Reaction mass of (Z)-3-(9-octadecenyloxy)propane-1,2-diol and alpha-(Z)-octadec-9-en-1-yl-omega-hydroxy-di[oxy[(hydroxymethyl)-1,2-ethanediyl]] and alpha-(Z)-octadec-9-en-1-yl-omega-hydroxy-tri[oxy[(hydroxymethyl)-1,2-ethanediyl]]

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:

13 March - 19 May 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 474 with minor deviation: relative humidity in the animal room was outside the range for three days during the acclimation period

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:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

relative humidity in the animal room was outside the range for three days during the acclimation period - lack of stability data in plasma (no GLP claimed for plasma sample analysis)

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:

Reference substance 001

Cas Number:

9022-76-8

Details on test material:

- Name of test material (as cited in study report): Chimexane NB
- Physical state: Yellow to brown viscous liquid
- Analytical purity: 98.5 %
- Lot/batch No.: 0129336
- Expiration date of the lot/batch: February 2007
- Storage condition of test material: Stored at room temperature, protected from light and humidity
- Stability: Stable

Test animals

Species:

rat

Strain:

other: OFA Sprague-Dawley

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: 155-187 g (males); 143-165 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: Carboxymethylcellulose 0.5 %
- Lot/batch no.: 034K0010 (Sigma)
- Concentration of test material in vehicle: 200 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item, Chimexane NB was suspended in a 0.5 % carboxymethylcellulose aqueous solution, using an ultra-turrax for mixing.

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:
2000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Preliminary toxicity assay: 3/sex/dose

Main assay:
- Vehicle, treated (2000 mg/kg bw/day) and positive control group: 5/sex/dose
- Treated group-2000 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 2000 mg/kg bw/day, in parallel, but were sacrificed and examined only in case of mortality observed in the five first 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 fetal 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.
- Clinical signs and mortality: No mortality and no clinical signs were observed.

RESULTS OF MAIN ASSAY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in the animals treated at 2000 mg/kg bw/day (2 consecutive days) with Chimexane NB, in males and females separately, or in both sexes combined when compared with the concurrent control groups. 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 female treatment group when compared to the vehicle control group.
- 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:
- After a single administration of 2000 mg/kg bw, concentrations of Chimexane NB measured in plasmas from Sprague-Dawley rats ranged from 863 to 1136 ng/mL and from 633 to 1305 ng/mL at 1 and 4 h sampling time, respectively in males and from 1100 to 2237 ng/mL and from 1002 to 1356 ng/mL at 1 and 4 h sampling time, respectively in females, clearly demonstrating the systemic exposure to Chimexane NB.

Any other information on results incl. tables

Table 7.6.2/1: 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
Vehicle	M	2.07 ± 0.37	-	0.60 ± 0.42	-	-	-
	F	1.97 ± 0.32	-	0.90 ± 0.42	-	-	-
	M + F	2.02 ± 0.33	-	0.75 ± 0.42	-	-	-
Test item 2000 mg/kg bw/day	M	2.00 ± 0.73	NS	0.40 ± 0.42	16	9	NS
	F	1.49 ± 0.22	<0.05	0.60 ± 0.65	16.5	8.5	NS
	M + F	1.74 ± 0.57	NS	0.50 ± 0.53	65.5	34.5	NS
Cyclophosphamide 25 mg/kg bw	M	1.00 ± 0.37	<0.01	8.00 ± 2.42	0	25	p<0.01
	F	0.62 ± 0.07	<0.001	6.90 ± 1.88	0	25	p<0.01
	M + F	0.81 ± 0.32	<0.001	7.45 ± 2.13	0	100	p<0.001

 

N.S.: Non-significant at the threshold of p=0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, Chimexane NB is not considered as clastogenic in bone marrow cells of rats according to the Annex VI of the Directive 67/548/EEC and the Regulation (EC) N° 1272-2008 (CLP).

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 NB in 0.5 % carboxymethylcellulose (CMC) at the following dose levels:

- 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.

The vehicle and positive control groups (5/sex/dose) received 0.5 % CMC and cyclophosphamide at 25 mg/kg bw, respectively. Bone marrow smears were obtained from the 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 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 mortality was observed. No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in the animals treated at 2000 mg/kg bw/day (2 consecutive days) with Chimexane NB, in males and females separately, or in both sexes combined when compared with the concurrent control groups. Statistically significant decrease in the ratio PCE to NCE was noted in the female treatment group when compared to the vehicle control group. Positive control induced a statistically significant increase in micronucleated polychromatic erythrocytes indicating the validity of the study.

 

Under the test conditions, Chimexane NB is considered as non clastogenic in bone marrow cells of rats according to the Annex VI of the Directive 67/548/EEC and the Regulation (EC) N° 1272-2008 (CLP).