Sucrose, glycerol and propane-1,2-diol, reaction products with C16-18(even numbered) fatty acids

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial mutation studies are negative for the registation substance and for read-across substances (structural analogues). Studies on mammalian cells are negative for a read-across substances (structural analogue) and for a source substance / constituent) with regard to in vitro testing for gene mutation and clastogenicity. For the source substance / constituent, also genetic toxicity in vivo is negative. General evaluation All experimental studies reported are GLP compliant guideline studies, the key studies have a Klimisch score 1 while the Klimisch scores of the supporting studies were changed from 1 to 2 to reflect the fact that they were conducted on read-across substances. The literature data reported in two cases for a source substance / constituent have a Klimisch score 2 and represent an acceptable, well-documented publication describing experimental results meeting basic scientific principles. Overall, Sucroglyceride C16 -18 is considered to be not mutagenic and to have no genotoxic potential.

Link to relevant study records

Reference

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:

2014-10-17 to 2014-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in THF
- Justification for choice of solvent/vehicle: resulting a homogeneous suspension

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal back¬ground growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol¬ic activation. Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strains TA 1537 and TA 98 at 5000 µg/plate in the presence of metabolic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Table1     Summary of Experiment I

Study Name: 1659600	Study Code: Harlan CCR 1659600
Experiment: 1659600 VV plate	Date Plated: 17/10/2014
Assay Conditions:	Date Counted: 20/10/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	THF			10 ± 0	9 ± 1	22 ± 3	214 ± 9	38 ± 6
	Untreated			11 ± 5	6 ± 2	17 ± 3	199 ± 14	39 ± 7
	PC-2014-568	3 µg		9 ± 2	9 ± 2	20 ± 5	200 ± 8	37 ± 2
		10 µg		10 ± 3	9 ± 4	23 ± 5	212 ± 20	39 ± 4
		33 µg		9 ± 1	11 ± 4	21 ± 2	178 ± 17	41 ± 8
		100 µg		14 ± 5	8 ± 1	28 ± 4	232 ± 38	38 ± 8
		333 µg		7 ± 2	12 ± 4	28 ± 6	169 ± 5	44 ± 3
		1000 µg		14 ± 1	7 ± 3	23 ± 2	182 ± 18	43 ± 14
		2500 µg		12 ± 5^P M	7 ± 2^P M	14 ± 3^P M	157 ± 11^P M	40 ± 8^P M
		5000 µg		14 ± 1^P M	6 ± 1^P M	13 ± 4^P M	174 ± 15^P M	22 ± 3^P M
	NaN3	10 µg		1140 ± 10			2184 ± 96
	4-NOPD	10 µg				303 ± 30
	4-NOPD	50 µg			67 ± 11
	MMS	2.0 µL						922 ± 34
With Activation	THF			10 ± 5	22 ± 2	35 ± 5	185 ± 17	42 ± 8
	Untreated			11 ± 4	19 ± 2	38 ± 3	184 ± 21	46 ± 11
	PC-2014-568	3 µg		11 ± 2	14 ± 2	34 ± 6	204 ± 15	46 ± 5
		10 µg		8 ± 2	10 ± 2	34 ± 7	197 ± 11	41 ± 9
		33 µg		12 ± 2	17 ± 3	26 ± 5	194 ± 2	59 ± 11
		100 µg		8 ± 2	13 ± 4	29 ± 7	190 ± 10	48 ± 8
		333 µg		13 ± 2	15 ± 3	36 ± 1	182 ± 5	52 ± 9
		1000 µg		16 ± 3	14 ± 1	21 ± 3	186 ± 20	63 ± 9
		2500 µg		12 ± 3^P M	11 ± 1^P M	20 ± 3^P M	188 ± 33^P M	34 ± 2^P M
		5000 µg		8 ± 2^P M	10 ± 2^P M	15 ± 3^P M	174 ± 25^P M	39 ± 6^P M
	2-AA	2.5 µg		495 ± 69	271 ± 17	3001 ± 1051	4565 ± 121
	2-AA	10.0 µg						247 ± 28

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

Table2     Summary of Experiment II

Study Name: 1659600	Study Code: Harlan CCR 1659600
Experiment: 1659600 HV2 Pre	Date Plated: 24/10/2014
Assay Conditions:	Date Counted: 27/10/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	THF			13 ± 3	13 ± 1	20 ± 2	115 ± 19	34 ± 4
	Untreated			8 ± 1	14 ± 2	29 ± 2	154 ± 19	35 ± 6
	PC-2014-568	33 µg		13 ± 4	14 ± 2	21 ± 4	124 ± 19	39 ± 5
		100 µg		13 ± 1	12 ± 3	21 ± 5	131 ± 4	34 ± 6
		333 µg		14 ± 4	13 ± 1	26 ± 3	116 ± 4	38 ± 6
		1000 µg		11 ± 2	12 ± 4	24 ± 3	104 ± 3	37 ± 4
		2500 µg		14 ± 2^P	10 ± 2^P M	17 ± 2^P M	108 ± 10^P	37 ± 4^P
		5000 µg		11 ± 3^P M	8 ± 1^P M	14 ± 4^P M	109 ± 6^P M	35 ± 3^P M
	NaN3	10 µg		1148 ± 77			1915 ± 103
	4-NOPD	10 µg				377 ± 48
	4-NOPD	50 µg			78 ± 5
	MMS	2.0 µL						714 ± 27
With Activation	THF			15 ± 4	17 ± 4	32 ± 4	126 ± 3	48 ± 1
	Untreated			16 ± 1	15 ± 2	25 ± 1	163 ± 22	45 ± 6
	PC-2014-568	33 µg		15 ± 2	16 ± 4	36 ± 1	117 ± 2	45 ± 6
		100 µg		13 ± 4	17 ± 5	35 ± 2	134 ± 13	38 ± 6
		333 µg		15 ± 5	13 ± 1	32 ± 8	132 ± 22	45 ± 6
		1000 µg		18 ± 2	19 ± 3	33 ± 8	120 ± 6	48 ± 1
		2500 µg		9 ± 2^P M	15 ± 4^P M	28 ± 3^P M	123 ± 8^P	49 ± 2^P
		5000 µg		10 ± 2^P M	16 ± 2^P M	34 ± 2^P M	109 ± 3^P M	49 ± 4^P M
	2-AA	2.5 µg		310 ± 95	212 ± 15	3589 ± 196	4393 ± 193
	2-AA	10.0 µg						312 ± 25

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

Conclusions:

Interpretation of results (migrated information):
negative with and without S9 mix

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item PC-2014-568 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and theEscheri­chia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                            33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation. Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strains TA 1537 and TA 98 at 5000 µg/plate in the presence of metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with PC-2014-568 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

In experiment I, the data in the solvent control of strain TA 100 without S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and had no detrimental impact on the outcome of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro – Ames Test

 

Key study on the registration substance Sucroglyceride C16 -18 (Evonik, 2014)

The test item PC-2014-568 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                            33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation. Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strains TA 1537 and TA 98 at 5000 µg/plate in the presence of metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with PC-2014-568 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

In experiment I, the data in the solvent control of strain TA 100 without S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and had no detrimental impact on the outcome of the study.

 

Supporting study on the read-across substance Sucroglyceride C12-18, C18unsatd. (Evonik, 1990)

The purpose of the study was to investigate the potential of the test substance Sucroglyceride C12-18, C18unsatd. to induce mutations using the bacterial reverse mutation assay.

The study was conducted in accordance with the OECD guideline 471 (Bacterial reverse mutation test) in the version dated 1983. The assay was performed in two independent experiments. Both took place as a plate incorporation test, using theS. typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100. All experiments were in the absence and in the presence of mammalian metabolic activation by a rat liver post mitochondrial fraction (S9). The number of replications was 4.

The current version of the OECD guideline 471 (adopted in 1997) requires at least 5 test strains and the use ofE. coliWP2 strains orS. typhimuriumTA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the test substance Sucroglyceride C12-18, C18unsatd. is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of the method withoutE. coliWP2 strains orS. typhimuriumTA 102 is considered sufficient to evaluate the mutagenic activity of Sucroglyceride C12-18, C18unsatd. in this bacterial test system.

 

The four tester strains ofS. typhimuriumwere exposed to Sucroglyceride C12-18, C18unsatd. in Methanol/H₂O = 1 : 2. Five concentrations were tested: 6.4-32-160-800-4000μg per plate in the presence and absence of metabolic activation.

Sucroglyceride C12-18, C18unsatd. was tested up to cytotoxic concentrations. No evidence of a mutagenic activity of the test substance was found. Neither a dose related doubling nor a biologically relevant increase in the mutant count with and without S9 mix in comparison with the negative control (solvent) was observed. The positive controls induced the appropriate responses in the corresponding strains.

The negative result from the first test was confirmed by a second independent experiment. The results of the positive control substances confirm the specificity of the tester strains as well as the full activity of the metabolising system.

Based on the results of this study it is concluded that Sucroglyceride C12-18, C18unsatd. is not mutagenic in theSalmonella typhimuriumreverse mutation assay.

 

Supporting study on the read-across substance Stearic acid, esters with methylα-D-glucoside (Evonik, 1990)

The purpose of the study was to investigate the potential of the read-across substance Stearic acid, esters with methylα-D-glucoside, a structural analogue of the registration substance, to induce mutations using the bacterial reverse mutation assay. The test substance is solid. Purity is 100%.

The study design was identical to the key study reported above.

The four tester strains ofS. typhimuriumwere exposed to Stearic acid, esters with methylα-D-glucoside in DMSO. 0.1 mL each of five concentrations were tested: 8-40-200-1000-5000μg per plate in the presence and absence of metabolic activation. The maximum concentration in the main experiments was set at 5000μg per plate since in the preliminary screening no certain cytotoxicity was observed up to this concentration.

 

Stearic acid, esters with methylα-D-glucoside did not induce a significant dose-related increase in the number of revertant colonies in all four tested strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 5000 µg/plate.

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. 

There was no evidence of induced mutant colonies over background.Based on these results it is concluded that Stearic acid, esters with methylα-D-glucoside is not mutagenic in theSalmonella typhimuriumreverse mutation assay.

 

Supporting study on the read-across substance Isostearic acid, esters with methylα-D-glucoside (Evonik, 2008)

The purpose of the study was to investigate the potential of the read-across substance Isostearic acid, esters with methylα-D-glucoside, a structural analogue of the registration substance, to induce mutations using the bacterial reverse mutation assay. The test substance is a yellow paste. Purity is 100%.

The study was conducted in accordance with the OECD guideline 471 (Bacterial reverse mutation test) in the current version dated 1997.

Strains ofS. typhimurium(TA 1535, TA 1537, TA 98 and TA 100) and ofE. coli(WP2 uvr A) were exposed to Isostearic acid, esterswith methylα-D-glucoside, in DMSO at concentrations of 33, 100, 333, 1000 and 3330 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method). Isostearic acid, esterswith methylα-D-glucosidedid not induce a significant dose-related increase in the number of revertant colonies in each of the five tester strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 1000 and 3330 µg/plate. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed.

There was no evidence of induced mutant colonies over background. Based on these results it is concluded that Isostearic acid, esterswith methylα-D-glucosideis not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

 

 

In vitro – Clastogenicity

 

Supporting study on the read-across substance Isostearic acid, esters with methylα-D-glucoside (Evonik, 2008)

The purpose of the study was to investigate the potential of the read-across substance Isostearic acid, esters with methylα-D-glucoside, a structural analogue of the registration substance, to induce chromosome aberrations with mammalian cells in vitro. The test substance is a yellow paste. Purity is 100%.

The study was conducted in accordance with the OECD guideline 473 (In VitroMammalian Chromosome Aberration Test).

In a mammalian cell cytogenetics assay, peripheral human lymphocyte cultures were exposed to Isostearic acid, esterswith methylα-D-glucosidein DMSO at concentrations of 0, 33, 100, 333 µg/mL culture medium (3 h exposure time , 24 h fixation time with and without metabolic activation in the first cytogenetic assay. In the second cytotgenetic assay concentrations of 0, 100, 300, 400, 500, 600, 700, 800 µg/mL (24 h and 48 h exposure time and fixation time) without S9-mix and 0, 33, 100, 300 µg/mL (3 h exposure time, 48 h fixation time) with S9-mix were tested, respectively.

Isostearic acid, esterswith methylα-D-glucosidewas tested up to cytotoxic and precipitating concentration. In the first cytogenetic assay, the test substance was tested up to 333 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Isostearic acid, esterswith methylα-D-glucosideprecipitated in the culture medium at this dose level. In the second cytogenetic assay, Isostearic acid, esterswith methylα-D-glucosidewas tested up to 800 µg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at 600 µg/mL. In the presence of S9-mix Isostearic acid, esterswith methylα-D-glucosidewas tested up to 300 µg/mL for a 3 h exposure time with a 48 h fixation time. The test substance precipitated in the culture medium at this dose level.

Isostearic acid, esterswith methylα-D-glucosidedid not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Isostearic acid, esterswith methylα-D-glucosideon the number of polyploid cells and cells with endo-reduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described.

Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

It is concluded that this test is valid and that Isostearic acid, esterswith methylα-D-glucosideis not clastogenic in human Iymphocytes.

 

Supporting study on the source substance and constituent Mono-Di-Glycerides (Hayes, 1994)

Relevant literature describes the potential of source substances and constituents belonging to the group of Mono-Di Glycerides to induce chromosome aberrations with mammalian cells in vitro.

Results were obtained with substances belonging to the SALATRIM family of triacylglycerols. They differ from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA.

SALATRIM 23CA Lot A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative in the chromosome aberration assay.

The percent cells with abnormal chromosomes was analyzed statistically and did not differ from the solvent control with either fat tested. The doses used in the presence of metabolic activation were identical to those used in the absence of metabolic activation. The positive controls (methylmethanesulfonate (-S9) and cyclophosphamide (+S9) produced the expected response. There was no change in the mitotic index with either fat compared to the solvent control. Neither fat produced increases in the percent cells with abnormal chromosomes or in any of the other criteria for chromosomal damage.

Therefore, both SALATRIM 23CA lot A014 and corn oil were neither clastogenic nor cytotoxic in the chromosomal aberration assay in the presence or absence of metabolic activation.

 

In vitro – Gene Mutation

 

Supporting study on the read-across substance Isostearic acid, esters with methylα-D-glucoside (Evonik, 2009)

The purpose of the study was to investigate the potential of the read-across substance Isostearic acid, esters with methylα-D-glucoside, a structural analogue of the registration substance, to induce gene mutations with mammalian cells in vitro. The test substance is a yellow paste. Purity is 100%.

The study was conducted in accordance with the OECD guideline 476 (In VitroMammalian Cell Gene Mutation Test).

 

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to Isostearic acid, esterswith methylα-D-glucosidein DMSO. The test was performed in two independent experiments in the absence and presence of S9-mix.

Experiment 1: 0 , 1, 3, 10, 33, 100, 150, 180, 200, 220, 240, 280, 300 µg/mL in absence of mammalian metabolic activation and 0, 1, 3, 10, 33, 100, 200, 250, 300, 350, 400, 425, 500 µg/mL with 8% metabolic activation.

Experiment 2: 0, 10, 25, 33, 50, 100, 115, 150, 175, 180, 200, 210, 220, 240, 260 µg/mL without metabolic activation and 0 , 1, 3, 10, 33, 100, 200, 250, 300, 350, 375 µg/mL with 12% metabolic activation.

Isostearic acid, esterswith methylα-D-glucosidewas tested beyond the limit of the solubility to obtain adequate mutagenicity data.

In the first segment of experiment 2, Isostearic acid, esterswith methylα-D-glucosidewas tested up to concentrations of 240 µg/mL in the absence of S9-mix and up to concentrations of 350 µg/mL in presence of 8% S9-mix with an incubation time of 3 hours. Isostearic acid, esterswith methylα-D-glucosidewas tested up to cytotoxic levels of 12 and 14% in the absence and presence of S9-mix, respectively.

In the second segment of experiment 2, Isostearic acid, esterswith methylα-D-glucosidewas tested up to concentrations of 210 µg/mL in the absence of S9-mix and up to concentrations of 375 µg/mL in the presence of 12% S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Isostearic acid, esterswith methylα-D-glucosidewas tested up to cytotoxic levels of 19 and 4% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix, the test substancedid not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix for metabolic activation.

The positive controls did induce the appropriate response. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Isostearic acid, esterswith methylα-D-glucosideis not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

Supporting study on the source substance and constituent Mono-Di-Glycerides (Hayes, 1994)

Relevant literature describes the potential of source substances and constituents belonging to the group of Mono-Di Glycerides to induce gene mutations with mammalian cells in vitro.

Results were obtained with substances belonging to the SALATRIM family of triacylglycerols. They differ from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA.

SALATRIM 23CA Lot A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the HPRT mammalian cell mutagenesis assay. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative in the HPRT assay.

Positive controls gave the expected results, validating the assay.

Corn oil did not alter either the relative cloning efficiency or the mutation frequency of the cells.

SALATRIM 23CA lot A014 presented no evidence of mutagenic potential under the conditions of the assay.

Therefore, both SALATRIM 23CA lot A014 and corn oil did not induce gene mutations in the HPRT assay in the presence or absence of metabolic activation.

 

In vivo – Chromosome Aberrations

 

Supporting study on the source substance and constituent Mono-Di-Glycerides (Hayes, 1994)

Relevant literature describes the potential of source substances and constituents belonging to the group of Mono-Di Glycerides to induce chromosome aberrations with mammalian cells in vivo.

Results were obtained with substances belonging to the SALATRIM family of triacylglycerols. They differ from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA.

SALATRIM 23CA Lot A014, a typical SALATRIM fat, was tested in mammalian cells in vivo in a rat bone marrow micronucleus assay. Corn oil also was tested as a reference fat.

Rats received these SALATRIM fats or corn oil at 10% (w/w) in the diet for 13 weeks. The in-life and bone marrow smear preparation portions of the study were part of a 13-week subchronic toxicity study conducted at Hazleton Wisconsin, Inc., Madison, WI, and the micronucleus assay portion of the study was conducted at Hazleton Washington, Vienna, VA. Detailed information on the experimental design and methods for the subchronic study can be found in the paper for the subchronic toxicology portion of that study (Hayes et al., 1994).

Both the SALATRIM fat and corn oil were negative in the in vivo micronucleus assay.The data confirm that SALATRIM fats lack genotoxic potential.

 

General evaluation of genetic toxicity

Sucroglyceride C16-18 is negative in an in vitro bacterial reverse mutation assay. Further to the registration substance Sucroglyceride C16-18 itself, also the three read-across substances, Sucroglyceride C12-18, C18unsatd., Stearic acid, esters with methylα-D-glucoside and Isostearic acid, esters with methylα-D-glucoside are negative in the Ames Test. This comparable profile in the bacterial test is taken as experimental evidence in support of the read-across approach applied to other test systems for genetic toxicity.

 

The read-across substance Isostearic acid, esters with methylα-D-glucoside, comparable to the registration substance Sucroglyceride C16-18 with regard to the Ames Test, has been examined for its potential clastogenicity and results negative in a chromosome aberration assay in peripheral human lymphocytes.  

Also results reported for Mono-Di-Glycerides, source substances and constituents of the registration substance Sucroglyceride C16-18, show the absence of a mutagenic potential in tests for chromosome aberrations in vitro.

 

The same read-across substance, Isostearic acid, esters with methylα-D-glucoside, has also been examined for its potential to cause gene mutations and results negative in a gene mutation assay in mouse lymphoma L5178Y cells.  

Results from gene mutation studies reported for Mono-Di-Glycerides, source substances and constituents of the registration substance Sucroglyceride C16-18, show the absence of a mutagenic potential in the HPRT mammalian cell mutagenesis assay.

 

Finally, the same Mono-Di-Glycerides, source substances and constituents of the registration substance Sucroglyceride C16-18, were shown not to be mutagenic, i.e. not to induce chromosome aberrations, in an in vivo rat bone marrow micronucleus test.

 

Based on the experimental evidence provided for Sucroglyceride C16-18 itself and extrapolating the read-across results for genetic toxicity to the registration substance, Sucroglyceride C16-18 is considered not to be a mutagen. The substance does not need to be classified for genetic toxicity according to CLP, EU GHS (Regulation (EC) No 1272/2008) and according to DSD (Directive 67/548/EEC) and labelling is not required.

Justification for selection of genetic toxicity endpoint
Only one in vitro study with Klimisch score 1 is available on the registration substance.

Justification for classification or non-classification

Sucroglyceride C16-18 is not mutagenic in the Salmonella typhimurium reverse mutation assay. A read-across substance and source substances / constituents are negative for clastogenicity in in vitro chromosome aberration tests and for gene mutation the mammalian cell gene mutation assay using mouse lymphoma L5178Y cells or in the HPRT test, respectively. In an in vivo micronucleus test, source substances / constituents proved not to be mutagens.

In conclusion, the full set of genotoxicity tests required by the REACH regulation is negative. According to CLP, EU GHS (Regulation (EC) No 1272/2008) and according to DSD (Directive 67/548/EEC), no classification for mutagenic toxicity is necessary for Sucroglyceride C16 -18 and no labelling is required.