D-Gluconic acid, δ-lactone, reaction products with propanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key information is based on state of the art in-vitro studies performed according to OECD guidelines.

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:

September-October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Samson 18
Lot No.: Z333-94-476
Purity: 50%
Molecular Weight: 253 g/mol
Description: Clear colorless liquid
Storage Conditions: Room temperature, protected from light
Receipt Date: 02 September 2015

Target gene:

Selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

initial toxicity-mutation assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate
confirmatory mutagenicity assay: 50.0, 150, 500, 1500 and 5000 μg per plate.

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Preparation of Tester Strains:
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.
Metabolic Activation System:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3503, Exp. Date: 28 July 2017) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
Initial Toxicity-Mutation Assay to Select Dose Levels:
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test article, in duplicate, in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon absence of post-treatment toxicity.
Confirmatory Mutagenicity Assay:
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and five dose levels of the test article, in triplicate, in the presence and absence of Aroclor-induced rat liver S9.

Evaluation criteria:

Scoring
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
Criteria:
- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

Not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Samson 18 did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment Start: April 03, 2017, Experiment Completion: May 27, 2017

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:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

Test Item Name FiberHance BM Solution
Batch/Lot Number SMSN-071916
CAS Name D-Gluconic acid, δ-lactone, reaction products with propanolamine
CAS Number 2060570-71-8
Purity 49.71% w/w
Supplied to JRF by Ashland Services B.V.
Date of Manufacture 19 July 2016
Date of Expiry 19 July 2017
Appearance Colourless clear liquid
pH 5.2
Storage Condition(at JRF) As per the instruction received from the Sponsor on storage of the test item, the test item was stored :
- Storage Temperature: 20-25°C
- Storage Container: In original container as supplied by the Sponsor
- Storage Location: Test Item Control Office, JRF

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: Human peripheral blood lymphocyte cultures
- Suitability of cells: recommended by the OECD and other regulatory authorities
- Sex, age and number of blood donors: Blood was drawn from two healthy, 27 and 28 years old male volunteers for the cytotoxicity test and main study, respectively, by venous puncture using heparinised syringe.
- Cell culture: The whole blood was cultured in RPMI-1640 (Roswell Park Memorial Institute) with L-glutamine and 25 mm HEPES (Sigma R4130) containing penicillin and streptomycin supplemented with 20% heat-inactivated (56 °C; 30 min) fetal bovine serum.:Cultures were prepared separately in centrifuge tubes by adding heparin sodium containing 0.5 mL of whole blood into 9.5 mL of complete medium [containing 20% serum and 2% phytohaemagglutinin (PHA-M)] in culture tubes and incubated at 37 ± 1 °C for approximately 48 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was buffered and supplemented with the essential co-factors -NADP, KCl, and D-Glucose-6-phosphate to form the “S9 mix”. The S9 fraction was used at a concentration of 2% in the final culture medium.

Test concentrations with justification for top dose:

CYTOTOXICITY TEST: 0.125, 0.25, 0.5, 1, 2 and 4 µL FiberHance BM Solution/mL of culture medium
CHROMOSOMAL ABERRATION TEST; 0.25, 0.5, 1, 2 and 4 µL FiberHance BM Solution/mL of culture medium

Vehicle / solvent:

Not applicable

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

CYTOTOXICITY TEST:
METHOD OF APPLICATION: in medium in culture tubes:
Volumes of 180 µL of relevant stock solutions were added into respective tubes containing 17.82 mL of medium to obtain the required test concentrations for the treatment in the absence and presence of metabolic activation system. To each culture tube 8 mL of the treatment medium was added. Serum free medium with KCl was used in the absence of metabolic activation while medium with S9 mix was used for treatment in the presence of metabolic activation system (2% v/v S9 mix).
DURATION:
The cultures were exposed for 4 hours both in the absence and presence of metabolic activation.
The cells were treated with metaphase arresting substance (colchicine, 0.3 µg/mL) 3 h and 18 minutes prior to harvesting:

CHROMOSOMAL ABERRATION TEST:
METHOD OF APPLICATION: in medium in culture tubes:
Serum free medium with KCl was used in the absence of metabolic activation while medium with S9 mix was used in the presence of metabolic activation.
DURATION:
Phase I: exposure for 4 h in absence and presence (2% v/v S9 mix) of metabolic activation
Phase II: exposure for 24 h in absence of metabolic activation
POST-TREATMENT:
In Phase I at the end of 4 hour treatment, cultures were centrifuged at 2000 rpm for 10 minutes and the supernatant was replaced with fresh complete medium and incubated at 37 ± 1 °C and 5% CO2 in a CO2 incubator until harvesting while in phase II treatment was continued for 24 in complete medium. The cells were harvested and processed for slide preparation approximately after 24 hour from the beginning of treatment.
As the phase II experiment was performed to confirm the negative results obtained in the absence of metabolic activation in Phase I, slide scoring data was evaluated after evaluating the data of phase I experiment.

CONTROLS:
Concurrent negative (RPMI-1640) and positive controls were maintained, in duplicate, along with each phase of the experiment, both in the absence and presence of metabolic activation system. Cyclophosphamide (40 µg/mL) was used as the positive control in the presence of metabolic activation and Mitomycin-C (0.3 µg/mL) was used as the positive control in the absence of metabolic activation.

HARVESTING OF CELLS:
The cells were treated with metaphase arresting substance (colchicine, 0.3 µg/mL) 3 h 20 minutes prior to harvesting in both phases.
The cultures were centrifuged at 2000 rpm for a period of 10 minutes. The supernatant was discarded and cells were suspended in 8 mL of freshly prepared 0.075 M potassium chloride solution. The cultures were incubated in the water bath at 37 ± 1 °C for a period of 30 minutes, centrifuged at around 2000 rpm for 10 minutes and supernatant was removed leaving approximately 1-2 mL. Freshly prepared chilled Carnoy's fixative (methanol and glacial acetic acid at the ratio of 3:1) was added and mixed vigorously. The tubes were then centrifuged at around 2000 rpm again for a period of 10 minutes and supernatant was removed. This procedure was repeated twice. At the final stage of centrifugation, supernatant was removed, leaving approximately 0.5 mL of fixative with cell pellet.

SLIDE PREPARATION:
From each culture tube, the fixed cell suspensionwas dropped on two pre-cleaned, ice-chilled slides. The slides were hen dried and labeled with study number, culture number, and slide number. Subsequently, they were stained with 5% Giemsa in phosphate buffer for 8 minutes. The slides were made permanent by mounting a cover slip with DPX mountant. Out of two slides, one was used for scoring and the other served as reserve. In order to prevent bias in the scoring procedure for chromosomal aberrations, the slide numbers were masked with code numbers and only decoded after scoring.

SCORING:
The slides were examined under a microscope and a minimum number of 1000 cells per slide were counted and numbers of metaphases were recorded in different fields to determine the mitotic index. Mitotic index was scored for all the five test concentrations.
Based on mitotic index; three suitable concentrations were selected for assessment of chromosomal aberrations. All slides, including those of positive and negative controls, were independently coded prior to microscopic analysis for chromosomal aberrations.
300 well spread metaphases (150/replicate) were scored for the structural chromosomal aberrations per concentration and controls.
Only cells containing 46 ± 2 chromosomes were examined for structural changes. A smaller number (e.g. 50 metaphases per slide) of metaphases were analyzed in slides showing higher frequency (≥20%) of aberrant cells. Gaps, breaks, fragments, exchanges and deletions, were recorded with their numbers and frequencies for all the treated and control cultures separately. In addition, 100 metaphases/replicate were examined for polyploidy. The number of metaphases with only gap or polyploidy were recorded but not considered in the calcualtion of total aberrations and percent aberrant cells.

Rationale for test conditions:

Assay Acceptance Criteria:
i. Slides to be assessed for aberrations should not have any evidence of contamination.
ii. Data of concurrent negative control cultures should be within the range of historical negative control database.
iii. Concurrent positive control should induce responses compatible with the historical data base and significantly higher than the concurrent negative control.
iv. Cell proliferation criteria in the negative control should be fulfilled.
v. All three experimental conditions should be tested unless one resulted in positive results.
vi. Adequate number of cells and concentrations should be analyzable.
vii. The criteria for the selection of the highest concentration should be consistent with the dose selection criteria (i.e. 55±10% reduction in mitotic index).

Evaluation criteria:

Assay Evaluation Criteria:
The test item was considered mutagenic if the results met the following criteria.
i. At least one of the test concentrations exhibits a statistically significant increase in % aberrant cells compared with the concurrent negative control.
ii. A dose related statistically significant (biologically relevant) increase in % aberrant cells in at least one concentration when evaluated with an appropriate trend test.
iii. Increases in % aberrant cells are not associated with large changes in pH or osmolality of the treated cultures.
iv. Any of the results are outside the distribution of the historical negative control data.
To assess the non-clastogenic activity of the test item following criteria were evaluated:
i. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii. There is no concentration-related increase when evaluated with an appropriate trend test.
An increase in the number of polyploid cells indicates that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. Biological relevance was considered first. The test item was considered non-mutagenic if the results did not meet the above mentioned criteria.

Statistics:

Gaps and polyploidy were not included in the calculation of total aberration frequency. Data on mitotic index, polyploidy, and percent aberrant cells were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad. and Weil, 1994). Where the data did not meet the homogeneity of variance, Student's t-test was performed to determine the level of significant difference between the negative control and three selected test concentrations (selected based on the mitotic index data) and positive controls.

Key result

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY TEST:
Precipitation was not observed at the tested concentrations up to 4 µL/mL of culture medium, which corresponds with a concentration of Reaction mass of 3-hydroxypropan-1-aminium D-gluconate and N-(3-hydroxypropyl)-D-gluconamide up to and including 2 mg/mL. Significant change in the pH or osmolality was not observed up to the highest concentration of 4 µL/mL, both in the absence and presence of metabolic activation. Significant reduction in mitotic index (i.e. 50 ± 5% reduction in mitotic index) was not observed at all the tested concentrations up to 4 µL/mL of FiberHance BM Solution, both in the absence and presence of metabolic activation.

CHROMOSOMAL ABERRATION TEST:
Phase I; FiberHance BM Solution did not induce statistically significant or biologically relevant increase in the number of cells with chromosomal aberrations or in the number of polyploid cells up to the highest concentration of 4 µL/mL of culture medium.
Phase II: In the absence of metabolic activation, FiberHance BM Solution did not induce a statistically significant or biologically relevant increase in the number of cells with chromosomal aberrations after 24 hours of exposure. A single incidence of polyploidy was observed at the dose level of 1 µL/mL in the absence of metabolic activation (24-h exposure) which was neither statistically nor biologically significant.
Hence, in neither of the two phases any dose related genotoxic response was observed up to the limit dose of FiberHance BM Solution at 4 µL/mL of culture medium, which corresponds with a concentration of Reaction mass of 3-hydroxypropan-1-aminium D-gluconate and N-(3-hydroxypropyl)-D-gluconamide up to and including 2 mg/mL.

Conclusions:

From the results of this study, it is concluded that FiberHance BM Solution did not show any potential to induce chromosome aberration, both in the absence and presence of metabolic activation under the present experimental conditions and is negative for clastogenicity up to 4 µL/mL (corresponding with a dissolved solid fraction of 2 mg/mL) .

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Forward Mutation Test at the Hprt Locus of the Chinese Hamster Ovary (CHO) - K1 Cell Line

Specific details on test material used for the study:

Test Item Name FiberHance BM Solution
Batch/Lot Number SMSN-071916
CAS Name D-Gluconic acid, δ-lactone, reaction products with propanolamine
CAS Number (CAS RN) 2060570-71-8
Purity 49.71% w/w
Manufactured by Not Provided
Supplied to JRF by Ashland Services B.V.
Date of Manufacture 19 July 2016
Date of Expiry 19 July 2017
Appearance Colourless clear liquid
Storage Condition (at JRF) As per the instruction received from the Sponsor on storage of the test item, the FiberHance BM Solution was stored as follows :
Storage Temperature : 20-25°C
Storage Container : In original container as supplied by the Sponsor
Storage Location : Test Item Control Office, JRF

Target gene:

Cells deficient in Hypoxanthine-guanine Phosphoribosyl Transferase (HPRT), due to mutation, are resistant to the cytotoxic effects of the purine analogue (6-thioguanine). HPRT proficient cells are sensitive to 6-thioguanine which causes the inhibition of cellular metabolism and halts further cell division. HPRT deficient cells are presumed to arise through mutation at the hprt locus; they cannot metabolize 6-thioguanine and thus survive and grow in its presence.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cell line; a sub-clone of Chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB), maintained in the Mutagenicity Section at Jai Research Foundation (Vapi, India).

Metabolic activation:

with and without

Metabolic activation system:

9 fraction procured from Defence Research and Development Organization, Nagpur, India buffered and supplemented with the essential co-factors betha-NADP, KCl and Glucose-6-phosphate, and used at a concentration of 2% in the final culture medium.

Test concentrations with justification for top dose:

The cytotoxicity test in the absence and presence of metabolic activation (2% v/v S9 mix): 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL
The Mutagenicity Experimentin the absence and presence of metabolic activation (2% v/v S9 mix): 0.25, 0.5, 1, 2 and 4 µL/mL

Vehicle / solvent:

Culture medium (α-MEM)

Untreated negative controls:

yes

Remarks:

culture media

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Test System:
CHO-K1 cell line (free from mycoplasma contamination), a sub-clone of Chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB), maintained in the Mutagenicity Section at Jai Research Foundation (Vapi, India) was used for this study. The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study. CHO-K1 cell line passage number 36 was used in cytotoxicity and mutagenicity tests.
Culture Medium:
α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides (Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was also supplemented with Penicillin (50 IU/mL of the medium) and Streptomycin (50 µg/mL)/ Penicillin (50 IU/mL of the medium), Streptomycin and Amphotericin B (0.25 µg/mL). At the time of selection Minimum Essential Medium Eagle -modification without nucleosides (-MEM w/o NS) with 10% dialyzed fetal bovine serum was used.
Selective Agent:
2-amino-6-mercaptopurine (6-thioguanine) was used as selective agent at a concentration of 5 µg/mL -MEM without nucleosides.
Culture Vessels:
Disposable tissue cultures flasks of 75 cm2 culture (Nunc) area with canted neck were used to culture the cell line and the treatment was given in the same flask. Disposable culture dishes (Corning) 60 mm were used to determine the cloning efficiency and 100 mm were used to select mutant colonies.
Culture Preparation:
HAT (Hypoxanthine Aminopterine Thymidine media supplement) treated cells showing normal growth were used for preparing cultures for treatment (Nestmann, E.R. et al., 1991). The preliminary cytotoxicity test indicated no effect on cell survival. Therefore to ensure sufficient number of cells at the end of treatment, approximately 3 million cell were used per flask. Approximately 24 hours prior to treatment, 28 culture flasks were prepared by inoculating 3015000 cells per flask. The culture flasks prepared for treatment were incubated at 37 ± 1 °C and 5% CO2 in humid air using a CO2 incubator (standard conditions). Two replicate flasks were maintained for each concentration. The day of preparation of culture was recorded as ‘day 0’.
Treatment
The day of treatment was recorded as day 1. Fourteen culture flasks for each treatment in absence and presence of metabolic activation were observed under an inverted microscope for their growth and culture conditions. Cultures free from contamination were used during main study. The untreated cultures maintained in the absence and presence of the metabolic activation system served as the negative control. Two culture flasks were maintained for each test concentration including negative and positive controls. The cultures were incubated at 37 ± 1 °C and 5% CO2 in humidified air for 4 hours. At the end of the exposure period, the treatment medium was removed from the flasks and the cell surface was rinsed using Dulbecco’s Phosphate Buffered Saline (DPBS). The washed cells were then trypsinized and suspended in complete medium (media containing serum) to obtain single cells. The cell concentration was determined by using a haemocytometer and adjusted accordingly with complete medium. The cell concentration in the flasks was adjusted to 1 - 2 x 105 cells/mL. A sample taken from the cell suspension was serially diluted with complete medium to approximately 1000 - 2000 cells/mL. An aliquot of 100 µL was then dispensed on the center of 60 mm tissue culture dishes and 5 mL of complete medium was added. The plates were used determine relative survival (RS) as a marker of cytotoxic effects of the selected test concentrations. Also, a convenient volume was transferred to fresh tissue culture flasks in such a way that the flasks received approximately 2 x 106 cells for each of treatment, negative and positive controls. These flasks were kept as expression flasks for mutation. All the plates and flasks were incubated in a CO2 incubator at 37 ± 1 °C and 5% CO2 for 8 days.
Subculture for Mutation Expression:
The cultures for mutation expression were subcultured on days 3 and 5, following the culture processing on day 1. During the expression period (7 - 9 days) the cell concentration in the flasks was adjusted by subculturing on days 3 (one flask per replicate) and 5 (two flask per replicate). The cells in each flask were trypsinized and the cell concentration was adjusted to approximately 2 x 10^6 cells/culture, provided with fresh complete medium and incubated under standard conditions.
Selection of Mutants and Cytotoxicity observation:
On day 8, the cells from the expression flasks were trypsinised for each test concentration, negative control and positive controls and were counted and plated for determination of survival and mutant frequency. The cell concentration was adjusted to around 1 - 2 x 10^5 cells/mL for survival plating and 2 x 10^5 cells/mL for selection of mutants. A sample taken from the cell suspension was diluted to approximately 1000 - 2000 cells/mL by serial dilution. An aliquot of 100 µL was then dispensed on the center of 60 mm culture plate and 5 mL of complete medium was added. Duplicate plates were maintained for each replicate of treatment, negative and positive controls. The plates were then incubated for 8 days for the determination of survival.
One mL of the suspension (2 x 10^5 cells/mL) was added into 100 mm culture plate with 10 mL of selective medium (α-MEM without nucleoside - complete medium containing 5 µg/mL of 6-thioguanine) (Gupta R.S., 1984). For each treatment 12 dishes were maintained per replicate, i.e. 24 dishes/concentration. The plates were incubated at 37 ± 1 °C and 5% CO2 in humidified air using a CO2 incubator for 8 days (i.e. up to Day 16). On day 9, the cytotoxicity plates were removed and the medium was decanted. The colonies were fixed, stained and washed. The colonies formed on each plate were counted and recorded for calculation of relative survival/cloning efficiency.
Enumeration of Colonies:
On day 16 the plates were removed from the incubator and the medium was decanted off and colonies were fixed using 3.7% formaldehyde for 10 minutes. The fixative was removed and the colonies were stained using 0.4% methylene blue for 10 minutes. The number of colonies formed on each replicate plate was counted and recorded. The number of colonies was used to calculate the cloning efficiency at the time of selection, number of clonable cells and the mutant frequency per 1 x 10^6 clonable cells.

Evaluation criteria:

Assay Acceptance Criteria
A mutation assay was considered acceptable if it met the following criteria:
a. The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
b. Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
c. Two experimental conditions (i.e., with and without metabolic activation) were tested unless one resulted in positive results.
d. At least four concentrations (not including the solvent and positive controls) with adequate number of cells and appropriate cytotoxicity are evaluated.
Assay Evaluation Criteria
A test item was considered positive in the present mutation assay if:
a. At least one of the test concentrations had exhibited a statistically significant increase in comparison with the concurrent negative control.
b. The increase was concentration-related when evaluated with an appropriate trend test.
c. Any of the results were outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit).
When all of these criteria were met, the test item was considered to be able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria were fulfilled, a test item was considered clearly negative if, in all experimental conditions examined:
a. None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
b. There were no concentration-related increase when evaluated with an appropriate trend test.
c. All results were inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit).

Statistics:

Weighted regression analysis was performed to evaluate the concentration response relationship on treatment groups against the negative control group (excluding positive controls).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxitiy:
The percent relative cloning efficiency (%RCE) was comparable to the control both in the absence of metabolic activation (75 -96 %) and in the presence of metabolic activation system (91 -104 %), at the tested concentration up to 2 µL/mL of culture medium. Based on the observed results, sponsors communication and guidelines limit a concentration of 4 µL/mL was proposed as the highest concentrations; considering the purity of the FiberHance BM Solution (49.71%).
Mutagenicity:
A significant concentration-related increase in the mutation frequency was not observed in any of the treated concentrations and the mutation frequency was comparable to that from the negative control group. The increased mutant frequency observed in the positive controls in main study demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Conclusions:

From the result of this study, it is concluded that FiberHance BM Solution does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation (2% v/v S9 mix) under the present experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The results of the Bacterial Reverse Mutation Assay according to OECD 471 indicate that, under the conditions of this study, the reaction mass did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

From the result of an OECD 476 study, it is concluded that the reaction mass, tested as FiberHance BM Solution, does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation (2% v/v S9 mix) under the present experimental conditions.

From the results of an OECD 473 study, it is concluded that the reaction mass, tested as FiberHance BM Solution, did not show any potential to induce chromosome aberration, both in the absence and presence of metabolic activation under the present experimental conditions and is negative for clastogenicity up to 4 µL/mL (corresponding with a dissolved solid fraction of 2 mg/mL).

Justification for classification or non-classification

The results of in-vitro tests performed according to OECD guidelines clearly show that Reaction mass of 3-hydroxypropan-1-aminium D-gluconate and N-(3-hydroxypropyl)-D-gluconamide fails to meet the criteria for classification as genotoxic or mutagenic.