erythrocytes. Two additional animals of each sex for the group treated at 1000, 1500 and 2000 mg/kg bw/day, in parallel, but were sacrificed and examined only in case of mortality observed in the first five treated animals. A preliminary toxicity study (3/sex/dose) was conducted before the main study at the dose of 2000 mg/kg bw/day for two consecutive days.

 

Bioanalytical data demonstrated a satisfactory systemic exposure of the test item at the doses tested. No clinical signs and no mortality were observed. Statistically significant decrease in the ratio of PCE to NCE was noted in the males treated at 1500 and 2000 mg/kg bw/day and in animals (both sexes combined) treated at 1500 mg/kg bw/day when compared to the concurrent negative control groups. Slight decrease (non statistically significant) in the PCE/NCE ratio were observed animals treated at 1000 mg/kg bw/day. No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in the animals treated up to 2000 mg/kg bw/day in either males or females. Positive control induced a statistically significant increase in micronucleated polychromatic erythrocytes indicating the validity of the study.

 

Under the test conditions, Chimexane HB is not considered as clastogenic in bone marrow cells of rats.