Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test material: the liquid marketed product "Proteol OAT" which contains ca.27.5% of the solid substance "Glutens, hydrozylates, reaction products with lauroyl chloride, sodium salts".

Three in vitro tests were performed according to OECD guidelines n° 471, 473 and 476 and are GLP studies. No mutagenic or genotoxic effect was detected according to these tests

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-04-29 to 1998-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch Number: 98 014 007
Description: extremely pale yellow viscous liquid
Storage conditions: room temperature in the dark

Target gene:

Histidine
Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Salmonella strains were obtained from the Uiversity of California at Berkeley
Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research association
All of the strains were stored at -196°C

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary study:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 pg/plate

Mutation Study
0 / 50 / 150 / 500 / 1500 / 5000 µg/plate

Vehicle / solvent:

Sterile distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 pg/plate for TA100, 5 pg/plate for TA1 535 and 2 pg/plate for E.coli WP2uvrA-

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 ug/plate for TA1537

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 pg/plate for TA98

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9-mix_ 1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10µg/plate for WP2uvrA-

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix_ 5 µg/plate for TA98

Details on test system and experimental conditions:

Preliminary toxicity study:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. A mixture of 0.1 mL of bacterial culture (TA100 or E.coli), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0,1 mL of test material formulation and 0.5 mL os S9-mix or phosphate buffer was overlaid onto sterile plates of Vogel-Bonner Minimal agar. Ten concentrations of the test material formulation and a vehicule control (streile distilled water) were tested. In addition, the sterility of the test material was assessed.
After approximately 48 hours incubation at 37°C the plates were assessed for numbres of revertatn colonies using a Domino colony counter.
Mutation Study-Experiment 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using direct plate incorporation method.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Mutation Study-Experiment 2:
The second experiment was performed using the same methodology as for the experiment 1.

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 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: all cell type tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

No significant 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.

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 five 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 50 to 5000 pg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (sterile distilled water) 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 and/or a reduction in the frequency of revertant colonies to all of the Saimonella strains at the maximum recommended dose both with and without metabolic activation. The test material was, therefore, tested up to the maximum recommended dose of 5000 ug/plate.

No significant 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.

Reference 2

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

Study period:

2009-04-25 to 2009-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test item
Name: LCE08119
Chemical charaterization: sodium lauroyl oat amino acids. anionic surfactant.
CAS: 161074-67-5
Appearance: clear and lighly viscous liquid
Batch: 0820000006
Storage: Required quantity of the test substance was received from the Test Substance Controller (TSC, IIBAT) and stored away from direct heat and sunlight at 15-25°C
Handling: The test substance was handled with necessary protective clothing and all recommended safety measures were followed

Target gene:

All chromosomes

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

Test concentrations with justification for top dose:

Solubility of the test substance
The test substance was found soluble in sterile millipore water and a homogenous solution was obtained.

Test substance preparation
A stock concentration of 0.5 ml of the test substance in 0.5 ml sterile millipore water was prepared to achieve a final concentration of 5 microliter/ml. Subsequently, desired concentrations were freshly preapred through serial dilutions.

Range finding studies
Before commencing the main study, the test substance was assessed for solubility, precipitation and cytotoxicity based on the mitotic index. Seven test concentrations viz., 0.079, 0.157, 0.313, 0.625, 1.25, 2.5, 5 μl/ml were tested with and without S9 and dosed in a final volume of 50 μl of the solvent. A second range finding study was performed as hemolysis was observed in 2.5 and 5 μl/ml and no indications of hemolysis was observed in 1.25 μl/ml. Also, no precipitation was observed in any of the test concentrations. So, three test concentrations between 1.25 and 2.5 μl/ml were chosen i.e. 1.28, 1.6 and 2 μl/ml to identify the high concentration exhibiting (> 50%) mitotic inhibition, if any. Concurrent solvent and positive controls were maintained.

Main study
Five test concentrations were chosen from the range finding studies for cytogenetic analysis i.e. 0.079, 0.157, 0.313, 0.625 and 1.25 μl/ml with and without S9. Since, there was no significant induction in chromosomal aberrations observed in any of the test concentrations with and without S9, an additional experiment continuous exposure – without S9 was performed with 1.5 cell cycle times. Concentrations chosen for the continuous exposure were 0.079, 0.157, 0.313, 0.625, 1.25 μl/ml.

Vehicle / solvent:

sterile millipore water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile millipore water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide monohydrate (Sigma, USA; N° CAS: 6055-19-2) and mitomycin C (Sigma, USA; N° CAS: 50-07-7) at concentrations of 20 μg/ml and 1 μg/ml with and without metabolic activation (S9) respectively, were used as the positive controls.

Details on test system and experimental conditions:

Description of the test procedure
Setting up of cultures
Heparinized whole blood obtained from healthy adult male donor was used to establish lymphocyte cultures in RPMI 1640 supplemented medium and the total volume of the culture medium was 5 ml. Cultures were incubated in PHA-M (GIBCO) containing medium for 48 h in a humidified atmosphere with 5% CO2 at 37 ± 0.5° C. The cultures were treated with the test substance at the chosen concentrations with and without S9 at 48 h from initiation of the cultures and subjected to two kinds of treatment i.e., 4 h exposure - 32 h expression period and continuous exposure - 36 h expression period. After 4 h of exposure in the presence of the test substance, cultures were washed of the test substance at 1600 rpm for 10 min and resuspended in freshly prepared supplemented medium (devoid of PHA-M and S9) and incubated upto 1.5 cell cycle times. For continuous exposure without S9, cultures were treated similarly as detailed above except that the cultures were incubated for 1.5 cell cycle times continuously in the presence of the test substance from the time of the test substance addition at 48 h. All the incubations were done in a humidified atmosphere with 5% CO2 at 37 ± 0.5° C. Duplicate cultures were maintained for all the treatment conditions.

Termination of cultures and preparation of slides
One hour prior to harvest, 0.4 μg/ml of colchicine (Sigma, USA) was added to the cultures. The cells were pelleted, hypotonically shocked with freshly prepared and pre incubated 0.075 M potassium chloride (Merck) solution at 37 ± 1° C, washed and fixed in 3:1 methanol and acetic acid. Slides were prepared from cells resuspended in fixative by dropping technique and heat fixed (SOP/GTX/005).
Staining and scoring of slides
Heat fixed metaphase preparations were stained in Giemsa (Merck), as per SOP/GTX/005. The mitotic index (MI) described as proportion of cells undergoing mitosis in 1000 cells was scored for each treatment condition. Two hundred well spread metaphases were scored (where possible) per concentration of the test substance and solvent control, equally divided amongst the duplicates. From the positive control 48 - 53 metaphases were scored. For control of bias, all slides were coded by QAU and blind scoring was performed.

Evaluation criteria:

The following parameters were reported; the number of cells scored, the percentage mitotic indices, frequencies of structural and numerical aberrations, percentage aberrant metaphases i.e., cells with aberration (inclusive of gaps) and total aberration data at different exposure times.
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.

Gaps were counted, but were not used in the final assessment since they are not considered as significant aberrations. Open breaks were considered as indicators of genetic damage, as were configurations resulting from the repair of breaks. Reunion figures were weighed slightly higher than breaks since they usually resulted from more than one break and may lead to stable configurations.
The number of cells bearing chromosomal aberrations per concentration, the type of aberration, the statistical significance of any increase and its correlation to the test concentration in a given time period, was considered for evaluating the mutagenic/genotoxic potential of the test substance. Though, the final decision was based upon scientific judgment.
There was no concentration related or reproducible increase in the number of cells bearing chromosomal aberrations in any of the concentrations tested and also no statistically significant dose-response relationship was observed. The negative result indicated that, under these experimental conditions the test substance did not induce any significant chromosomal aberration in human peripheral blood lymphocytes.

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.

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For the main study cytogenetic analysis (4 h exposure) five test concentrations were chosen from the range finding studies 0.079, 0.157, 0.313, 0.625, 1.25 μl/ml. The percentage (%) frequency of structural aberrations in the solvent control group and the test concentrations 0.079, 0.157, 0.313, 0.625, 1.25 μl/ml with S9 were 1.50, 1.00, 1.50, 3.50, 1.50, 2.00 respectively and 1.50, 1.50, 1.00, 2.00, 2.00, 2.50 without S9. The results exhibited a lack of significant induction in chromosomal aberrations in the concentrations tested with and without S9, and found to be in consensus with the solvent control.
In the main study - continuous exposure without S9, the percentage mitotic indices of the four test concentrations viz., 0.079, 0.157, 0.313, 0.625 μl/ml were 4.8, 4.2, 3.5 and 1.5 respectively. Corresponding values in the duplicate cultures were 4.5, 4.4, 3.6, and 2.2. This was found to be comparable with the solvent control with a mean percentage mitotic index of 5.45, though a mild inhibition in MI was observed in concentrations 0.157 and 0.313 μl/ml and a (> 50%) mitotic inhibition in 0.625 μl/ml. Hemolysis was observed in 1.25 μl/ml. This inhibition in MI was observed to be a dose response. The percentage (%) frequency of structural aberrations in the solvent control group and the test concentrations 0.079, 0.157, 0.313, 0.625 μl/ml were 2.00, 1.00, 1.00, 1.00, 5.88 respectively. The results exhibited a lack of significant induction in chromosomal aberrations in test concentrations 0.079, 0.157, 0.313, 0.625 μl/ml without S9 and found to be in consensus with the solvent control, though a mild insignificant induction in aberration was observed in 0.625 μl/ml, which was towards the toxic concentration also indicated by a (> 50%) mitotic inhibition. Owing to the low mitotic index only 51 metaphases were analyzed for aberrations in 0.625 μl/ml by blind scoring.
The cultures treated with known mutagens in the 4 h exposure with and without S9 exhibited a percentage (%) frequency of structural aberration 79.25 and 100.00 respectively and in the continuous exposure without S9 were 80.00. A total of 48 - 53 metaphases were scored in the positive controls. A statistically significant (p < 0.05) increase was observed compared to the cultures treated with test substance and solvent alone, confirming the sensitivity of the assay.

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions employed, LCE08119 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. Hence, LCE08119 may be considered non genotoxic in the in vitro chromosomal aberration assay in human lymphocytes

Executive summary:

The ability of LCE08119, 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 Sodium lauroyl oat amino acids, anionic surfactant (as certified by the sponsor) was found soluble in sterile millipore water and the homogenous solution obtained 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. 0.079, 0.157, 0.313, 0.625, 1.25, 2.5, 5 μl/ml. No precipitation was observed in any of the test concentrations; though hemolysis was observed in 2.5 and 5 μl/ml and no indications of hemolysis was observed in 1.25 μl/ml. So, three test concentrations between 1.25 and 2.5 μl/ml were chosen i.e. 1.28, 1.6 and 2 μl/ml to identify the high concentration exhibiting (> 50%) mitotic inhibition, if any. Hemolysis was observed in 2 and 1.6 μl/ml. A mild mitotic inhibition was seen in 1.28 and 1.25 μl/ml though not (> 50%) and their mitotic indices were comparable. Other concentrations i.e. 0.079, 0.157, 0.313 and 0.625 μl/ml exhibited comparable MI to that of solvent control. Hence, from the results of the range finding studies 1.25 μl/ml was chosen as the high dose for the main study analysis.

For the main study cytogenetic analysis five concentrations i.e. 0.079, 0.157, 0.313, 0.625 and 1.25 μl/ml with (10% S9) and without S9 were chosen from the range finding studies. Concurrent solvent and positive controls were maintained. The solvent control received 1% sterile millipore water. 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. 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 0.079, 0.157, 0.313, 0.625 and 1.25 μl/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 the test concentrations 0.079, 0.157, 0.313 μl/ml without metabolic activation in continuous exposure. A mild insignificant induction in aberration was observed in 0.625 μl/ml, which was towards the toxic concentration also indicated by a (> 50%) mitotic inhibition and owing to the low MI observed in 0.625 μl/ml less than 100 metaphases were analyzed for aberrations by blind scoring Hemolysis was observed in 1.25 μl/ml. A mild inhibition in MI was also observed in concentrations 0.157 and 0.313 μl/ml. However, the positive control exhibited statistically increased frequencies of cytogenetic anomalies validating the sensitivity of the assay.

Thus, LCE08119 was considered non genotoxic in the in vitro chromosomal aberration assay in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-02 to 2009-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Test item
Name: LCE08119
Chemical characterization: sodium lauroyl oat amino acids. Anionic surfactant
CAS: 161074-67-5
Storage: required quantity of the test substance was received from the Test Substance Controller (TSC, IIBAT) and stored away from direct heat and sunlight at 15-25 °C
Handling: The test substance was handled with necessary protective clothing and all recommended safety measures were followed

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

exogenous mammalian (S9) activation system.

Test concentrations with justification for top dose:

range finding study:
0.02, 0.04, 0.079, 0.157, 0.313, 0.625, 1.25, 2.5, 5 μl/ml

main study:
0.157, 0.313, 0.625, 1.25 and 2.5 μl/ml

Vehicle / solvent:

millipore water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

methylmethanesulfonate (without S9); benzo(a)pyrene (with S9)

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Test System : The established cell line L5178Y TK+/- is TK competent heterozygous mouse lymphoma cells
Source : American Type Culture Collection (ATCC), U.S.A
Rationale for selection of the test system : To meet the requirements of OECD Guidelines for Testing of Chemicals (Section 4, No.476, Adopted 21st July 1997)

Preparation of Cultures:
An aliquot of frozen cells from storage was revived and washed off the freezing medium (SOP/GTX/012). Cells were handled with care giving appropriate time to recover before use in the lymphoma assay, and subsequently were checked for contamination. No contamination was found.

To make roughly 10 ml of RPMI 20, 7 ml of sterile 1X RPMI 1640 medium (GIBCO) was supplemented with 2 ml of sterile heat inactivated foetal bovine serum (GIBCO).

Solvent Control
Cultures were treated with 0.1 ml sterile millipore water alone.

Solubility of the test substance
The test substance was found miscible in sterile millipore water and a homogenous solution was obtained.
Test substance preparation
A stock concentration of 0.5 ml of the test substance in 0.5 ml sterile millipore water was prepared to achieve a final concentration of 2.5 μl/ml. Subsequently, desired concentrations were freshly prepared through serial dilutions. To achieve the highest concentration 5 μl/ml, the cells were directly treated with 0.1 ml of the test substance.

Treatment of Target Cells:
• 1 x 107 cells were collected in a sterile disposable 50 ml centrifuge tube. The complete growth media was supplemented with 5% v/v foetal bovine serum and the final treatment culture volume was made up to 20 ml.
• Cultures were treated with test substance/known mutagen/sterile millipore water, of known concentrations.
• Those requiring metabolic activation received 1 ml of S9 mix and those without metabolic activation received 1 ml (150 mM) KCl solution.
• The treatment times for cultures in the presence of the test substance were 3 h and 24 h, in humidified 5% CO2 atmosphere at 37 ± 1° C.
• At the end of the treatment periods, cultures were gently agitated, washed twice with RPMI A medium and centrifuged at 1600 rpm for 5 minutes.
• The pelleted cells were resuspended in 50 ml of RPMI 10 complete growth medium and incubated in tissue culture flasks, in a humidified 5% CO2 atmosphere at 37 ± 1° C.
• The solubility of the test substance was assessed visually, at the beginning and end of the treatment.
• Duplicate cultures were set for the test concentrations, solvent and positive controls in the main study.

Expression of the Mutant Phenotype
Cultures were maintained in flasks for a total of 2 days. The cell density was maintained as per the method described in the range finding study. During this time, the tk- mutation was expressed. While subculturing, care was taken such that the cell count did not exceed 1 × 106 cells/ml or retaining at least a total of 1 × 107 cells/flask.

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.

Suspension Growth (SG) is a measure of growth in suspension during treatment and the expression period
Viability is the measure of the cells ability to clone i.e. Cloning efficiency (CE).
Relative Total Growth
Mutant frequency

Statistics:

The number of mutant colonies obtained in the test substance treated cultures and controls was statistically compared employing Chi-square test (NCSS, 2000 statistical software) for significance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

With 3 h treatment
The mutant frequency (MF) (Experiment 1) of concentrations 0.157, 0.313, 0.625, 1.25 and 2.5 μl/ml ranged 58.51 to 82.86 with S9 and 61.93 to 94.25 without S9. Also, a similar trend was observed in the duplicates (Experiment 2) 64.18 to 96.06 with S9 and 66.64 to 87.72 without S9. These values were on par with the solvent control with MF values of 57.00 & 54.86 with S9 and 60.14 & 58.14 without S9 and met the criteria requirement for the assay.
The cultures treated with MMS at concentrations 10 and 20 μg/ml without S9 (Experiment 1 & 2) exhibited MF of 430.92 & 517.67 and 437.37 & 571.63 respectively. The MF for BP treated cultures at concentrations 2 and 3 μg/ml with S9 (Experiment 1 & 2) was 491.28 & 484.77 and 415.16 & 563.57 respectively.
There was no significant increase in the number of small and large colonies in the solvent and treated cultures. However, a significant increase in the total mutant frequencies was seen in the positive control, with an increase in both the small and large colonies, confirming the sensitivity of the experimental conditions and in line with our historical data.
A detailed account on the cell counts, viability and mutant plate counts is given. The cloning efficiencies of the test concentrations with S9 ranged 71.66 to 96.50 (Experiment 1) and 64.87 to 89.30 (Experiment 2), and found comparable to the solvent control which exhibited 96.50 & 102.91. The cloning efficiencies of the test concentrations without S9 ranged 69.65 to 96.50 (Experiment 1) and 67.69 to 93.52 (Experiment 2), and found comparable to the solvent control which exhibited 106.38 & 99.62. Though, an evident decrease was observed in 2.5 μl/ml when compared to the solvent control indicating a propensity for cytotoxicity.
With 24 h treatment
The mutant frequency (MF) of concentrations 0.157, 0.313, 0.625, 1.25 and 2.5 μl/ml ranged 63.48 to 80.52 (Experiment 1) and 59.73 to 86.48 (Experiment 2) without S9. These values were on par with the solvent control which exhibited 50.83 & 52.57 respectively and met the criteria requirement for the assay.
The cultures treated with MMS at concentrations 10 and 20 μg/ml without S9 exhibited MF values 417.73 & 502.86 (Experiment 1) and 469.26 & 561.03 (Experiment 2) respectively. The data on large colonies and small colonies are given. The values obtained from the positive control confirmed the sensitivity of the experimental conditions. A detailed account on the cell counts, viability and mutant plate counts is given. The cloning efficiencies of the test concentrations ranged 73.75 to 87.96 (Experiment 1) and 68.66 to 94.99 (Experiment 2), and found comparable to the solvent control which exhibited 108.20 & 104.62 without S9. Though, an evident decrease in 2.5 μl/ml was observed when compared to the solvent control indicating a propensity for cytotoxicity.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance LCE08119 did not induce any gene mutations under the experimental conditions employed in this study, and therefore can be concluded that the test substance is non - mutagenic in L5178Y TK+/- cell line - mouse lymphoma assay.

Executive summary:

A study in mouse lymphoma L5178Y TK+/- cell line was performed to measure mutation at thymidine kinase (TK) locus with and without exogenous mammalian (rat liver S9) microsomal metabolic activation in order to evaluate the ability of LCE08119, sponsored by SEPPIC, France, in inducing gene mutation. The test substance, constituted of Sodium lauroyl oat amino acids, anionic surfactant (as certified by the sponsor), was found miscible in millipore water and the homogenous solution obtained was used for testing.

A range finding study was performed - 3 h and 24 h treatments with and without S9 to fix the high dose for the main study based on precipitation and cytotoxicity of the test substance. The concentrations chosen were 0.02, 0.04, 0.079, 0.157, 0.313, 0.625, 1.25, 2.5, 5 μl/ml, and the maximum possible dose being 5 μl/ml. Precipitation was not observed in any of the tested concentrations. The % Relative Total Growth (RTG) for the above mentioned concentrations was 75.62, 62.81, 70.03, 62.97, 54.81, 43.52, 38.43, 17.36 and 9.98 respectively with S9 and 72.28, 69.34, 65.60, 55.01, 48.98, 44.64, 34.27, 16.07 and 9.99 respectively without S9 in the 3 h treatment. The percentage (%) Relative Total Growth (RTG) for the above mentioned concentrations in the 24 h treatment without S9 was 71.32, 66.91, 70.36, 57.72, 45.73, 41.32, 32.02, 13.42 and 6.35 respectively. The % RTG of concentration 2.5 μl/ml was found in the range 10 - 20% and for concentration 5 μl/ml it was below the criteria requirement for the high dose (<10%).

Hence, the high concentration chosen for the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was 2.5 μl/ml.

Based on the results of the range finding study, the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was performed employing concentrations 0.157, 0.313, 0.625, 1.25 and 2.5 μl/ml. Two positive controls, methylmethanesulfonate (MMS) in DMSO at concentrations 10 and 20 μg/ml without S9 and benzo(a)pyrene (BP) at concentrations 2 and 3 μg/ml with S9 were used. The solvent control received 0.1 ml of sterile millipore water alone.

The mutant frequency (MF) in the 3 h treatment (Experiment 1) for concentrations 0.157, 0.313, 0.625, 1.25 and 2.5 μl/ml ranged 58.51 to 82.86 with S9 and 61.93 to 94.25 without S9, and the solvent control exhibited MF of 57.00 with S9 and 60.14 without S9. Similarly, the mutant frequency (MF) in the 24 h treatment (Experiment 1) for 0.157, 0.313, 0.625, 1.25 and 2.5 μl/ml ranged 63.48 to 80.52 without S9, and the solvent control exhibited MF of 50.83. These values were on par with the solvent control and met the criteria requirement for the assay. A similar trend was observed in the respective duplicates (Experiment 2) in the 3 h and 24 h treatments.

The positive controls in 3 h treatment (Experiment 1), BP at concentrations 2 and 3 μg/ml exhibited MF 491.28 and 484.77 and MMS at concentrations 10 and 20 μg/ml exhibited MF 430.92 and 517.67. The positive control in 24 h treatment (Experiment 1), MMS at concentrations 10 and 20 μg/ml exhibited MF 417.73 and 502.86. A statistically significant increase, both small and large colonies, in the mutant frequencies of known mutagen treated cultures confirmed the sensitivity of the assay.

Thus, from the above data no apparent evidence of test substance induced gene mutation in L5178Y TK+/- mouse lymphoma cell line was observed under the given test conditions. Hence, LCE08119 was considered non-mutagenic in the mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available