Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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:
2012-05-10/2012-10-18
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose for cross-reference:
reference to same 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)
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Reference substance name:
dodecane-12-lactam, manufacturing of, by-products from, distillation residues
EC Number:
923-400-5
IUPAC Name:
dodecane-12-lactam, manufacturing of, by-products from, distillation residues
Details on test material:
. Test item
TEST ITEM : AMINOCAL
NAME ON IDENTIFICATION TAG : Dodécane-12-lactame, fabrication, sous-produits, résidus de distillation
OTHER NAME : Dodecane-12-lactam, manufacturing of, by-products from, distillation residues
IPL REGISTRATION NUMBER : 120321
BATCH NUMBER : April 2011
EXPIRY DATE : April 2013
APPEARANCE : Brown solid with strong odour
PURITY : Unknown, test item used as supplied
SALT / BASE RATIO : Not applicable
WATER CONTENT : Unknown
CORRECTION FACTOR : No correction factor was applied
MOLECULAR WEIGHT : Not applicable
QUANTITY SUPPLIED : 80 g + 50 g
STORAGE CONDITIONS : Room temperature, protected from light and humidity
STABILITY UNDER
STORAGE CONDITIONS : up to April 2013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Young adult male and female OFA Sprague-Dawley rats (Charles River France origin, Saint-Germain-sur- l’Arbresle; FRANCE) were used for the study.
The period of acclimatization was of 6 and 8 days for male and female rats, respectively. The animals received a clinical examination in order to retain only those which were healthy.
The animals were identified by numbered ear rings.

As recommended, at the start of the main study, the weight range of animals did not exceed ± 20 % of the mean weight when compared to the vehicle group. Indeed, the weight homogeneity of the animals used in this test after random-distribution was checked, by comparing the mean weight in each treatment group with that in the control group using Student’s t test (Table 5). There was no statistically significant difference between the weights of animal treated with the test item and those of control rats, except for the high and low dose male groups. However, it was considered that this slight deviation did not affect the quality of the current study.
Body weight in male rats of the main assay ranged between 165 g and 226 g, and body weight in female rats of the main assay ranged between 155 g and 202 g.

The bedding consists of dust-free, irradiated softwood pellets.
The animals were dispatched in polypropylene cages by random-distribution.
The cages were placed in a ventilated system in the animal room, which was ventilated 20 times per hour. A timer provides lighting 12 hours a day (8 a.m. - 8 p.m.) in all the animal room. The temperature in the ventilated animal cupboard was 22 ± 3 °C, and humidity was 55 ± 15 %, with minor exceptions (see § 13
Deviations).

The animals were not fasted at the treatment time. Drinking water, softened by reverse osmosis and filtered on 0.20 µm membrane, was provided ad libitum. The feedstuff used was No. 811020 RM1(E)SQC 25KGy irradiated from Special Diets Services (ENGLAND), batch 8572, for the preliminary toxicity assay, and No. A04C10 irradiated rat/mouse feed from SAFE, batch 11286, for the confirmatory toxicity assay and the main assay.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle
In accordance with the Study Plan, the test item AMINOCAL was suspended in Carboxymethylcellulose
(Sigma, batch 039K0040) at 0.5% in distilled water (Fresenius, batches 13EH8231 for the toxicity assays,
13ELP281 and 13HAP221 for the main assay, respectively).


The stability of the test item in the solvent was unknown but preparations for treatments were performed just before use.
Details on exposure:
For the preliminary and confirmatory toxicity assays, suspensions at a maximum concentration of 200 mg/mL were prepared and administered to the animals at the dose volume of 10 mL/kg, giving a final dose of 2000 mg/kg. In the main genotoxicity assay, three suspensions at the initial concentrations of 200 - 100 and 50 mg/mL were prepared, giving final doses of 2000 - 1000 and 500 mg/kg, respectively when administered at 10 mL/kg.


Stability in the vehicle was unknown, but the preparations for treatments were performed just before use.


. PRELIMINARY TOXICITY TEST AND CHOICE OF DOSES
Treatments
The procedure described below is in accordance with the latest official recommendations of the principal regulatory authorities (OECD No.474, 1997; Hayashi et al., 1994, 2007) concerning the micronucleus test in rat.
Treatments took the form of 3 successive administrations at 24-hour intervals by the oral route, i.e. under the same conditions than the genotoxicity assay.

Toxicity assay
The EC and OECD guidelines recommend that the test be carried out at the maximum tolerated dose. The maximum tolerated dose is described as the highest dose which causes no mortality, but which may give rise to the appearance of signs of toxicity (OECD No. 474, 1997; Hayashi et al., 1994).

In an effort to reduce the number of laboratory animals required and in order to obtain more accurate information, the toxicity test was performed according to the improved experimental design recommended by Fielder et al. (1992). For the preliminary assay, groups of 2 male and 2 female rats weighing about 200 g and 5 to 6 weeks old received the test item by oral route. Then, for the confirmatory assay, the maximum non-lethal dose was confirmed using a group of 10 animals of both sexes (5 males and 5 females).

The results concerning the symptomatology and the occurrence of death are given in the form of a table, indeed, results of the toxicity test (preliminary and confirmatory assays) by the oral route in rat are reported in Table 1, Appendix No. 1.

Animals were treated three times with 2000 mg/kg/day. The results of the toxicity assays indicated that this dose induced neither mortality nor clinical signs, as confirmed hereafter.

TREATMENTS FOR THE DEFINITIVE GENOTOXICITY ASSAYS
After random-distribution, 4 groups of animals from Charles River (3 groups of 5 male and 5 female rats, and one group of 7 male and 7 female rats, for a total of 22 males and 22 females) were treated with either the test item or the vehicle (negative control groups). Negative control animals were treated under the same conditions as treated animals.

Treatment took the form of 3 successive administrations at 24-hour intervals by oral route. Samples were
taken at 3 to 6 hours after the last treatment.
In addition, 1 group of 5 male and 5 female rats were treated with the reference substance cyclophosphamide for the micronucleus test,
Cyclophosphamide was administered by the intraperitoneal route under a volume of 10 mL/kg as a single injection 24 hours before sampling, at a dose of 25 mg/kg.


Duration of treatment / exposure:
Treatment took the form of 3 successive administrations at 24-hour intervals by oral route. Samples were
taken at 3 to 6 hours after the last treatment.
Frequency of treatment:
One treatment by day
Post exposure period:
Samples were taken at 3 to 6 hours after the last treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 , 2000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
CYCLOPHOSPHAMIDE: 25 mg/kg via intraperitoneal route

Examinations

Tissues and cell types examined:
Bone marrow: polychromatic (PE) and normochromatic (NE) erythrocytes
Details of tissue and slide preparation:
Slide preparation and counting
At the sampling time, the females of all groups, and the males treated with the positive control were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrows were extracted with foetal calf serum (1 mL per animal).

The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant were removed. The centrifugate were spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.

After blind coding the slides by a person not involved in the study, two slides per animal were read by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined from the microscopic examination of 2000 polychromatic erythrocytes.
The polychromatic/normochromatic erythrocyte ratio was determined from the microscopic examination of
1000 erythrocytes per animal.
Evaluation criteria:
. Criteria of validity for the test
The mean number of micronuclated PCE observed in the negative control animals was within the range of the historical values for control animals.
Furthermore, statistically significant increases in the frequency of micronucleated PCE were noted in the groups treated with cyclophosphamide (Mann-Whitney), demonstrating the sensitivity of the animal strain used to a clastogenic agent (Tables 2 and 11).


Otherwise, the results for the ratio PCE/NCE obtained on negative control animals and those treated with the positive reference substance were similar to those generally obtained in the laboratory.


The validity criteria for the test were fulfilled and the test was validated.
Statistics:
The results are shown in the form of tables giving the number of micronucleated PCE per 1000 polychromatic erythrocytes for each animal, together with the total or micronucleated PCE for both genders and the statistical analysis. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), calculated from 1000 polychromatic erythrocytes was also established for both treated and control animals.

The statistical comparison for the polychromatic/normochromatic erythrocyte ratio and for the weight homogeneity within the sex of each group was performed using Student's t test.

Statistical analysis was performed for micronucleated PCE number using a non-parametric test, the Mann- Whitney U-rank test. An analysis of a large number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution. This makes it necessary to use a non-parametric statistical test, and the Mann-Whitney U-rank test is recommended by UKEMS (Lovell et al., 1989). Statistical analysis for micronucleus number was conducted, males and females separately and two sexes combined as the results within the sex of each group were homogeneous.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:

PRELIMINARY TOXICITY ASSAY
A slight decrease in spontaneous motor activity was noted in the 2 male an females rats between 2 and 4 hours after the first treatment. No other clinical sign was observed after up to 6 hours after the third treatment.

CONFIRMATORY TOXICITY ASSAY
No clinical signs were observed after up to 6 hours after the third treatment.

The highest dose retained for the micronucleus assay was set at 2000 mg/kg/day (x2). Two inferior doses of 1000 and 500 mg/kg/day (x2) were also tested.

PCE / NCE ratio
No statistically significant decrease in the ratio PCE to NCE was noted in the 3 AMINOCAL treatment groups when compared to the negative control group . In consequence, no proof of systemic exposure was evidenced.


Frequency of micronucleated PCE
Regarding the frequency of micronucleated polychromatic erythrocytes, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with AMINOCAL at any dose, both sexes combined or males and females separately, when compared with the control group.


Therefore, the test item was considered as not genotoxic under these experimental conditions

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after three oral administrations,
at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.
Executive summary:

The potential clastogenic activity of AMINOCAL (batch April 2011) was tested using the in vivo micronucleus test in female and male rats, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 3 successive daily treatments (see ICH S2(R1) and scientific developments and experience of the regulation of chemical compounds, a working group of the GUM), at the maximum dose recommended by OECD guidelines, i.e. 2000 mg/kg/day (x3), followed by one sampling time 3 to 6 hours after the last treatment. The two lower doses of 1000 and 500 mg/kg/day (x3) were also analysed. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 AMINOCAL treatment groups when compared to the negative control group. No statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with AMINOCAL at any dose, both sexes combined or males and females separately, when compared with the control group. Therefore, the test item was considered as not genotoxic under these experimental conditions.

Categories Display