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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registration substance was not mutagenic in a guideline conform Salmonella typhimurium reverse mutation assay (Ames test) using five tester strains with and without metabolic activation (S9-mix from induced rat liver). Likewise, the submission substance was also not mutagenic in a guideline compliant mammalian cell gene (HPRT) mutation assay in V79 Chinese hamster cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from induced rat liver

Test concentrations with justification for top dose:

Experiment I: 3.16, 10.0, 31.6, 1000, 316, 1000, 2500 and 5000 µg / plate
Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg / plate (additionally 1.0 and 3.16 µg/plate for TA 100, TA 1535 and TA 1537 without S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility reasons

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: Three plates for each strain and dose level including controls

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of background lawn or reduction in number of revertants down to a mutation facot < 0.5 in relation to solvent control

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Glucamide OL RM did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.Therefore, Glucamide OL RM is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to Glucamide OL RM at concentrations of 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate(experiment I) and 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II). Glucamide OL RM was tested up to the limit concentration of 5000 μg/plate in all tester strains used. The positive controls induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-08 to 2013-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.00, 3.16 and 10 mM

Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
5, 10, 15, 20, 22.5, 25, 30 and 35 µM

Experiment I
without metabolic activation: 0.10, 0.25, 0.5, 1, 4, 6, 8, 10, 12 and 14 µM
and with metabolic activation: 1.1, 2.8, 5.6, 11, 28, 56, 111, 167 and 222 µM
Experiment II
without metabolic activation: 15.0, 20.0, 27.5, 35.0, 42.5, 50.0, 90.0, 100 and 140 µM
and with metabolic activation: 0.75, 1.5, 4.0, 7.5, 15, 40, 100, 150 and 210 µM

Vehicle / solvent:

Vehicle (Solvent) used: dimethylsulfoxide (DMSO). The test item was heated to around 50 °C until it was liquid, dissolved in dimethylsulfoxide (DMSO) and added to cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) prior to treatment.

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: 300 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

1 and 1.5 µg/mL; with metabolic activation Migrated to IUCLID6: 1 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: DMSO
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 6 μM; experiment I with S9: ≥ 111 μM; Experiment II without S9: ≥ 90 μM; Experiment II with S9:≥ 150 μM

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Glucamide OL RM is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to Glucamide OL RM dissolved in DMSO at concentrations of

- 0.10, 0.25, 0.5, 1, 4, 6, 8, 10, 12 and 14 µM (without metabolic activation, Experiment I)

- 1.1, 2.8, 5.6, 11, 28, 56, 111, 167 and 222 µM (with metabolic activation, Experiment I)

- 15.0, 20.0, 27.5, 35.0, 42.5, 50.0, 90.0, 100 and 140 µM (without metabolic activation, Experiment II)

- 0.75, 1.5, 4.0, 7.5, 15, 40, 100, 150 and 210 µM (with metabolic activation, Experiment II).

Glucamide OL RM was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 10.0% for the highest concentration (14 µM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 222 µM with a relative growth of 13.4%. In experiment II without metabolic activation the relative growth was 11.5% for the highest concentration (140 µM) evaluated. The highest concentration evaluated with metabolic activation was 210 µM with a relative growth of 12.9%.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 3.31 was found at a concentration of 14 µM with a relative growth of 10.0%.

The exceedance of the threshold value of 3.0 could not be confirmed in the second experiment (without metabolic activation), which was performed under stronger conditions (long term exposure). Therefore, this finding of experiment I was considered to be not biologically relevant.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.56 was found at a concentration of 5.6 µM with a relative growth of 84.2%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.92 was found at a concentration of 42.5 µM with a relative growth of 82.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.23 was found at a concentration of 210 µM with a relative growth of 12.9%.

The positive controlsdidinduce the appropriate response. 

There was no evidence of a concentration related positive responseof induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The registration substance did not induce chromosome aberrations (micronuclei) or clastogenic effects in a guideline conform micronucleus test in vivo.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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.

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

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Glucamide OL was tested for potential point mutation in a guideline conform bacterial reverse mutation assay (Ames test) according to OECD TG 471 in the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 in two independent experiments. There were no mutagenic responses in any of the tester strains up to test concentrations of 5000 µg per plate, neither with nor without metabolic activation (S9-mix from induced rat liver). Therefore the registration substance is considered to be non-mutagenic in this bacterial reverse mutation assay. This study is selected as key study.

Glucamide OL was tested for potential gene mutation in a guideline conform mammalian cell gene mutation assay (HPRT locus) according to OECD TG 476 in V79 cells of the Chinese hamster. Using independent experiments, no biologically relevant increase of mutants was found after treatment with the test item, neither with nor without metabolic activation. No dose-response relationship was observed. Therefore, the registration substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster. This study is selected as key study.

Likewise, Glucamide OL was also not mutagenic when tested inan oral (gavage)in vivomicronucleus assay in mice up to the limit dose of 2000 mg/kg body weight. Therefore, the registration substance is considered to be non-mutagenic in the micronucleus test in mice. This study is selected as key study.

In conclusion, Glucamide OL is not mutagenic in the bacterial reverse mutation assay, the mammalian (HPRT) mutation test in V97 cells and in the in vivo micronucleus test in mice.

Justification for classification or non-classification

Based on all availabe data, the registered substance is not consiered to be mutagenic. Accordingly, classification & labelling does not apply.