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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation

Mouse Lymphoma Assay (OECD 490): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 20-23, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light.
- Stability under test conditions: Expected to be stable for duration of testing.
- Solubility and stability of the test substance in the solvent/vehicle: Precipitation was noted at the 1580 and 5000 µg/plate concentrations.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was ground, then made into solutions for the appropriate concentrations.
- Final preparation of a solid: Solutions were ground, vortexed, and sonicated in a water bath prior to use.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution

Target gene:
his, rfa, uvrA/B, R-factor, trp
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:
chemically-induced rat liver S9
Test concentrations with justification for top dose:
1.58, 5.0, 15.8, 50, 158, 500, 1580, 5000 µg/plate
The top dose was the OECD standard limit dose.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: ICR 191 Acridine 10 µg/mL, Daunomycin 60 µg/mL, 2-Aminoanthracene 100 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1 x 10^9 bacteria/mL

DURATION
- Preincubation period: 30 minutes (confirmatory test)
- Exposure duration: 65 hrs
- Expression time (cells in growth medium): 65 hrs

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
OECD guideline
Evaluation criteria:
Validity: Vehicle controls should appear normal with mean revertant colony counts should be close to historical controls or published values. Positive controls should produce a substantial increase in revertant colonies.
Toxicity: Partial or complete absence of background lawn of non-revertant bacteria, or a substantial dose-related reduction in revertant colony counts.
Mutagenicity: Mutation factor 2 or greater for Salmonella strains TA98, TA100, and E. coli strain WP2 uvrA; mutation factor 3 or greater for Salmonella strains TA1535 and 1537. These increases must be dose-related and/or reproducible.
Statistics:
Means and standard deviations.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 ug/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was seen in the some tests at the 1580 and 5000 µg/plate dose levels.
- Other confounding effects: Contamination was also observed in some tests at concentrations of 158 µg/plate and above.



Conclusions:
The test substance in not mutagenic to bacteria.
Executive summary:

The test substance Sucrose Palmitate Stearate MDT Grade was tested for mutagenicity in the Ames test according to OECD Guideline 437. The strains Salmonella TA98, TA100, TA1535, and TA1537, in addition to E. coli strain WP2 uvrA were tested with and without metabolic activation to doses of 1.58, 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate. Negative controls and positive controls were also tested. The results showed the test substance is not mutagenic to bacteria.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 28 - September 12, 2017
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)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay in mouse lymphoma L5178Y cells
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: 5 mg/mL in 0.25% THF

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of the test item was made with 0.25% THF. This stock solution was then diluted in RPMI and 5% HS.
- Final preparation of a solid: The test solution was then soniticated for 10 minutes at 37 degree C.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich cell bank
- Cell cycle length, doubling time or proliferation index: 10-12 hr doubling time
- Whether whole blood or separated lymphocytes were used if applicable: lymphoma cells
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in RPMI 1640 complete medium.
- Modal number of chromosomes: 40

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
10, 20, 50, 100, 200, and 300 µg/mL
Top dose based on results of toxicity pre-experiment which found precipitation at concentrations of 400 µg/mL and above.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: THF was chosen due to the solubility of the test item in that solvent as compared to other solvents.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension of 11 mL RPMI medium and 5% horse serum
- Cell density at seeding (if applicable): 1 x 10E07

DURATION
- Preincubation period: 1-3 days
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days (8 days for cloning efficiency experiment)

SELECTION AGENT (mutation assays): RPMI with 20% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: all

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test substance is considered mutagenic if the following criteria were met:

induced mutant frequency of >= 126 mutants per 10E06 cells
dose-dependent increase in mutant frequency
increased occurrence of small colonies >= 40% of all colonies
Statistics:
Mean mutant frequency
Mean induced mutant frequency
p-value
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at hightest dose level of 300 µg/mL

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed at concentrations of 10, 30, 100, 200, 400, and 1000 µg/mL with and without metabolic activation. Precipitation was seen at the 400 and 1000 µg/mL concentrations both with and without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Ethylmethanesulfonate (300 µg/mL): range - 318.7-2919.0, mean - 726.5, standard deviation - 203.5
Methylmethanesulfonate (10 µg/mL): range - 376.4-2416.1, mean - 763.4, standard deviation - 421.6
Benzo[a]pyrene (2.5 µg/mL): range - 303.6-1267.2, mean - 635.9, standard deviation - 167.8
- Negative (solvent/vehicle) historical control data:
Negative without S9: range - 50.1-170.3, mean - 87.9, standard deviation - 25.5
Negative with S9: range - 50.1-165.9, mean 85.1, standard deviation - 24.3

Mouse Lymphoma Assay Results – Without Metabolic Activation

Concentration

Relative Cloning Efficiency (%)

Relative Total Growth (%)

Mutant Frequency (mutants/10E06 cells)

Induced Mutant Frequency (mutants/10E06 cells)

Control 1

102.9

106.4

69.5

--

Control 2

98.0

100.3

69.5

--

Solvent Control

100.0

100.0

64.9

--

10 µg/mL

106.5

109.8

71.8

6.9

25 µg/mL

85.1

86.7

64.6

-0.3

50 µg/mL

102.9

97.3

86.5

21.6

100 µg/mL

87.7

81.2

73.9

9.0

200 µg/mL

104.7

86.6

69.0

4.2

300 µg/mL (Precipitate observed)

93.4

84.1

61.6

-3.3

Ethylmethanesulfonate*

91.9

73.0

649.0

584.2

Methylmethanesulfonate*

62.8

46.6

631.5

566.7

* Global Evaluation Factor exceeded and statistically significant increase in mutant frequency seen

Mouse Lymphoma Assay Results – With Metabolic Activation

Concentration

Relative Cloning Efficiency (%)

Relative Total Growth (%)

Mutant Frequency (mutants/10^6 cells)

Induced Mutant Frequency (mutants/10^6 cells)

Control 1

94.4

89.4

63.7

--

Control 2

107.0

105.6

63.7

--

Solvent Control

100.0

100.0

80.2

--

10 µg/mL

89.0

96.6

91.8

11.6

25 µg/mL

112.3

119.5

71.2

-8.9

50 µg/mL

91.6

84.8

68.4

-11.8

100 µg/mL

100.4

95.5

49.6

-30.6

200 µg/mL

98.9

96.2

72.7

-7.4

300 µg/mL (Precipitate observed)

114.2

102.7

60.4

-19.8

Benzo[a]pyrene*

90.3

64.4

702.5

622.4

* Global Evaluation Factor exceeded and statistically significant increase in mutant frequency seen

Conclusions:
The test substance is non-mutagenic.
Executive summary:

The mutagenicity of the test substance was determined in an OECD Guideline 490 study. The mutagenicity of the test substance was determined at concentrations of 10, 20, 50, 100, 200, and 300 µg/mL both with and without metabolic activation. THF was used as a solvent. Negative, solvent, and positive controls were used. Precipitation was seen at the highest test concentration of 300 µg/mL. The test substance was not cytotoxic, nor was it mutagenic.

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

Genetic toxicity in vivo

Description of key information

Micro nucleus test (OECD 474): negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 27 - November 9, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: Expected to be stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was ground until it passed through a 425 micron sieve.
- Final dilution of a dissolved solid, stock liquid or gel: The test substance was dissolved in distilled water. A separate dilution was prepared for each dose level in order to maintain a constant dose volume.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution
Species:
mouse
Strain:
ICR
Details on species / strain selection:
Mice were selected as micronucleated erythrocytes are not readily removed from the blood.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo Laboratories, Inc.
- Age at study initiation: 8 weeks (preliminary test), 7 weeks (main test)
- Weight at study initiation: Preliminary test - 31-39.7 g males, 24.8-28.5 g females; Main test - 36-39 g males, 23-27 g females
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Plastic solid bottom cages
- Diet: Harlan Teklad Global 16% Protein Rodent Diet #2016, ad libitum
- Water: Filtered tap water ad libitum
- Acclimation period: 5-18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 42-55
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 27, 2015 To: November 19, 2015
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
- Concentration of test material in vehicle: Preliminary test - 500 and 2000 mg/kg bw, Main test - 2000 mg/kg bw
- Amount of vehicle (if gavage or dermal): Enough vehicle for constant volume dose of 5 ml.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared on day of dosing. The test substance was ground until it passed through a 425 micron sieve. The test substance was dissolved in distilled water. A separate dilution was prepared for each dose level in order to maintain a constant dose volume.
Duration of treatment / exposure:
Preliminary test: two doses administered at a 24 hr interval
Main test: two doses administered at a 24 hr interval, positive control single dose on Day 2
Frequency of treatment:
Once daily
Post exposure period:
2 days
Dose / conc.:
500 mg/kg bw/day
Remarks:
Preliminary test only
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Preliminary and main test
No. of animals per sex per dose:
Preliminary test: 3 animals per group per sex
Main test: 5 animals per group per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw/day
Tissues and cell types examined:
Blood, erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on preliminary test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling performed 44-48 hrs after final treatment.

DETAILS OF SLIDE PREPARATION: Duplicate samples of 60-120 microliters of blood per sample were collected. The blood was fixed by rapid pipetting in chilled methanol, and stored for at least three days. The samples were centrifuged, the methanol removed, and the cells suspended in shipping buffer. One set of samples was sent to Litron Laboratories, 3500 Winton PI, Rochester, New York 14623, United Sates of America, for analysis. Cells were stained with fluorescent labeled anti-CD71, fluorescent labeled anit-CD61 antibody, and propidium iodide following RNAse treatment.

METHOD OF ANALYSIS: The cells were analyzed by flow cytometry for a minimum of 4000 immature erythrocytes per animals, and DNA content in both immature and mature erythrocytes.

Evaluation criteria:
Micronucleated immature erythrocytes (MIE) values within expected range based on published values and laboratory control values for negative controls. Clear increase in MIE values outside historical control range for negative control animals in the positive controls. A statistically significant dose-related increase in MIE values as compared to the negative control group with at least two individual animals outside the laboratory control range was considered a positive result.
Statistics:
Analysis of variance followed by Bonferroni-corrected multiple comparison test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: No
- Evidence of cytotoxicity in tissue analyzed: No
- Harvest times: 44-48 hrs after final exposure

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant difference from negative controls.
- Appropriateness of dose levels and route: The route chosen (oral gavage) is the recommended route. The dose levels were chosen based on the preliminary test. The highest dose in the preliminary test (2000 mg/kg bw/day) was chosen based on the lack of toxicity and cytotoxocity.
- Statistical evaluation: No test substance related change in reticulocyte fraction (% RET) was observed. No statistically significant increase in micronucleus frequency (% MN-NCE) was observed.
Conclusions:
The test substance is not genotoxic with respect to micronucleus induction.
Executive summary:

An In Vivo Mouse Erythrocyte Micronucleus Test (Flow Cytometry) was performed using the test substance Sucrose Palmitate Stearate MDT. A dose of 2000 mg/kg bw was administered to 5 female and 5 male mice on two consecutive days. The mice were sacrificed two days later, and blood samples drawn for analysis of micronuclei. Other groups of mice were used as negative and positive controls. The test substance Sucrose Palmitate Stearate MDT Grade is not genotoxic with respect to micronucleus induction.

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

Additional information

In vitro

Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (Merril, 2016). The Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and E. coli strain WP2 were exposed to test substance concentrations of 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 µg/plate in water with and without metabolic activation using the plate incorporation method. Precipitation of the test substance was observed at 1580 and 5000 µg/plate. The test substance was bacteriotoxic towards TA 100 at 500 µg/plate and above with and without metabolic activation. In the absence and presence of metabolic activation the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains. The revertant frequencies of the vehicle and negative control were within the expected range and the positive control chemicals induced marked increases in revertant colonies, demonstrating the effective performance of the experiments. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected bacterial strains in the presence and absence of metabolic activation.

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed with the test substance according to OECD guideline 490 and under GLP conditions (Schreib, 2017). Based on a pre-experiment, mouse lymphoma L5178Y cells were exposed for 4 h to test substance concentrations of 10, 20, 50, 100, 200, and 300 µg/mL, both with and without metabolic activation. Tetrahydrofuran (THF) was used as a solvent. Negative, solvent, and positive controls were used. Precipitation was seen at the highest test concentration of 300 µg/mL. The test substance was not cytotoxic, nor mutagenic. In conclusion, the test substance did not induce gene mutation in mouse lymphoma cells.

In vivo

Cytogenicity in mammalian cells

A micronucleus assay in bone marrow cells of ICR mice was performed with the test substance according to OECD Guideline 474 and in compliance with GLP (Koetzner, 2016). In an preliminary test 3 animals each received 500 and 2000 mg/kg bw/day test substance oral by gavage, respectively. Since no systemic toxicity or cytogenicity was observed, in the main test 5 male and 5 female mice received 2000 mg/kg bw/day test substance oral by gavage on two consecutive days. 44 – 48 h after final exposure animals were killed, blood was collected and erythrocytes were isolated. Cells were stained with fluorescent labeled anit-CD71 antibody and propidium iodide. At least 4000 immature cells per animal were analysed by flow cytometry. No clinical signs of toxicity were observed in the animals during the study period. No test substance related change in reticulocyte fraction (% RET) and micronucleus frequency (% MN-NCE) was observed. The data indicate that the test substance is not genotoxic in mammalian bone marrow cells with respect to micronucleus induction under in vivo test conditions.

 

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.