D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: clastogenicity/aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-12 to 2013-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

EC Number:

940-373-5

Cas Number:

1591782-99-8

Molecular formula:

C25H49NO6 (C18 unsatd.) C25H51NO6 (C18 satd.)

IUPAC Name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

additive 1

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

Test material form:

other: waxy paste

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: minimum 7 weeks
- Assigned to test groups randomly: yes
- Fasting period before study: Four hours before treatment all animals were under fasting condition.
- Housing: 5 animals of identical sex per cage, IVC cage (Polysulphone), Type II L
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x per hour
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Aqua ad iniectabilia
- Justification for choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics.
- Concentration of test material in vehicle: 200 mg/mL (1 MTD), 100 mg/mL (0.5 MTD), 40 mg/mL (0.2 MTD)
- Amount of vehicle (if gavage or dermal): 10 mL per kg body weight
- Lot/batch no.: 26210S1_2 AlleMan Pharma

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared by adding the required volume of Aqua ad iniectabilia and further short vortexing. Afterwards the test item-water mixture was heated to 50°C and vortexed again. After cooling to RT, the test item was applied to the animals. When crystallization occured, the test item-water mixture was again heated to 50°C and homogenized by vortexing. All animals received a single volume orally of 10 mL/kg bw.

Frequency of treatment:

The animals received the test item once orally.

Post exposure period:

Sampling of the peripheral blood was carried out on animals 44 h (all control and dose groups) and 68 h (negative control, 1 MTD group) after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
three dose groups: 1 MTD (2000 mg/kg bw), 0.5 MTD (1000 mg/kg bw), 0.2 MTD (400 mg/kg bw)
Basis:
other: solution in Aqua ad iniectabilia

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control: clastogenic control substance, good stability at room temperature, broad basis of historical laboratory data
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:

peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preceding study on toxicity was performed with the same strain and under identical conditions as in the main study. Three animals of each sex were treated orally for detection of the maximum tolerable dose. The maximum dose that was applied was 2000 mg/kg bw according the OECD 474 guideline. The maximum volume which was administered was 10 mL/kg bw. The highest dose group evaluated in the main experiment (2000 mg/kg bw) was based on the toxicity observed in the pre-experiment.

METHOD OF ANALYSIS:
Evaluation of all samples, including those of positive and negative controls, was performed using a flow cytometer (FACScan, BD Biosciences). Anti-CD71 antibodies were labelled with Fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensity were recorded on the FL1, FL2 and FL3 channels for FITC, PE and PI respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test item the ratio between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Evaluation criteria:

There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.

Statistics:

For the statistics the nonparametric Mann-Whitney Test was used. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. Three male and three female mice received a single dose of 2000 mg/kg bw orally and showed toxicity such as reduction of spontaneous activity, piloerection, bradykinesia and half eyelid closure.

RESULTS OF DEFINITIVE STUDY
- Toxicity in the main experiment:
2000 mg/kg bw was tested as the maximum tolerable dose (1 MTD) in the main experiment. The volume administered was 10 mL/kg bw orally. All animals treated with the highest dose (1 MTD) showed mild toxic effects. The animals treated with 1000 mg/kg bw (0.5 MTD) and 400 mg/kg bw (0.2 MTD) showed no toxic effects after the treatment with the test item at all.


- Induction of micronuclei (for Micronucleus assay):
The negative controls (44 h and 68 h) evaluated were within the range of the historical control data of the negative control (0.10 – 0.34%). The mean values of micronuclei observed for the negative control (44 h) were 0.22% (male mice) and 0.21% (female mice). The mean values for the 68 h negative control was 0.24% (male and female mice). The mean value of micronuclei observed after treatment with 0.2 MTD was 0.19% (male and female mice). The values in the male and female group were within the range of the corresponding negative control and within the range of the historical negative control data. The mean values noted for the 0.5 MTD dose group were 0.23% (male mice) and 0.25% (female mice). The values were within the range of the corresponding negative control and within the range of the historical negative control data. The dose group treated with 1 MTD (44 h treatment) showed mean values of 0.23% (male mice) and 0.32% (female mice). The value observed in the male group was within the range of the corresponding negative control. The value observed in the female group was increased compared to the corresponding negative control, but this increase was not statistically significant. Additionally, both values were within the range of the historical negative control data. The mean values observed for the 1 MTD 68 h treatment were 0.29% (male and female mice). These values were within the range of the corresponding negative control and within the range of the historical negative control data. No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.


- Ratio of PCE/NCE (for Micronucleus assay):
The negative controls (44 h, 68 h) were within the range of the historical control data of the negative control (1.19 - 4.30). The mean values noted for the 44 h negative control were 2.31 (male mice) and 2.08 (female mice). The mean values detected for the 68 h negative control were 3.48 (male mice) and 3.79 (female mice). The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.63 (male mice) and 1.52 (female mice). The mean value observed in the male group was statistically significant decreased compared to the corresponding negative control. However, the values observed in the male and in the female group were within the range of the historical negative control data. The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 1.98 (male mice) and 1.69 (female mice). The mean values observed in the male and in the female group were within the range of the historical negative control data. Moreover, in comparison to the corresponding negative control no statistically significant decreases were observed. The animals who received 1 MTD (44 h treatment) showed mean values of 1.85 (male mice) and 2.36 (female mice). The mean values observed in the male and in the female group were within the range of the historical negative control data. Moreover, in comparison to the corresponding negative control no statistically significant increase/decrease were observed. The animal group which was treated with 1 MTD (68 h treatment) showed mean values of the relative PCE of 2.31 (male mice) and 3.96 (female mice). The value observed in the male group was statistically significant decreased compared to the corresponding negative control. However, the values observed in the male and in the female group were within the range of the historical negative control data.

- Statistical evaluation:
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item Glucamide OL RM did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item Glucamide OL RM is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

In a NMRI mouse peripheral blood micronucleus assay, five male and female animals per dose group were treated orally with Glucamide OL RMat doses of 2000, 1000 and 400 mg/kg bw.  Peripheral blood cells were harvested at 44 h (all dose and control groups) and 68 h (negative control and 1 MTD group) post-treatment.  The vehicle was Aqua ad iniectabilia. The animals received the test item once orally.
There were signs of toxicity during the study.  The animals treated with doses of 0.2 and 0.5 MTD showed no signs of systemic toxicity. The animals treated with 1 MTD showed mild signs of systemic toxicity such as reduction of spontaneous activity, piloerection and half eyelid closure. Glucamide OL RM was tested at an adequate dose based on OECD 474. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.