Glycine, N-methyl-N-(1-oxododecyl)-, 1-methylethyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

This study was performed to investigate the potential of the test item to induce gene mutations according to the pre-incubation test (experiment I and experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Experiment I: 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

In experiment I, minor toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 1535 with and without S9 mix and in strain TA 1537 without S9 mix. In experiment II, toxic effects were observed in the presence of metabolic activation in strains TA 1537 and TA 100 all at the upper concentrations.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive controls in experiment I with metabolic activation, where only 50 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (3400 µg/mL) chosen with regard to the molecular weight and the purity of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data.

In both cytogenetic experiments, distinct toxic effects indicated by reduced cell numbers and/or mitotic indices were observed. In the presence of S9 mix, at both preparation intervals statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.

The potential of the test item for inducing chromosomal aberrations was assessed in cultured mammalian cells (CHL cells) by the direct method with 24- and 48-hour treatment and by the metabolic activation method with 6-hour treatment in the presence and absence of S9 mix. Prior to a chromosomal aberration test, a cell growth inhibition test was performed to determine the test concentrations for the chromosomal aberration test. As a result, it was found that the 50% cell growth inhibition concentration of the test item 58.2 µg/mL for the test by the direct method with 48-hour treatment and 1969.2 µg/mL for the test by the metabolic activation method. Based on the results of the cell growth inhibition test, the chromosomal aberration test was performed at a total of 5 test concentrations; specifically, the highest test concentration was set at 120 µg/mL for the test by the direct method and at 4000 µg/mL for the test by the metabolic activation method, and 4 lower concentrations were calculated by dividing 4 times by 2 and rounding the results. The results were negative in the tests by the direct method and by the metabolic activation method. The positive control articles used for the present study produced markedly positive results in each test.

From the above results, it is concluded that the test item has no potential for inducing chromosomal aberrations under the conditions of the present study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001 - 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21-07-1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity: ISOPROPYL N-LAUROYLSARCOSINATE
Description: pale yellow liquid
Batch number: 002013
Purity: 92.9 %
Stability of test item: stable under storage conditions
Expiry date: 31-03-2003

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

According to guideline

Evaluation criteria:

Acceptability of the Assay
This assay is considered acceptable if it meets the following criteria:
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
-the positive control substances should produce a significant increase in mutant colony frequencies.

Evaluation of Results
A test item is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

Not required

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 µg/plate

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks on result:

other:

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the pre-incubation test (experiment I and experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Experiment I: 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

In experiment I, minor toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 1535 with and without S9 mix and in strain TA 1537 without S9 mix. In experiment II, toxic effects were observed in the presence of metabolic activation in strains TA 1537 and TA 100 all at the upper concentrations.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001 - 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21-07-1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: ISOPROPYL N-LAUROYLSARCOSINATE
Description: pale yellow liquid
Batch number: 002013
Purity: 92.9 %
Stability of test item: stable under storage conditions
Expiry date: 31-03-2003

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments I and II: 0.8 - 150 µg/mL
A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. In a quantitative assessment, exponentially growing cell cultures were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. 24 hrs after start of treatment the cells were stained and counted.
The highest concentration used in the pre-test was chosen with respect to the current OECD Guideline for in vitro mammalian cytogenetic tests and 3400 µg/mL were applied as top concentration for treatment of the cultures. Test item concentrations between 26.6 and 3400 µg/mL (+/- S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item after 4 hrs treatment was observed at >= 106 µg/mL (+/- S9 mix). Clear toxic effects were observed after treatment with 26.6 µg/mL (-S9 mix). In addition, after 4 hrs treatment with 106.3, 212.5 and 425 µg/ml (+S9 mix) reduced cell numbers were observed. Higher concentrations (850 – 3400 µg/mL) showed reduced toxicity in comparison to the described concentrations below. 25 µg/mL (-S9 mix) and 150 µg/ml (+S9 mix) were chosen as top concentrations in the main experiment I. Due to missing toxicity the experimental part without S9 mix was repeated with a top test item concentration of 75 µg/ml. Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with >= 26.6 µg/mL. Therefore, 25 µg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 150 µg/mL were chosen as top treatment concentration with respect to the results obtained in experiment I.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

-S9 mix +S9 mix
Exp. I Exp. II Exp. I Exp. II
Exposure period 4 hrs 18 hrs 28 hrs 4 hrs 4 hrs
Recovery 14 hrs - - 14 hrs 24 hrs
Preparation interval 18 hrs 18 hrs 28 hrs 18 hrs 8 hrs

Rationale for test conditions:

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate and a reasonable plating efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22. Lacking metabolic activities of cells under in vitro conditions are a disadvantage of tests with cell cultures as many chemicals only develop a mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations.

Evaluation criteria:

Acceptability of the test
The chromosome aberration test performed in the laboratory was considered acceptable if it meets the following criteria:
-The number of structural aberrations found in the negative and/or solvent controls falls within the range of the historical laboratory control data: 0.0 - 4.0 %
-The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data.

Evaluation of results
A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps)
and/or
-no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
-the number of induced structural chromosome aberrations are not in the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps)
and
-either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Fisher's exact test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

It can be stated that the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive controls in experiment I with metabolic activation, where only 50 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (3400 µg/mL) chosen with regard to the molecular weight and the purity of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data.

In both cytogenetic experiments, distinct toxic effects indicated by reduced cell numbers and/or mitotic indices were observed. In the presence of S9 mix, at both preparation intervals statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

N-Isopropyl lauroyl sarcosinate (Lot No. 980303) is a clear liquid and stable at room temperature.

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung fibroblasts (CHL)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Direct method: 7.5, 15, 30, 60 or 120 µg/mL
Metabolic activation method: 250, 500, 1000, 2000 or 4000 µg/mL (+/- S9 mix)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

mitomycin C

Details on test system and experimental conditions:

Comparable to guideline

Rationale for test conditions:

This study was part of a series of studies to evaluate the safety of the test item. This chromosomal aberration with cultured mammalian cells was in accordance with the guidelines for toxicity studies of cosmetics.

Evaluation criteria:

Criteria for Judging the Validity of the Study
The present study was considered valid when it met the following conditions: the incidence of cells with chromosomal aberrations was lower than 5% in the cells treated with the negative control article; the incidence of cells with chromosomal aberrations other than gaps was 10% or higher in the cells treated with the positive control article; the incidence of chromosomal aberrations noted in the cells treated with each control article was within the range of the background data obtained at the testing facility; and no factors that might affect the test system existed.

Evaluation Criteria
The final judgment on the outcome of each test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5%, the result was assessed as negative; from 5 to 10% (10% excluded), equivocal; and 10% or higher with a concentration-dependent rise, positive. The incidence of cells having structural aberrations with gaps and the incidence of those without gaps were calculated separately, and cells with gaps were assessed for chromosomal aberrations.

Statistics:

No significance tests were performed.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung fibroblasts (CHL)

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

Conclusions:

It is concluded that the test item has no potential for inducing chromosomal aberrations under the conditions of the present study.

Executive summary:

The potential of the test item for inducing chromosomal aberrations was assessed in cultured mammalian cells (CHL cells) by the direct method with 24- and 48-hour treatment and by the metabolic activation method with 6-hour treatment in the presence and absence of S9 mix. Prior to a chromosomal aberration test, a cell growth inhibition test was performed to determine the test concentrations for the chromosomal aberration test. As a result, it was found that the 50% cell growth inhibition concentration of the test item 58.2 µg/mL for the test by the direct method with 48-hour treatment and 1969.2 µg/mL for the test by the metabolic activation method. Based on the results of the cell growth inhibition test, the chromosomal aberration test was performed at a total of 5 test concentrations; specifically, the highest test concentration was set at 120 µg/mL for the test by the direct method and at 4000 µg/mL for the test by the metabolic activation method, and 4 lower concentrations were calculated by dividing 4 times by 2 and rounding the results. The results were negative in the tests by the direct method and by the metabolic activation method. The positive control articles used for the present study produced markedly positive results in each test.

From the above results, it is concluded that the test item has no potential for inducing chromosomal aberrations under the conditions of the present study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 and 48 hrs after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 m / 5 f) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic (PCE) and normochromatic (NCE) erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide used as positive control and administered orally showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Ten male Wistar rats were treated once via gavage with 2000 mg/kg test item in corn oil. As vehicle controls ten male rats were treated with corn oil. Five of these rats were sacrificed 3 hours and the remaining five rats were sacrificed 24 hours after administration. Thereafter hepatocytes and stomach cells were isolated and investigated in the comet assay. As positive control 400 mg/kg EMS in corn oil was administered once via gavage to 10 male rats which were sacrificed for the examination of hepatocytes and stomach cells in the comet assay 3 hours after administration. Doses for the comet assay were selected based on the results of a dose range finding study, revealing no tissue toxicity in liver and stomach. Treated rats of the main study showed no signs of toxicity. The tail lengths of hepatocytes and stomach cells of positive control rats were clearly and statistically significant increased, thus demonstrating the sensitivity of the test system for the detection of genotoxic effects.

After administration of 2000 mg/kg test item the tail lengths of hepatocytes and stomach cells were comparable to those of vehicle controls. In conclusion, the test item is considered to be non-genotoxic in the comet assay in vivo to hepatocytes and stomach cells of male Wistar rats.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21-07-1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Identity: ISOPROPYL N-LAUROYLSARCOSINATE
Description: pale yellow liquid
Batch number: 002013
Purity: 92.9 %
Stability of test item: stable under storage conditions
Expiry date: 31-03-2003

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source: RCC Ltd., Füllinsdorf / Switzerland
Number of animals: 72 (36 m / 36 f)
Age at start of acclimatization: 8 – 10 weeks
Body weight at start: 27.6 – 33.8 g

Acclimatization: About one week under test conditions after health examination.
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with target ranges for temperature 22 +/- 3°C and for relative humidity between 30-70 %. 12 hours fluorescent light/12 hours dark, music during the light period.
Accommodation: Individually in Makrolon type I cages
Diet: Pelleted standard Altromin 1324 ad libitum
Water: Community tap-water ad libitum

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Study Procedure
Test Groups: 6 m / 6 f were assigned to each test group. Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h and 24 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Duration of treatment / exposure:

One oral dosing via gavage and sacrifice after 24 h or 48 h

Frequency of treatment:

One oral dosing via gavage and sacrifice after 24 h or 48 h

Post exposure period:

24 h or 48 h

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

dosing for 24 h preparation interval

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

dosing for 24 h preparation interval

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

dosing for 24 h and 48 h preparation interval

No. of animals per sex per dose:

5 m / 5 f

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Tissues and cell types examined:

At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides and 10 animals (5 m / 5 f) per test group were evaluated.

Details of tissue and slide preparation:

According to guideline

Evaluation criteria:

Acceptance Criteria
The study was considered valid as the following criteria are met:
-the negative controls are in the range of the historical control data (0.01 - 0.15 %; mean = 0.066 ± 0.032 PCEs with micronuclei)
-the positive controls are in the range of the historical control data (0.91 - 2.975 %; mean = 1.644 ± 0.446 PCEs with micronuclei)
-at least 80 % of animals are evaluable

Evaluation of Results
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. The primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Whitney test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg: reduced spontaneous activity, eyelid closure, ruffled fur

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

It can be stated that the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 and 48 hrs after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 m / 5 f) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic (PCE) and normochromatic (NCE) erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000 and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide used as positive control and administered orally showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Identity: ELDEW SL-205
Description: pale yellow liquid
Batch number: 412035
Purity: 100 %
Stability of test item: stable under storage conditions
Expiry date: 09-12-2007

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

The animals used were young adult male rats, strain CRL:(WI) BR Wistar, bred and supplied by Charles River, Sulzfeld, Germany. At application they weighed 186-291 g, and were thus approximately 6 to 8 weeks of age.
On the day of arrival, the health of the animals was appraised before acclimatizing them to the housing conditions for a period of at least five days. Only healthy animals without symptoms were used in the study.
The animals were kept individually in type IIA cages, they were identified by cage marks, and bedding of soft wood granules was used.
Husbandry was standardized, with twelve hours of electrical lighting daily, 21.5-22.0°C room temperature, and 42-55% mean relative humidity.
The fixed-formula feed was NAFAG 9441 and tap water were available to the rats ad libitum.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Rational for Dose Selection
The selection of the doses was based on a dose range finding study. The test substance was administered in a volume of 10 mL/kg. Because there is no difference in toxicity between the sexes the study was performed in males. Six male rats were treated at 2000 mg/kg and observed regularly. Three rats were killed about 24 hours after administration, the other three rats were sacrificed on day 3 post administration; samples of the liver and stomach were fixed in 10% buffered formalin. There were no changes of general condition or behaviour of the animals at any time. The fixed tissues of the rats sacrificed the day after administration were examined histopathologically. No treatment related findings were seen in the forestomach or gastric mucosa of the glandular stomach. In the liver no indication of hepatocellular degeneration was found in any of the rats. The number of hepatocellular mitotic figures was slightly increased in one of the rats. Since mitoses are a regular finding in hepatocytes of young rats this finding is most probably incidental. Based on these results the limit dose of 2000 mg/kg was chosen for the comet assay in male rats.

Duration of treatment / exposure:

Single dosing via gavage and sacrificed 3 or 24 hours after treatment

Frequency of treatment:

Single dosing via gavage and sacrificed 3 or 24 hours after treatment

Post exposure period:

3 or 24 hrs

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

sacrifice 3 or 24 hrs after treatment

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

sacrifice 3 or 24 hrs after treatment

No. of animals per sex per dose:

5 m

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulfonate

Tissues and cell types examined:

Hepatocytes and stomach cells

Details of tissue and slide preparation:

Comparable to guideline

Evaluation criteria:

Assay Acceptance Criteria
An assay is considered acceptable if the following criteria are met:
-Tail length data obtained for a given treatment are acceptable as part of the evaluation if obtained from at least two slides and 100 cells per animal
-The positive control EMS should induce a mean tail length increase of at least 30% compared to the mean tail length value of the vehicle control group

Assay Assessment Criteria
A test was considered positive if there was a biologically relevant and statistically significant increase of the evaluated parameter(s) in tissue cells of treated animals in comparison to the negative control.
A test was considered negative, if there were no biologically relevant and statistically increases of evaluated parameters. A test was also considered negative if there was a significant increase of investigated parameters, which according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of mean data of evaluated parameters above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, normally a second test will be performed.

Statistics:

Yes, the one-sided Wilcoxon rank test was applied for comparison of the treatment groups

Conclusions:

The test item is considered to be non-genotoxic in the comet assay in vivo to hepatocytes and stomach cells of male Wistar rats.

Executive summary:

Ten male Wistar rats were treated once via gavage with 2000 mg/kg test item in corn oil. As vehicle controls ten male rats were treated with corn oil. Five of these rats were sacrificed 3 hours and the remaining five rats were sacrificed 24 hours after administration. Thereafter hepatocytes and stomach cells were isolated and investigated in the comet assay. As positive control 400 mg/kg EMS in corn oil was administered once via gavage to 10 male rats which were sacrificed for the examination of hepatocytes and stomach cells in the comet assay 3 hours after administration. Doses for the comet assay were selected based on the results of a dose range finding study, revealing no tissue toxicity in liver and stomach. Treated rats of the main study showed no signs of toxicity. The tail lengths of hepatocytes and stomach cells of positive control rats were clearly and statistically significant increased, thus demonstrating the sensitivity of the test system for the detection of genotoxic effects.

After administration of 2000 mg/kg test item the tail lengths of hepatocytes and stomach cells were comparable to those of vehicle controls. In conclusion, the test item is considered to be non-genotoxic in the comet assay in vivo to hepatocytes and stomach cells of male Wistar rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genetic toxicity of the test item was investigated in different in vitro and in vivo studies, which in total gave no indication for a mutagenic/genotoxic potential. Therefore, there is no need for classification and labelling of the test item according to CLP Regulation 1272/2008/EG.