acetalization product between glucose and C14 alcohol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: No amendment to the study plan was requested. No incident, which could have affected the quality or the interpretation of the results obtained, was observed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2005/06/25

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

LCE08120

IUPAC Name:

LCE08120

Details on test material:

- Chemical Characterization : Fatty alcohol and glucoside. It is derived from vegetable raw materials
- Appearance : white colour pellets
- Lot No. : T82925
- Retest Date : 15.07.2010
- Storage : stored away from direct heat and sunlight below 40° C.
- Handling : The test substance was handled with necessary protective clothing and all recommended safety measures were followed.
- Test substance identity : The test substance identity was confirmed by Department of Analytical Chemistry, IIBAT.

Method

Target gene:

numerical and structural aberrations for chromosome or chromatid

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes obtained from a healthy adult male non- smoking donor without any recent history of illness and under no medication was used for the study.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (10% S9)

Test concentrations with justification for top dose:

range finding study : 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 μg/ml
Main study: 78.13, 156.25, 312.5, 2500 and 5000 μg/ml

Vehicle / solvent:

The test substance formed an emulsified mixture that got saturated in sterile millipore water and found miscible in DMSO (dimethyl sulphoxide). The homogenous solution obtained in DMSO was used for testing.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile millipore water and DMSO (dimethyl sulphoxide)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: and Mitmomycine C

Details on test system and experimental conditions:

The assay was performed in cultured human periphal blood lymphocytes obtained from peripheral blood of a healthy male.
A range finding study, with (10% S9) & without metabolic activation system (S9) was performed to fix the high dose for the main study based on solubility, precipitation and cytotoxicity by assessing the mitotic index (MI).
For the main study cytogenetic analysis five test concentrations i.e. 78.13, 156.25, 312.5, 2500 and 5000 μg/ml with (10% S9) and without S9 were chosen from the range finding study.
Solvent control, 1% (of culture medium) DMSO was tested, and Cyclophosphamide monohydrate and mitomycin C at concentrations of 20 μg/ml and 1 μg/ml with and without metabolic activation (S9) respectively, were used as the positive controls.
The cultures were tested in duplicates for all treatment conditions.
The PHA-M (GIBCO) stimulated cultures were maintained in a humidified atmosphere with 5% CO2 at 37 ± 0.5° C. After the test substance addition at 48 h from the initiation of culture with and without S9, cultures were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical - colchicine (Sigma, USA) at a concentration of 0.4 μg/ml. Metaphase preparations were made from cells in fixative, after hypotonic shock with pre incubated 0.075 M KCl (Merck) at 37° C and a series of washes with fixative (methanol: acetic acid at 3:1) at 1600 rpm for 10 min. Heat fixed preparations were stained with Giemsa (Merck). Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases (where possible) with 46 ± 2 centromeres were analyzed for aberrations in the solvent control and test concentrations and for the positive controls around 50 metaphases were scored. The observations were recorded and summarized as percentage structural and numerical aberrations.

Evaluation criteria:

The following factors were taken into account during evaluation:
- The overall aberration frequencies
- The percentage of cells with aberrations
- The percentage of cells with more than one aberration
- Any evidence for increased amounts of damage with increasing dose, (i.e.,) a positive dose response.
- The estimated number of breaks involved in production of the different types of aberrations observed (i.e.,) complex aberrations, may have more significance than simple breaks.

Statistics:

The percent structural and numerical aberrations and percent mitotic indices of the test substance treated cultures and controls were statistically compared using Student's t – test (NCSS 2000, statistical software) for significance.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A range finding study - 4 h exposure with (10% S9) & without metabolic activation system (S9) and 32 h expression period was performed to fix the high dose for the main study based on solubility, precipitation and cytotoxicity by assessing the mitotic index (MI). Seven concentrations were employed i.e. 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 μg/ml. Heavy precipitation was observed in concentrations 5000 and 2500 μg/ml in the final culture medium, though increased solubility was seen on time and found non-interfering on analysis. Mild hemolysis was observed in 625 and 1250 μg/ml at the end of expression period. Concentrations 78.13 and 156.25 μg/ml were found to have a comparable MI to that of the solvent control. A mild mitotic inhibition was seen in 312.5 and 2500 μg/ml. Cytotoxicity in 5000 μg/ml was observed as a decreased cellularity and a (>50%) inhibition in MI with S9 and (<50%) inhibition in MI without S9. Complete cytotoxicity was observed in 625 and 1250 μg/ml seen as a drastic reduction in cellularity and metaphases, also attributed to the mild hemolysis observed. Hence, from the results of the range finding study 5000 μg/ml was chosen as the high dose for the main study analysis.
For the main study cytogenetic analysis five test concentrations i.e. 78.13, 156.25, 312.5, 2500 and 5000 μg/ml with (10% S9) and without S9 were chosen from the range finding study. Concurrent solvent and positive controls were maintained. The solvent control received 1% DMSO. Positive controls employed for with and without S9 were 20 μg/ml cyclophosphamide (Sigma, USA) and 1 μg/ml mitomycin C (Sigma, USA) respectively.
The cultures were maintained in duplicates for all the treatment conditions. The PHA-M (GIBCO) stimulated cultures were maintained in a humidified atmosphere with 5% CO2 at 37 ± 0.5° C. After the test substance addition at 48 h from the initiation of culture with and without S9, cultures were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical - colchicine (Sigma, USA) at a concentration of 0.4 μg/ml. Metaphase preparations were made from cells in fixative, after hypotonic shock with pre incubated 0.075 M KCl (Merck) at 37° C and a series of washes with fixative (methanol: acetic acid at 3:1) at 1600 rpm for 10 min. Heat fixed preparations were stained with Giemsa (Merck). Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases (where possible) with 46 ± 2 centromeres were analyzed for aberrations in the solvent control and test concentrations and for the positive controls around 50 metaphases were scored. The observations were recorded and summarized as percentage structural and numerical aberrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Peripheral blood lymphocytes obtained from a healthy adult male non- smoking donor without any recent history of illness and under no medication was used for the study.

Applicant's summary and conclusion

Conclusions:

Under the test conditions employed, LCE08120 did not induce any significant structural or numerical aberrations both in the presence and absence of an exogenous mammalian metabolic activation system (S9) in the 4 h and continuous exposures. An interfering precipitation was observed in 2500 and 5000 μg/ml after exposure and thereon an increased solubility was observed which was non-interfering on analysis in 4 h exposure. On the other hand, no evident precipitation was observed in 625 and 1250 μg/ml, though a mild hemolysis was seen as decreased cellularity and metaphases, the definitive reason of which cannot be adjudged. Hence, LCE08120 may be considered non genotoxic in the in vitro chromosomal aberration assay in human lymphocytes.

Executive summary:

The ability of LCE08120, sponsored by SEPPIC, France, in inducing structural and numerical cytogenetic anomalies was evaluated by enumerating the incidence of chromosomal aberrations in cultured human peripheral blood lymphocytes. The peripheral blood was obtained from a healthy adult male non-smoking donor without any recent history of illness. The test substance, constituted of fatty alcohol and glucoside and derived from vegetable raw materials (as certified by the sponsor), formed an emulsified mixture that got saturated in sterile millipore water and found miscible in DMSO (dimethyl sulphoxide). The homogenous solution obtained in DMSO was used for testing.

A range finding study - 4 h exposure with (10% S9) & without metabolic activation system (S9) and 32 h expression period was performed to fix the high dose for the main study based on solubility, precipitation and cytotoxicity by assessing the mitotic index (MI). Seven concentrations were employed i.e. 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 μg/ml. Heavy precipitation was observed in concentrations 5000 and 2500 μg/ml in the final culture medium, though increased solubility was seen on time and found non-interfering on analysis. Mild hemolysis was observed in 625 and 1250 μg/ml at the end of expression period. Concentrations 78.13 and 156.25 μg/ml were found to have a comparable MI to that of the solvent control. A mild mitotic inhibition was seen in 312.5 and 2500 μg/ml. Cytotoxicity in 5000 μg/ml was observed as a decreased cellularity and a (>50%) inhibition in MI with S9 and ( 50%) inhibition in MI without S9. Complete cytotoxicity was observed in 625 and 1250 μg/ml seen as a drastic reduction in cellularity and metaphases, also attributed to the mild hemolysis observed. Hence, from the results of the range finding study 5000 μg/ml was chosen as the high dose for the main study analysis.

For the main study cytogenetic analysis five test concentrations i.e. 78.13, 156.25, 312.5, 2500 and 5000 μg/ml with (10% S9) and without S9 were chosen from the range finding study. Concurrent solvent and positive controls were maintained. The solvent control received 1% DMSO. Positive controls employed for with and without S9 were 20 μg/ml cyclophosphamide (Sigma, USA) and 1 μg/ml mitomycin C (Sigma, USA) respectively.

The cultures were maintained in duplicates for all the treatment conditions. The PHA-M (GIBCO) stimulated cultures were maintained in a humidified atmosphere with 5% CO2 at 37 ± 0.5° C. After the test substance addition at 48 h from the initiation of culture with and without S9, cultures were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical - colchicine (Sigma, USA) at a concentration of 0.4 μg/ml. Metaphase preparations were made from cells in fixative, after hypotonic shock with pre incubated 0.075 M KCl (Merck) at 37° C and a series of washes with fixative (methanol: acetic acid at 3:1) at 1600 rpm for 10 min. Heat fixed preparations were stained with Giemsa (Merck). Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases (where possible) with 46 ± 2 centromeres were analyzed for aberrations in the solvent control and test concentrations and for the positive controls around 50 metaphases were scored. The observations were recorded and summarized as percentage structural and numerical aberrations.

Under the test conditions employed, no significant induction in structural or numerical aberrations was observed in any of the concentrations tested with and without metabolic activation in 4 h exposure. The inhibition in MI was observed to be a dose response. However, the positive controls exhibited statistically increased frequencies of cytogenetic anomalies validating the sensitivity of the assay.

Based on the negative results from 4 h exposure, an additional experiment was performed with continuous exposure in the presence of the test substance for 1.5 cell cycle time without S9. The concentrations employed were 78.13, 156.25, 312.5, 2500 and 5000 μg/ml and the experiment was performed in a similar pattern as detailed in the 4 h exposure.

Under the test conditions employed, no significant induction in structural or numerical aberrations was observed in any of the test concentrations without metabolic activation in continuous exposure. While, considering the % aberrant metaphases (inclusive of gaps) a mild induction in aberrations was observed in 2500 μg/ml, though concluded insignificant as gaps were not excluded in the final assessment. Cytotoxicity was observed in 5000 μg/ml observed as a drastic reduction in cellularity and metaphases and hence not considered for analysis.A (>50%) inhibition in MI was observed in 312.5 and 2500 μg/ml, and a mild inhibition in 78.13 and 156.25 μg/ml. Hence, reduced number of metaphases was scored in 2500 μg/ml. This inhibition in MI was observed to be a dose response. However, the positive control exhibited statistically increased frequencies of cytogenetic anomalies validating the sensitivity of the assay.

Thus, LCE08120 was considered non genotoxic in the in vitro chromosomal aberration assay in human lymphocytes.