Fatty acids C16-18 (even numbered), mono and di esters with Sucrose

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro bacterial mutagenicity test is available for the target substance:

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

In the absence of data on gene mutation in mammalian cells for Fatty acids C16 -18 (even numbered), mono and diesters with sucrose (no CAS) read-across approach was conducted on source substance

Fatty acids C16 -18 (even numbered), mono, di and triesters with sucrose (no CAS) and Sucrose laurate as explained in the analogue justification (chapter 13):

Gene mutation in mammalian cells (OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

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:

16 - 25 April

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

missing 5th strain (TA 102 or E. coli WP2)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

yes

Remarks:

missing 5th strain (TA 102 or E. coli WP2)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

Sep 1985

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Test substance is well soluble in DMSO at approx. 48 °C and is stabil for 4 h.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in 2 different experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of colonies or a thinning of the bacterial background lawn

Evaluation criteria:

A test compound is classified as mutagenic if it has either of the following effects:
a) a test compound produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of
revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test compound induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of
revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
lf the test substance does not produce reproducible increases of at least 2 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this system.
The test results must be reproducible.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: +S9: at and above 2500 µg/plate; -S9: at and above 500 µg/plate Exp 2: +S9: at and above 500 µg/plate; -S9: at and above 100 µg/plate

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:

cytotoxicity

Remarks:

Exp 1: +S9: at and above 2500 µg/plate; -S9: at and above 100 µg/plate Exp 2: +/- S9: at and above 500 µg/plate

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:

cytotoxicity

Remarks:

Exp 1 and 2: +S9: at and above 2500 µg/plate; -S9: at and above 500 µg/plate

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:

Exp 1: +S9: at and above 5000 µg/plate; -S9: at and above 500 µg/plate Exp 2: +/- S9: at and above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Visible precipitation of the test substance was observed at 2500 µg/plate and above in both experiments.

RANGE-FINDING/SCREENING STUDIES: In a toxicity test with a dilution of tester strain TA 100, which was performed in parallel with the second experiment, toxicity was found at a concentration of 2500 μg/plate in the presence of metabolic activation and 100 μg/plate in the absence of metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both mutagenicity experiments strain dependent cytotoxicity was observed in a dose range of 100 to 2500 μg/plate and above without metabolic activation and in a dose range of 500 to 5000 μg/plate withmetabolic activation. Thinning of bacterial
lawn and in most cases also a reduction in the number of colonies were observed at these doses.

Table 1: Results of Experiment 1

	Number of revertant colonies (mean of 3 plates±SD)
With S9
Test substance (µg/plate)	TA 1535	TA 1537			TA 98	TA 100
Solvent control	10.7±1.2	14.7±1.2			26.7±5.5	115.3±5.0
Negative control (untreated)	10.3±1.5	17.0±4.6			22.3±1.5	148.3±4.0
0	10.7±1.2	14.7±1.2			26.7±5.5	115.3±5.0
4	13.3±1.5	14.3±2.5			26.7±4.7	123.7±6.7
20	11.7±3.5	14.3±1.5			21.0±1.7	132.7±8.0
100	9.3±2.3	13.0± 1.0			22.7±2.1	126.0±2.6
500	9.7±0.6	14.7±3.5			19.7±2.5	121.7±3.2
2500	7.0±1.7 r	2.7±1.2 r			16.3±6.7 r	93.3±10.1
500	3.0±2.0 r	1.0±0.0 c			8.3±2.5 r	102.0±15.6 r
Positive Control	2-AA	2-AA			2-AA	2-AA
Dose (µg/plate)	1.0 µg/plate	1.0 µg/plate			0.5 µg/plate	0.5 µg/plate
Number of revertant colonies/plate	194.3±9.3	288.7±33.2			2211.7±0.5	1757.7±69.6
Without S9
Test substance (µg/plate)	TA 1535		TA 1537		TA 98	TA 100
Solvent control	8.3±0.6		12.0±2.0		19.7±3.2	116.3±15.3
Negative control (untreated)	11.7±0.6		12.7±1.2		25.0±3.6	119.3±15.2
0	8.3±0.6		12.0±2.0		19.7±3.2	116.3±15.3
4	12.7±0.6		8.3±3.1		22.7±2.9	107.7±11.9
20	10.3±2.9		8.0±3.0		19.3±2.5	129.0±17.6
100	9.7±2.1		5.0±3.6 r		21.3±2.9	117.0±5.3
500	10.0±2.0 r		5.0±1.7 r		12.7±2.1 r	83.7±7.6
2500	1.7±0.6 r		2.7±1.2c		6.3±0.6 r	93.0±9.5
5000	1.3±0.6 r		1.0±0.0 c		6.7± 3.1 c	83.3±14.4 r
Positive Control	NaN3		9-AA		2-NF	NaN3
Dose (µg/plate)	2.5		2.5		2.5	2.5
Number of revertant colonies/plate	461.3±2.3		150.7±43.1		564.3±67.5	636.3±10.1
2AA: 2-Aminoanthracene 9-AA: 9-Aminoacridine 2-NF: 2-Nitrofluorene NaN3: sodium azide				r: reduced background growth c: clearing of background growth

Table 2. Results of Experiment 2

	Number of revertant colonies (mean of 3 plates±SD)
With S9
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100
Solvent control	12.3±1.2	9.7±2.5	31.3±5.1	130.3±0.6
Negative control (untreated)	12.3±1.5	10.3±0.6	26.0±2.6	163.3±13.3
0	12.3±1.2	9.7±2.5	31.3±5.1	130.3±0.6
4	9.3±7.6	9.0±1.0	32.7±6.5	137.7±12.4
20	8.3±5.5	8.3±1.2	23.7±2.1	139.0±11.5
100	10.7±3.1	10.3±3.2	28.0±3.0	149.3±5.1
500	12.3±2.9 r	4.3±2.5 r	30.3±12.1	143.7±10.1
2500	6.3±2.9 r	3.0±1.7 r	12.3±2.1 r	129.7±0.6 r
500	6.3± 6.7 r	2.0±1.0 c	10.3±1.2 r	135.3±14.2 r
Positive Control	2-AA	2-AA	2-AA	2-AA
Dose (µg/plate)	1.0 µg/plate	1.0 µg/plate	0.5 µg/plate	0.5 µg/plate
Number of revertant colonies/plate	172.7±13.1	272.3±13.8	2199.7±150.5	1897.7±114.2
Without S9
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100
Solvent control	13.7±0.6	8.0±0.0	20.3±1.2	130.3±0.6
Negative control (untreated)	13.7±2.1	7.3±1.2	26.0±4.4	163.3±13.3
0	13.7±0.6	8.0±0.0	20.3±1.2	125.3±15.9
4	11.0±2.6	7.0±1.0	24.0±1.7	129.3±7.0
20	9.0±1.7	8.3±1.2	24.3±1.2	124.0±8.2
100	11.3±3.1 r	7.0±1.0	26.7±3.8	123.7±23.6
500	11.3±2.5 r	2.3±0.6 r	13.0±2.6 r	105.3±19.5
2500	1.7±1.5 r	1.3±0.6 c	6.7±0.6 r	95.0±3.6 r
5000	0.3±0.6 r	1.0±0.0 c	5.0±1.0 r	83.0±7.9 r
Positive Control	NaN3	9-AA	2-NF	NaN3
Dose (µg/plate)	2.5	2.5	2.5	2.5
Number of revertant colonies/plate	431.3±55.0	215.3±33.1	520.3±89.2	1897.7±114.2
2AA: 2-Aminoanthracene 9-AA: 9-Aminoacridine 2-NF: 2-Nitrofluorene NaN3: sodium azide			r: reduced background growth c: clearing of background growth

Conclusions:

Under the test conditions used, the substance was not mutagenic in any of the four strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) tested with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

growth inhibition was seen at all concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Source: Fatty acids, C16-18 (even numbered), mono di and triesters with sucrose (no CAS), Schreib, 2017

Conclusions:

Based on the results of the mouse lymphoma assay in L5178Y cells, both source substances were non-mutagenic in mammalian cells with or without metabolic activation. Therefore and by analogy, the target substance is expected to be non-mutagenic in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In the absence of data on gene mutation in mammalian cells in vivo for Fatty acids C16 -18 (even numbered), mono and diesters with sucrose (no CAS) read-across approach was conducted on source substance

Fatty acids C16 -18 (even numbered), mono, di and triesters with sucrose (no CAS) as explained in the analogue justification (chapter 13):

Micronucleus test (OECD 474): negative in mouse erythrocytes

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

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

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 or test system 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

Only data on the gene mutation in bacteria of Fatty acids, C16-18 (even numbered), mono and diesters with sucrose are available. The assessment was therefore additionally based on QSAR modelling and studies conducted with an analogue substance as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

In vitro gene mutation in bacteria

Fatty acids, C16-18 (even numbered), mono and diesters with sucrose (target)

A bacterial gene mutation assay with the target substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (Müller, 1996). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to test substance concentrations of 4, 20, 100, 500, 2500 and 5000 µg/plate in DMSO with and without metabolic activation using the plate incorporation method. A toxicity experiment using histidine enriched agar plates and a dilution of the tester strain TA 100 was performed in parallel with the second experiment. Precipitation of the test substance was observed at 2500 µg/plate and above in both experiments. The test substance was bacteriotoxic towards all tester strains in a dose range of 500 to 5000 µg/plate with metabolic activation and 100 to 2500 µg/plate and above 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 S. typhimurium strains in the presence and absence of metabolic activation.

 

Genetic toxicity (mutagenicity) in bacteria profiling in silico

Fatty acids, C16-18 (even numbered), mono and diesters with sucrose

As only four Salmonella strains were tested for bacterial mutagenicity, the target substance Fatty acids, C16-18 (even numbered), mono and diesters with sucrose (no CAS) was further assessed for its mutagenicity potential by QSAR modelling (OECD Toolbox, 2018 and VEGA, 2018). Four constituents of the UVCB substance, which were considered representative for the UVCB substance, were selected for evaluation by means of (Q)SAR models included in the free available OECD QSAR Toolbox v4.1 and VEGA software v1.1.4. The models or profilers included in VEGA and the OECD QSAR Toolbox did not provide any results indicative for mutagenic potential of evaluated components of the target substance. Only two alerts (“H-acceptor-path3-H-acceptor” and “Oxolane”) were identified as possibly related to micronucleus mutagenicity. H-acceptor-path3-H-acceptor is triggered by a multitude of different substances, and was therefore considered as negligible. The alert “Oxolane” resulted from the sucrose moiety of the target substance. Experimental data available in the data base of OECD QSAR Toolbox for sucrose showed no mutagenicity. Therefore this alert was also considered negligible for the target substance. Absence of mutagenic potential was also supported by the findings of the VEGA software. The four evaluated constituents were consistently predicted as “Non-Mutagenic” by the VEGA models for Mutagenicity (Ames test). Therefore following a weight of evidence approach and in good congruence with the experimental results of the Ames test, Fatty acids, C16-18 (even numbered), mono and diesters with sucrose is considered as not mutagenic in bacteria.

 

Gene mutation in mammalian cells

Fatty acids, C16-18 (even numbered), mono, di and triester with sucrose

An in vitro mammalian cell gene mutation assay was performed with source substance Fatty acids, C16-18 (even numbered), mono di and triesters with sucrose 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.

Sucrose laurate

An in vitro mammalian cell gene mutation assay was performed with source substance Sucrose laurate to OECD guideline 490 and under GLP conditions (Schreib, 2016). Based on a pre-experiment, mouse lymphoma L5178Y cells were exposed for 4 h to test substance concentration of 0.005, 0.010, 0.025, 0.050, 0.075, 0.100, 0.125 and 0.150 mg/mL without metabolic activation, and 0.075, 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, 0.250 mg/mL with metabolic activation. Growth inhibition was seen at all concentrations with and without metabolic activity. No biologically relevant increase in mutants was seen after exposure to the test substance. No dose-response relationship was observed. In conclusion, the test substance did not induce mutations in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions used.

In vivo

Cytogenicity in mammalian cells

Fatty acids, C16-18 (even numbered), mono, di and triesters with sucrose

A micronucleus assay in bone marrow cells of ICR mice was performed with source substance Fatty acids, C16-18 (even numbered), mono di and triesters with sucrose 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 anti-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.

Overall conclusion for genetic toxicity

Data from a gene mutation study in bacteria (Ames) for Fatty acids, C16-18 (even numbered), mono and diesters with sucrose (no CAS) are available. The OECD QSAR toolbox did not predict bacterial mutagenicity of the target substance and therefore confirms the results of the Ames test. In the absence of in vitro mammalian cell data, read-across from the analogue substances Fatty acids, C16-18 (even numbered), mono, di and triesters with sucrose and Sucrose laurate was applied. Infact, both substances were tested for in vitro mammalian cell gene mutation according to OECD 490; results of both read-across substances were negative. 

Similarly, in the absence of in vivo genotoxicity data, read-across from the analogue substance Fatty acids, C16-18 (even numbered), mono, di and triesters with sucrose was applied. In fact, Fatty acids, C16-18 (even numbered), mono, di and triesters with sucrose was tested negative in the in vivo MNT according to OECD 474.

Based on the available data and following the analogue approach, Fatty acids, C16-18 (even numbered), mono and diesters with sucrose is not expected to be mutagenic and/or clastogenic.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Fatty acids C16 -18 (even numbered), mono and diesters with sucrose (no CAS), data will be generated from data available for reference source substance(s) to avoid unnecessary testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.