Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2012-06-06 until 2012-09-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study according to OECD 476; the study was conducted with the potassium salt instead of sodium. Details of the read-across justification are summarized in the attached read-acorss report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts

IUPAC Name:

Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts

Constituent 2

Reference substance name:

91744-38-6

Cas Number:

91744-38-6

IUPAC Name:

91744-38-6

Test material form:

solid: compact

Details on test material:

- Name of test material (as cited in study report): Glyceryl Stearate Citrate (Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts)
- Molecular weight (if other than submission substance): Not applicable (mixture)
- Substance type: Industrial Chemical
- Physical state: Pale yellow solid block
- Analytical purity: 100%
- Purity test date: Not indicated
- Lot/batch No.: 105618
- Expiration date of the lot/batch: May 11, 2014
- Stability under test conditions: Not indicated
- Storage condition of test material: Room temperature

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. .


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control with THF			-	100.0	100.0	100.0	12.3	1.0	100.0	100.0	100.0	13.8	1.0
Positive control (EMS)	150.0		-	84.1	132.6	113.8	70.9	5.8	80.4	76.7	63.8	114.8	8.3
Test item	4.7		-	97.9	culture was not continued^#				87.8	culture was not continued^#
Test item	9.4		-	84.6	104.9	118.8	13.4	1.1	79.6	106.1	55.7	24.3	1.8
Test item	18.8		-	88.6	142.2	100.7	9.8	0.8	98.8	105.7	31.0	47.0	3.4
Test item	37.5		-	80.4	155.1	139.6	12.3	1.0	83.9	76.0	39.0	25.3	1.8
Test item	75.0	P	-	68.5	70.8	113.2	8.3	0.7	82.0	96.4	42.7	25.1	1.8
Test item	150.0	P	-	80.7	90.3	126.2	7.4	0.6	69.8	80.6	35.6	84.9	6.1
Experiment I / 4 h treatment				culture I					culture II
Solvent control with THF			+	100.0	100.0	100.0	11.9	1.0	100.0	100.0	100.0	10.7	1.0
Positive control (DMBA)	1.1		+	58.3	70.4	70.2	802.4	67.3	85.0	48.2	119.1	274.5	25.5
Test item	4.7		+	81.4	culture was not continued^#				113.4	culture was not continued^#
Test item	9.4		+	77.8	120.4	92.1	28.2	2.4	96.0	60.9	109.9	13.3	1.2
Test item	18.8		+	80.5	105.1	100.7	10.3	0.9	119.3	87.5	92.3	17.5	1.6
Test item	37.5		+	78.5	130.0	100.3	11.5	1.0	119.0	97.9	105.2	8.5	0.8
Test item	75.0	P	+	72.4	100.9	119.0	9.5	0.8	128.4	77.6	109.0	7.4	0.7
Test item	150.0	P	+	72.6	98.2	107.7	6.7	0.6	131.9	76.2	121.7	15.1	1.4
Experiment II / 24 h treatment				culture I					culture II
Solvent control			-	100.0	100.0	100.0	20.4	1.0	100.0	100.0	100.0	9.7	1.0
Positive control (EMS)	150.0		-	108.1	79.6	86.7	381.1	18.7	105.1	126.4	103.5	422.4	43.7
Test item	4.7		-	109.1	culture was not continued^#				106.8	culture was not continued^#
Test item	9.4		-	98.6	108.0	84.6	26.2	1.3	105.6	95.6	117.2	23.1	2.4
Test item	18.8		-	98.8	92.3	92.4	34.1	1.7	109.3	75.7	98.4	20.2	2.1
Test item	37.5	P	-	102.2	93.8	85.5	14.7	0.7	106.1	93.8	99.1	9.9	1.0
Test item	75.0	P	-	97.2	104.0	84.7	24.0	1.2	105.8	144.5	113.3	13.2	1.4
Test item	150.0	P	-	99.0	115.4	79.0	30.3	1.5	103.9	94.1	95.9	2.6	0.3
Experiment II / 4 h treatment
Solvent control with THF			+	100.0	100.0	100.0	35.2	1.0	100.0	100.0	100.0	21.0	1.0
Positive control (DMBA)	1.1		+	103.0	120.7	67.8	732.9	20.8	100.0	105.0	93.9	348.7	16.6
Test item	4.7		+	104.4	culture was not continued^#				101.6	culture was not continued^#
Test item	9.4		+	123.9	109.0	98.6	14.8	0.4	108.7	107.6	95.2	4.5	0.2
Test item	18.8		+	133.2	89.6	93.2	29.6	0.8	112.2	109.0	98.7	10.8	0.5
Test item	37.5		+	94.0	127.6	79.8	26.9	0.8	100.9	93.4	101.3	16.4	0.8
Test item	75.0	P	+	97.8	95.3	74.5	23.4	0.7	105.7	123.3	107.4	15.7	0.7
Test item	150.0	P	+	106.3	104.3	74.7	15.7	0.4	105.3	99.5	94.7	14.2	0.7

^#   culture was not continued since a minimum of only four analysable concentrations is required

P  precipitation observed at the end of treatment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance (glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item (glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.