Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to Guidelines: OECD n° 471 Dir. 92/69/CEE : B14 EPA (TSCA):


Salmonella Typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co factors).


This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese regulatory Authorities including MITI, MHW, MOL and MAFF. it also meets the requirements of the OECD, EC and USA, EPA (TSCA guidelines).


The dose range was determined in a preliminary toxicity assay and was 15 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as in experiment 1 , fresh cultures of the bacterial strains and fresh test material formulations. An extra dose level was included in both experiments to allow for the toxicity for the test material to the bacterial strains used and to ensure that there were at least four non-toxic dose level plated out.


 


The test material caused a visible reduction in the growth of the bacterial lawn to all of the tester strains at the maximum recommended dose tboth with and without metabolic activation (except for TA98 without S9 -mix). The test material was, therefore, tested up to the maximum recommended dose.


 


No sigificant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.


 


According to guideline OECD 473: 


It is concluded from the present experiment that Lipacide UG does not induce mutagenicity (chromosomal aberration potential) up to a concentration of 0.0 12 mg/ml of culture media in the presence and absence of metabolic activation system (5% as well as 15% v/v S9 mix).


 


According to guideline OECD 490: 


The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.


Methods :


One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (dimethyl sulfoxide (DMSO)), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.


Results:  


The maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that achieved optimum levels of toxicity in all three of the exposure groups.


Conclusion:


The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.


 


 


 


 


 


 


 


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03 June 2019 to 25 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Identification: Undecylenoyl Glycine
Chemical Name:Undecylenoyl Glycine
CAS Number: 54301-26-7
Physical State / Appearance: Off white solid
Batch: SS 655
Molecular Weight: 241
Purity: 95.6%
Expiry Date: 01 May 2020
Storage Conditions: Room temperature, in the dark
Target gene:
Thymidine kinase (tk) locus of L5178Y mouse lymphoma tk ( +/- ) cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium.

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes








Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Lot No. PB/βNF S9 mix used in this study, was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a certificate of S9 efficacy is available.

- method of preparation of S9 mix :S 9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM)

- concentration or volume of S9 mix and S9 in the final culture medium : 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.
Test concentrations with justification for top dose:
- Preliminary Cytotoxicity Test Range finding study: 0; 7.81; 15.63; 31.25; 62.5; 125; 250; 500; 1000; 2000 (μg/mL)
- Main study: 0; 62.5;125; 250; 300; 350; 400; 450; 500 (μg/mL)
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity).The molecular weight of the test item was 241 therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level, and a correction for the purity of the test item of 95.6% was applied to the dose formulations.The dose range used in the preliminary toxicity test was 7.81 to 2000 μg/mL for all three of the exposure groups. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.Based on the %RSG values observed, the maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Solvent DMSO was used.
The test item formed a solution in DMSO at 200 mg/mL considered acceptable for dosing. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (DMSO) exposure groups were used as the vehicle controls.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethanesulphonate (EMS) , was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (DMSO) exposure groups were used as the vehicle controls.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide was used as the positive control in the presence of metabolic activation.
Details on test system and experimental conditions:
Cell Line:

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture:

The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing:

The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to 10% donor horse serum medium.

Test Item Preparation:

The molecular weight of the test item was 241 therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level, and a correction for the purity of the test item of 95.6% was applied to the dose formulations. The test item was found to be insoluble in RPMI 1640 medium at 20 mg/mL. The test item formed a solution in DMSO at 200 mg/mL considered acceptable for dosing. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings are in the following table (see more details on Any other information on materials and methods incl. tables).

Control Preparation:

Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBW8635, purity treated as 100%, expiry 10/01/24) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0389646, purity 99.9%, Expiry 01/10/22) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

EXPOSURE:

Preliminary Toxicity Test:

A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.81 to 2000 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

Mutagenicity Test:

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (62.5 to 500 μg/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 12.5 to 300 μg/mL for the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Plate Scoring:

Microtitre plates were scored using a magnifying mirror box after ten days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for two hours.


Evaluation criteria:
Cytotoxicity was measured by relative survival growth percentage (relative cloning efficiency) or RTG. The highest concentration for the main study was chosen on the basis that it exhibited a 10 - 20% RTG. Scoring of large and small colonies was done visually and microscopically in the test and control plates to understand the mechanistic action of the test substance. Small colonies were defined as less than a quarter of the diameter of the well.
Statistics:
The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Preliminary Cytotoxicity Test:


The dose range of the test item used in the preliminary toxicity test was 7.81 to 2000 μg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:







































































Dose
(μg/mL)
% RSG (-S9)
4-Hour Exposure
% RSG (+S9)
4-Hour Exposure
% RSG (-S9)
24-Hour Exposure
0100100100
7.818810796
15.638911788
31.259911176
62.5938363
125949932
25074645
500110
1000100
2000200

There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups, when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at 2000 μg/mL at the end of the exposure period in the 4-hour exposure groups in both the absence and presence of metabolic activation. Based on the %RSG values observed, the maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline.


Main Experiment results:
































































































































Concentration (μg/mL)4-Hours -S9Concentration
(μg/mL)
4-Hours+S9
% Relative Suspension GrowthRelative Total Growth5-TFT resistant mutants/106 viable cells 2 days after exposure % Relative Suspension GrowthRelative Total Growth5-TFT resistant mutants/106 viable cells 2 days after exposure
01001.00155.1301001.00140.86
62.5870.99152.2062.5950.92150.12
125910.97160.68125870.91125.96
250600.68148.69250580.55152.51
300410.48134.95     300   Ø170.12153.71
350100.08149.57     350   Ø2  
     400   Ø1       400   Ø0  
     450   Ø0      450   Ø1  
    500   Ø0      500    Ø1  
Mutant Frequency threshold for a positive response = 281.13Mutant Frequency threshold for a positive response = 266.86
Positive controlPositive control
EMS

400

 


72



 


0.59



 


1534.07



CP


1.5



 


74



 


0.51



 


1235.67



 

















































































Concentration
(μg/mL)
24-Hours-S9
 %RSGRTGMF§
01001.00141.88
12.5950.96140.40
25700.88139.04
50791.01121.28
100540.77120.11
150320.52141.03
200120.20191.03
       250      Ø4  
        300       Ø1  
MF threshold for a positive response = 267.88
Positive control

Ethylmethanesulphonate



150



 


52



 


0.46



 


1589.23



Ø = Not plated due to excessive toxicity


 

Conclusions:
The maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that achieved optimum levels of toxicity in all three of the exposure groups as recommended by the OECD 490 guideline.

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

Introduction. 


The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.


Methods :


One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (dimethyl sulfoxide (DMSO)), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.


Results:  


The maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that achieved optimum levels of toxicity in all three of the exposure groups.


Conclusion:


The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.


 


 


 


 


 


 




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:
from 1998-04-29 to 1998-05-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP study
Qualifier:
according to
Guideline:
other: OCDE n° 471 Dir. 92/69/CEE : B14 EPA (TSCA)
GLP compliance:
yes (incl. certificate)
Remarks:
1996-02-27
Type of assay:
bacterial reverse mutation assay
Target gene:
for S. typhimurium: histidine gene
for E. coli: tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (10 % liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 15 / 50 / 150 / 500 / 1500 / 5000 µg/plate
Concentration range in the main test (without metabolic activation): 15 / 50 / 150 / 500 / 1500 / 5000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA 100, TA 1535, TA 1537, WP2uvrA-
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
for TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
for TA 100, TA 1535, WP2uvrA-
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
the test material should have induced a reproductible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: preliminary test
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella Typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese regulatory Authorities including MITI, MHW, MOL and MAFF. it also meets the requirements of the OECD, EC and USA, EPA (TSCA guidelines). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as in experiment 1 , fresh cultures of the bacterial strains and fresh test material formulations. An extra dose level was included in both experiments to allow for the toxicity for the test material to the bacterial strains used and to ensure that there were at least four non-toxic dose level plated out.

The vehicle, dimethylsulphoxide, control plates gave counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The test material caused a visible reduction in the growth of the bacterial lawn to all of the tester strains at the maximum recommended dose tboth with and without metabolic activation (except for TA98 without S9 -mix). The test material was, therefore, tested up to the maximum recommended dose.

No sigificant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

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: OECD guideline, GLP study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: human lymphocyte
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.003, 0.006 and 0.0 12 mg of test substance per ml ofthe culture media
Vehicle / solvent:
dimethylsulphoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix activation
Details on test system and experimental conditions:
5.0 mg Lipacide UG was dissolved in 2 ml of the vehicle dimethyl suiphoxide (DMSO) (i.e. 2.5 mg/ml). From this 1.25 mg and 0.625 mg Lipacide UG per ml of DMSO were prepared by serial dilution. These three concentrations of low, mid and high doses i.e. 0.625, 1.25 and 2.5 mg Lipacide UG perm! ofDMSO respectivelv ere used in the test. 37.5 il each ofthese solutions ere added to the celi cultures. These dilutions will result in 0.003, 0.006 and 0.0 12 mg of test substance per ml ofthe culture media, respectively. The experiments were conducted without as well as with metabolic activation using 5% v/v S9 mix for assessment ofchromosomal aberration. The above concentrations of the test substance were selected based on the test substance induced changes in mitotic index and cytotoxicity in the range finding study. The results obtained in the first phase were confirmed by experiments of a second phase comprising of independent assay with increased exposure period but using the same concentrations (but without metabolic activation) as that of the first phase. A third phase was conducted by the same procedure using same test substance concentrations excepting that there concentration ofS9 mix was increased in test system (15% instead of 5% of phase I). At each phase appropriate positive and negative controls were used.

Sterile procedures were followed throughout the experimental period. Blood was drawn from a healthy volunteer by venous puncture using heparinized syringe. A total number of 24 culture vials were maintained in phase I and 12 culture vials each in phase II and III were maintained. To each culture vial containing 7 ml of RPMI 1640 supplemented with 10% fetal calf serum, 0.1 ml of phytohaemagglutinin and an amount of about 0.5 ml of heparinized blood was added for getting the cells(lv phocv tes in cultures). The cultures ere alloed to gro at 37°C for a period of 48 hour. At the end of 48 h the required concentrations of test substances was added as described previously.

At the end of respective treatment periods the ceil division was arrested by adding 30 tl of aqueous colchicine per culture. Afler 2 h the cultures were hypotonically treated with 0.075 M potassium chloride solution followed by fixation and repeated wash in Carnoys fixative. Slides were prepared by dropping celi suspension from a distance on clean slides pre-stored in chilled methanol and distilled water. The siides were dried on a slide warmer and stained with 5% Giemsa in buffer and coded.
Species / strain:
other: human lymphocyte
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no chromosomal aberration induced
Cytotoxicity / choice of top concentrations:
other:
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
cytotoxicity:
Increased mitotic index observed in the low doses (0.003 and 0.006 mg Lipacide UG/mI of culture media) indicate the enhancing ability ofthe test substance for the celi development at low doses. However, decrease observed in mitotic index at higher dose may or may not be indicator of cytotoxicity though there were fewer spreads showing probable cytotoxicity at these concentrations, because it is known that MI is not a good indicator of cytotoxicity (Galloway loc cil). Its relationship to DNA damage is further confounded by the observation that mutagens and carcinogens concentrations that induce in celis high levels of DNA damage did not seem to reduce mitotic index at these concentrations (Slamenova et al 1999). So we do not know the significance of test substance induced decrease in mitotic index at higher concentrations because our experiments on finding effect of Lipacide UG on in vitro chromosomal aberrations were not designed to get that information.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
It is concluded from the present experiment that Lipacide UG does not cause chromosomal aberration in human lymphocytes in cultures up to a concentration of 0.012 mg/ml of culture media, the highest concentration employed in presence or absence of metabolic activation system (5% as well as 15% v/v S9 mix).
Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of Lipacide UG

(supplied by Seppic 75, Quai d’Orsay, F-75321 Paris, Cedex 07, France) in human lymphocyte cultures.

The methods followed were as per the guideline of:

The Organisation for Economic Co-operation and Development (OECD) for testing of chemicals N° 473 (adopted on 2l July, 1997) entitled, “In vitro Mammalian Chromosome Abenation Test”.

The required test concentrations of Lipacide UG was prepared as follows:

- 5.0 mg Lipacide UG was dissolved in 2 ml of the vehicle dimethyl suiphoxide (DMSO) (i.e. 2.5 mg/ml). From this 1.25 mg and 0.625 mg Lipacide UG per ml of DMSO were prepared by serial dilution. These three concentrations of low, mid and high doses i.e. 0.625, 1.25 and 2.5 mg Lipacide UG perm! ofDMSO respectivelv ere used in the test. 37.5 il each ofthese solutions ere added to the celi cultures. These dilutions will result in 0.003, 0.006 and 0.0 12 mg of test substance per ml ofthe culture media, respectively. The experiments were conducted without as well as with metabolic activation using 5% v/v S9 mix for assessment ofchromosomal aberration. The above concentrations of the test substance were selected based on the test substance induced changes in mitotic index and cytotoxicity in the range finding study. The results obtained in the first phase were confirmed by experiments of a second phase comprising of independent assay with increased exposure period but using the same concentrations (but without metabolic activation) as that of the first phase. A third phase was conducted by the same procedure using same test substance concentrations excepting that there concentration ofS9 mix was increased in test system (15% instead of 5% of phase I). At each phase appropriate positive and negative controls were used.

- Sterile procedures were followed throughout the experimental period. Blood was drawn from a healthy volunteer by venous puncture using heparinized syringe. A total number of 24 culture vials were maintained in phase I and 12 culture vials each in phase II and III were maintained. To each culture vial containing 7 ml of RPMI 1640 supplemented with 10% fetal calf serum, 0.1 ml of phytohaemagglutinin and an amount of about 0.5 ml of heparinized blood was added for getting the cells(lv phocv tes in cultures). The cultures ere alloed to gro at 37°C for a period of 48 hour. At the end of 48 h the required concentrations of test substances was added as described previously.

Phase I

In phase I, the medium was changed after 6 h exposure and cells were allowed to grow for about 1.5 normal cell cycle from the time of treatment.

Phase II

In phase II, the exposure period was for about 1.5 normal celi cycle without metabolic activation (to cover at least the period of one whole celi cycle).

Phase III

In phase III, the exposure period was 6 h with higher concentration of S9 (15% v/v instead of 5%) to confirm the negative results observed in the previous phases.

At the end of respective treatment periods the ceil division was arrested by adding 30 tl of aqueous colchicine per culture. Afler 2 h the cultures were hypotonically treated with 0.075 M potassium chloride solution followed by fixation and repeated wash in Carnoys fixative. Slides were prepared by dropping celi suspension from a distance on clean slides pre-stored in chilled methanol and distilled water. The siides were dried on a slide warmer and stained with 5% Giemsa in buffer and coded.

The mitotic index was obtained by screening a minimum of 1000 cells in different fields and the number of metaphases in each field were recorded. This was followed by observing metaphase spreads at 100 X oil immersion for structural and numerical aberrations. Chromosomal and chromatid changes were recorded separately for each aberrant celI and per cent aberration was calculated. Gaps were recorded separately. The total aberrations were considered without gaps and recorded. Comparison of different treatment groups was made with the respective control groups.

In the I phase, the positive control group exhibited significant increase in the per cent aberration as compared to the respective control. The mitomycin-C treated ceils and the cyclophosphamide treated groups showed mean aberration (without gap) 7.5 and 6.5 per cent, respectively. No treatment related changes in the mitotic index as well as the per cent aberrations were observed up to the dose level of 2.5 mg Lipacide UG/ml of vehicle (i.e. 0.0 12 mg of Lipacide UG/ml ofthe culture media) with and without metabolic activation.

In the II’ phase, the negative results obtained in the ft phase were confirmed. No significant increase in per cent aberrations were observed by increasing the exposure period.

In the 3 phase, the negative results obtained in the II’ phase of assay were confirmed in an independent experiment with variation in one parameter i.e., the S9 concentration being increased from 5% to 15%. All the remaining conditions were kept same as in the Ist phase experiment. The per cent aberrant cells were not altered in any treatment groups, confirming the negative results obtained in the earlier experiment.

It is concluded from the present experiment that Lipacide UG does not induce mutagenicity (chromosomal aberration potential) up to a concentration of 0.0 12 mg/ml of culture media in the presence and absence of metabolic activation system (5% as well as 15% v/v S9 mix).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No further data necessary

Additional information

Justification for classification or non-classification

Three in vitro tests data are available (OECD 471, 473 and 490) with the registered substance and showed no significant effect. The substance is not mutagenic and does not need to be classified according to the GHS.