Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Genotoxic activity using micronucleus in rat
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
from 2008-01-28 to 2008-04-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: in compliance with OECD and GLP guideline (as the data is used in a read-across approach, a maximal reliability score of 2 was attributed).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
SPONSOR : ROQUETTE
TEST ITEM : LAB3822
BATCH NUMBER : FAL 07/25
I.P.L. REGISTRATION NUMBER : 070713
EXPIRY DATE : March 2009
APPEARANCE : slightly yellowish liquid
PURITY* : 87%
DENSITY* : 1.02
STORAGE CONDITIONS : at room temperature
STABILITY UNDER STORAGE CONDITIONS : up to its expiry date (i.e. March 2009)
QUANTITY SUPPLIED : 250 mL
*: A purity and a density correction were made. The doses are expressed in mg/kg of pure LAB3822.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Male and female OFA Sprague Dawley rats (Charles RIVER origin, Saint -Germain-sur-l'Arbresle- FRANCE) are used for the study. The period of acclimatization is at least 5 days. The animals receive a clinical examination in order to retain only the healthy ones.
The animals are housed in polypropylene cages measuring 42,5 x 26,6 x 15 cm, covered by a stainless steel netted lid, in which they are placed in groups of 3 or 2 by random-distribution.
The cages are placed in a ventilated cupboard slightly overpressurized relative to animal room also ventilated.
The bedding consists of dust-free, sterilized wood shavings.
The feedstuff used is No. A04C10 irradiated rat/mouse feed from SAFE batch 70718.
The animals are not fasted at the treatment time. Drinking water softened, treated by osmosis and filtered on 0.2 μm membrane is provided ad libitum.
The temperature in the ventilated animal cupboard is 22 ± 3 °C, and humidity is 55 ± 15 %.
Ventilation renews the air 20 times an hour. A timer provides lighting 12 hours a day (8 a.m. - 8
p.m.) in all the animal rooms.
Animals : rat: OFA Sprague Dawley sex: male and female ; weight: 159 g to 192 g (males); 129 g to 157 g (females) ;

Administration / exposure

Route of administration:
oral: feed
Vehicle:
None
Details on exposure:
After random-distribution, 5 groups of animals OFA Sprague Dawley rats from Charles River (4 groups of 10, and one group of 14, for a total of 27 males and 27 females), weighing approximately 200 g and 5 to 6 weeks old, are used for this test. Ears rings identify animals.
Treatment takes the form of 2 successive administrations at 24-hour intervals by the route specified in the specific study plan. As the test item is administered as supplied, no vehicle is used in the present study and the negative control thus received no treatment (neither solution or suspension).
The reference substance, cyclophosphamide, is administered by the intraperitoneal route in a single injection 24 hours before sampling.
Samples are taken at 24 hours after the last treatment.
The animals are treated according to the experimental program below:
Negative control (no treatment) :5males ; 5 females
Test item: High dose, maximum tolerated dose (MTD) or 2000 mg/kg/day (x2)(If the maximum tolerated dose is equal or higher than 2000 mg/kg, this dose is administered according to OECD (1997) and HAYASHI et al. (2007) : 5(+2) males ; (+2)females (For the group treated with the high dose, 2 animals of each sex are treated, in parallel, but are sacrificed and examined only in case of mortality observed in the five first treated animals.
Test item: Mid dose, MTD/2 or 1000 mg/kg/day (x2) (If the maximum tolerated dose is equal or higher than 2000 mg/kg, this dose is administered according to OECD (1997) and HAYASHI et al. (2007)) : 5males ; 5 females
Test item: Low dose, MTD/4 or 500 mg/kg/day (x2)(If the maximum tolerated dose is equal or higher than 2000 mg/kg, this dose is administered according to OECD (1997) and HAYASHI et al. (2007)) : 5males ; 5 females


Doses / concentrations
Remarks:
Doses / Concentrations:
2000 – 1000 – 500 mg/kg/day (x 2)
Basis:

Control animals:
yes
Positive control(s):
The reference substance is cyclophosphamide

- Route of administration: intraperitoneal
- Doses / concentrations:25mg/kg IP (positive control).The animal treatment with the positive control is realized, only once, after the treatment with the studied test item.
Number of animals : 5 males ; 5 females

Examinations

Details of tissue and slide preparation:
At the sampling time, the animals are sacrificed by CO2 asphyxia; the femurs are removed, and the bone marrow is extracted with foetal calf serum (1 mL per animal).
The cell suspensions are centrifuged for 5 minutes at 1000 rpm. The supernatant is eliminated. The centrifugate is spread on slides. The smears are stained using a technique, derived from the May Grunwald Giemsa technique (SCHMID, 1975), which makes it possible to distinguish between polychromatic and normochromatic erythrocytes.
After coding the slides by a person not involved in the study, two slides per animal are read by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) is determined for at least 2000 polychromatic erythrocytes.
(In case of divergence, 2000 new polychromatic erythrocytes are examined; the retained value is the mean of all readings).
The polychromatic/normochromatic erythrocyte ratio is determined by analyzing at least 1000 erythrocytes per animal.

Results and discussion

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item LAB3822 (batch FAL 07/25) was investigated by means of the in vivo micronucleus test in male and female OFA Sprague Dawley rats treated orally with 2000 – 1000 and 500 mg/kg/day (x2), followed by one sampling time 24 hours after the last treatment. As a conclusion, LAB3822 induced no genotoxic activity under these experimental conditions.
Thirty minutes, 2 or 4 hours after oral administration of 500, 1000 or 2000 mg/kg of LAB3822, plasmatic concentrations of Isosorbide measured in OFA Sprague-Dawley rats clearly demonstrated a systemic exposure to Isosorbide, and thus to LAB3822.
(The determination of Isosorbide concentrations in plasma was performed in a GLPcompliant laboratory using a validated analytical method (CERB/Reference No. 20080183B). The dosing results were thus considered as reliable).
Executive summary:

STUDY OF GENOTOXIC ACTIVITY USING THE MICRONUCLEUS TEST IN RAT

(2 treatments, 1 sampling time)

SUMMARY

SPONSOR : ROQUETTE

TEST ITEM : LAB3822

BATCH NUMBER : FAL 07/25

STUDY LOCATION : INSTITUT PASTEUR DE LILLE

Genetic Toxicology Laboratory

1, rue du Professeur Calmette - B.P. 245

59019 LILLE CEDEX

THIS STUDY WAS CARRIED OUT IN COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS

METHODS

 Animals (ex, strain, species)  male and female OFA Sprague Dawley rats
 Number of animals per group  5 males and 5 females
 Form administered  test item administered as supplied
 Route  oral
 Dose volume  test item administered as supplied: the dose volume was dependant on the weight of the rat
 Vehicle  test item administered as supplied
 Stability in vehicle  not applicable
 Treatment schedule  negative control and treated: 2 daily treatments positive control: one treatment
 Number of sampling times  1: negative control and treated: 24 hours after the last treatment 1: positive control: 24 hours after treatment

TOXICITY ASSAY

In accordance with the Amendment No. 1, no preliminary toxicity assay was performed. Indeed, previous acute toxicity study by oral route (CERB study number 20070258 TR) demonstrated that the DMT was higher than 2000 mg/kg in male and female Sprague Dawley rats. The genotoxicity assay was thus performed at 2000 – 1000 and 500 mg/kg/day (x2). No clinical signs were observed during the mean genotoxicity assay.

GENOTOXICITY ASSAY

 Number of sampling times 1: negative control and treated: 24 hours after the second treatment 1: positive control: 24 hours after single treatment
 Reference substance  cyclophosphamide, 25 mg/kg, intraperitoneal
Number of polychromatic erythrocytes observed for each animal  2000

                                                                                                                                                                                                                                                                                                                                                     

                                                                                                                                                                                                                                                                                                                                                             

RESULTS:

Genotoxicity assay

Number of sampling times : 1: negative control and treated: 24 hours after the second treatment

1: positive control: 24 hours after single treatment

Reference substance

Number of polychromatic erythrocytes observed for each animal: 200

    SAMPLING TIME(24 h after last treatment)  TEST ITEMDOSES in mg/kg/day (x2)     SEX        PCE/NCE RATIO     MICRONUCLEI FOR 1000 PCE
 Mean  Student's t Test (p)  Mean  Mann Whitney(p)
       Negative control Group        VEHICLE  M  1.38    0.70  
 F  1.01    0.40  
 M+F  1.20    0.55  
Positive control Group        Cyclophosphamide25 mg/kg/day (x1)         M  0.72  <0.05 7.60   p<0.01
 F  0.60  <0.05  6.60  p<0.01
 M+F  0.66  <0.001  7.10  p<0.001
LAB 3822TREATEDGROUPS                                 2000  M  1.04  N.S.  0.80  N.S.
 F  1.21  N.S.  0.90  N.S.
 M+F  1.12  N.S.  0.85  N.S.
1000         M  0.93  N.S.  0.60  N.S.
 F  0.94  N.S.  0.30  N.S.
 M+F  0.93  N.S.  0.45  N.S.
       500  M  1.06  N.S.  0.20  p<0.05
 F  0.89  N.S.  0.20  N.S.
 M+F  0.97 N .S.  0.20  p<0.05

No statistically significant increase in the number of micronuclei was noted in male and in female rats dosed with 2000 - 1000 and 500 mg/kg/day (x2).

CONCLUSION:

The test item LAB3822 (batch FAL 07/25) provided by ROQUETTE was investigated by means of the in vivo micronucleus test in male and female OFA Sprague Dawley rats treated orally with 2000 – 1000 and 500 mg/kg/day (x2), followed by one sampling time 24 hours after the last treatment. As a conclusion, LAB3822 induced no genotoxic activity under these experimental conditions.

Thirty minutes, 2 or 4 hours after oral administration of 500, 1000 or 2000 mg/kg of LAB3822, plasmatic concentrations of Isosorbide measured in OFA Sprague-Dawley rats clearly demonstrated a systemic exposure to Isosorbide, and thus to LAB3822. (The determination of Isosorbide concentrations in plasma was performed in a GLP-compliant laboratory using a validated analytical method (CERB/Reference No. 20080183B). The dosing results were thus considered as reliable).