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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

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

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.