Glycerol monomyristate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative

Chromosome aberration in mammalain cells: negative

Gene mutation in mammalian cells: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium supplemented with 10% calf serum

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9 mix: 891, 1783, 3565 µg/mL
24h treatment without S9 mix: 55.7, 111, 223, 446 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of vehicle: due to limited solubility of the test substance in water, DMSO was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: mitomycin C (MMC), 0.1 µg/mL (6h exposure), 0.05 µg/mL (24h exposure); +S9: cyclophosphamide (CM), 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h (short treatment) and 24 h (continuous treatment)
- Expression time (cells in growth medium): 18 h (after 6 h treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR: 0.2 μg/mL of colcemid (added 2 h prior to harvest)
STAIN: 1.2% Giemsa stain (in sodium phosphoric acid buffer 1/100 mol/L pH 6.8) for 12 min

NUMBER OF REPLICATIONS: 2 replications in one experiment

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative cell growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

100 cells/plate were observed and classified into gap, ctd (chromatid break), cte (chromatid exchange), csb (chromosome break), cse (chromosome exchange) and oth (others). The total number of cells with aberrations excluding gaps was calculated. The number of polyploid cells was also counted.
The test results were considered positive, if a statistically significant increase in the frequency of chromosome aberrations was observed, the increase showed dose-dependency and it was reproducible.

Statistics:

Differences in chromosome aberration frequencies between treated and control cells were analysed with the Fisher's exact test (p < 0.025). Dose-dependency was assessed using the Cochran Armitage trend test.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 446 µg/mL (24 h treatment, without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was not soluble in water.
- Precipitation: visible precipitation was noted at 223 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
To find the appropriate concentration range, a cytotoxicity test was performed at test concentrations of 3565, 1783, 891, 446, 223, 111, 55.7, 27.9, 13.9, 6.96 µg/mL. Cytotoxicity was evaluated both after a continuous 24 h exposure (-S9 mix) and a short-term 6 h exposure (± S9 mix) followed by an 18 h recovery period prior to harvest.
The test substance was not cytotoxic after short-term exposure at any concentration in the presence of S9 mix. Without metabolic activation, relative growth was reduced to about 71% of the control value.
After 24 h continuous treatment without S9 mix, the test substance was not cytotoxic up to 1783 µg/mL. At 3565 µg/mL, relative growth was decreased to ca. 13 % of the control value. The concentration leading to 50% cytotoxicity was calculated to be 2738 µg/mL.
Based on these results, the main test was conducted using the following test substance concentrations:
6 h treatment (± S9 mix): 891, 1783 and 3565 µg/mL
24 h treatment (-S9 mix): 55.7, 111, 223 and 446 µg/mL

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main test, no cytotoxic effects were observed after short-term exposure. The test substance was cytotoxic at 446 µg/mL after the 24 h treatment.

Table 1. Results of main study (short-term treatment) and additional study (continuous treatment)

Test item	Concentration	Relative cell growth	Aberrant cells in %
	in μg/mL	in %	with gaps	without gaps
Exposure period 6h, fixation time 18h, without S9 mix
DMSO		100	1	1
Glycerol monooleate	891 a)	121.8	0	1
	1783 a)	114.8	1	0
	3565 a)	101.1	1.5	0.5
MMC	0.1	77.5	4.5	36*
Exposure period 6h, fixation time 18h, with S9 mix
DMSO		100	0.5	0
Glycerol monooleate	891 a)	135.9	2	2
	1783 a)	123.9	1	0.5
	3565 a)	111.4	1	0.5
CP	12.5	83.4	3.5	19.5*
Exposure period 24h, without S9 mix
DMSO		100	1	1
Glycerol monooleate	55.7	99	0.5	0
	111	105.9	1	1
	223 a)	87.0	0.5	0
	446 a)	91.6	Toxic
MMC	0.05	104.4	6.5	25*

*: Significant difference from control (Fisher's exact test, p < 0.025)

a): Visible precipitation was observed at the end of exposure period.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2010 - 12 Jun 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix Migrated to IUCLID6: 0.1 µg/mL ( 6 h treatment), 0.05 µg/mL (24 and 48 h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix Migrated to IUCLID6: 7 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy

Evaluation criteria:

The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more

Key result

Species / strain:

other: CHU/IU

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: at concentrations of 0.078 µg/mL and higher

RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Table 1. Effects of the test substance on viability and chromosome aberrations.

Test item	Concentration	Cell viability	Aberrant cells in %
	in µg/mL	in %	Numerical	Structural including gaps
Exposure period 6h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.0	1.5
MMC	0.010	105.5	0.0	39.5
Test substance	0.020	101.0	0.5	0.0
	0.039	98.0	1.0	1.5
	0.078	88.0	0.0	0.0
	0.156	82.5	0.5	1.0
Exposure period 6h with S9 mix
Untreated	--	--	0.0	2.0
Solvent	--	100	0.0	0.5
B(a)P	7.000	96.5	0.0	28.5
Test substance	0.020	96.0	0.0	2.0
	0.039	92.0	0.0	0.0
	0.078	86.0	1.0	0.5
	0.156	84.5	0.5	1.0
Exposure period 24h without S9 mix
Untreated	--	--	1.0	0.5
Solvent	--	100	0.5	0.0
MMC	0.050	102.0	0.0	41.5
Test substance	0.078	90.5	0.0	0.5
	0.156	82.5	0.0	1.0
	0.313	76.0	1.0	1.0
	0.625	66.0	0.0	0.5
	1.250	66.0	0.5	2.0
	2.500	84.5	0.5	0.0
	5.000	92.0	0.0	1.0
Exposure period 48h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.5	0.0
MMC	0.050	94.0	0.0	58.5
Test substance	0.078	99.0	0.0	1.0
	0.156	86.5	0.0	0.5
	0.313	74.5	1.0	0.0
	0.625	70.0	0.0	0.0
	1.250	79.5	0.5	0.0
	2.500	87.5	0.5	0.0
	5.000	92.5	0.5	1.0

MMC: Mitomycin

B(a)P: Benz(a)pyrene

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication meeting basic scientific principles. Test compound was a mixture of short- and long-chain acyl triglyceride.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

exposure duration not reported;

Principles of method if other than guideline:

The SALATRIM family of triacylglycerols differs 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. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative in the three assays.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) CHO-K1 cells obtained from the American Type Culture Collection
- Type and identity of media: in Ham’s F12 medium with 31 µg/mL penicillin, 50 µg/mL streptomycin sulfate, and 5 % heatinactivated
fetal bovine serum (F12/5).

Metabolic activation:

with and without

Metabolic activation system:

final S-9 concentration of 1 %

Test concentrations with justification for top dose:

0, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL
The high dose was limited by the low solubility of the fats in the assay medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
Stock solutions of SALATRIM 23CA lot A014 and corn oil were made in acetone such that the acetone never exceeded 1 % of the culture.

Untreated negative controls:

yes

Remarks:

Corn oil doses were 327.7-1000 μg/mL.

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Positive controls were EMS (200 μg/mL of culture) without metabolic activation and 3-MC (5 μg/mL of culture) with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: not reported
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14-17 days

Phenotypic expression of induced mutants was conducted by incubating the cells for 7 days. Cells were subcultured every 2-3 days by trypsinizing the flask, counting the cells, and replating 2 x 10(6) cells. After the expression period, approximately 3 x 10(6) cells from each culture were seeded in 100 mL of cloning medium supplemented with TG for selection of resistant cells. Approximately 600 cells were seeded in 100 mL of TG-free medium to determine the percentage of viable cells. The cells were incubated for 14-17 days and cell colonies counted with an ARTEK colony counter. Mutant frequency (ratio of mutant cells to nonmutant cells) was calculated by dividing the number of resistant colonies by the number of unselected viable colonies.

SELECTION AGENT (mutation assays): The selective cloning medium contained 30 µM 6-thioguanine (TG).

NUMBER OF REPLICATIONS: The definitive study was done in duplicate using duplicate cultures for each replicate.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by detaching the cells from the culture flask with 0.05% trypsin-0.02% EDTA. A Model 2F Coulter counter was used to determine cell numbers, and an aliquot containing at least 1-2 x 10(6) cells was added to 20 mL of F12/5 to determine phenotypic expression. The remaining cell suspension was diluted in approximately 35 mL of medium so that 500 cells were plated into two Petri dishes. After incubation for 14-17 days, cell colonies were determined. Survival was expressed as the cloning efficiency (CE) relative to the solvent control.

Evaluation criteria:

The test results were considered positive if a dose-related increase in the number of mutant colonies occurred and the mutant frequencies of duplicate cultures treated with one or more concentrations of the test article were at least 3 times the average of those from the solvent control cultures.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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
- Water solubility: Although the test material was soluble in the acetone, turbidity was noted in the cultures, indicating solubility had been exceeded.

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were done to determine cytotoxicity and aid in selection of the dose range.

Table 3: Results of the HPRT Mammalian Cell Gene Mutation Assay of SALATRIM 23CA lot A014

with and without Metabolic Activation*

SALATRIM Dose (μg/mL)	without metabolic activation		with metabolic activation
	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)
0	100	15	100	10
31.3	96	7	94	7
62.5	100	18	84	6
125	89	11	93	10
250	90	17	97	15
500	78	12	69	9
1000	86	17	75	10
corn oil 1000**	91	6	79	15
EMS200	56	170	-	-
3MC 5	-		79	170

 

 

*Data represent the mean of two cultures with the exception of the 0 dose (solvent control), which represents the mean of triplicate cultures.

**Data from a independent experiment with corn oil as test substance instead of SALATRIM

EMS= Ethyl methanesulfonate; positive control for the assay without metabolic activation.

3MC = 3-Methylcholanthrene; positive control for the assay with metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 Jan - 10 Feb 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test system included 5 S. typhimurium strains, but neither the tester strain TA102 nor an additional E. Coli strain were tested.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1992

Deviations:

no

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

First experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Second experiment: 6.25, 12.5, 25, 50 and 100 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80/bidistilled water (0.1 mL/plate)
- Justification for choice of solvent/vehicle: The suspension medium Tween 80/bidist. water was chosen according to the solubility properties tested preliminary before start of the study.

Untreated negative controls:

yes

Remarks:

untreated fresh cell suspensions

Negative solvent / vehicle controls:

yes

Remarks:

suspension medium Tween 80/bidist. water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide (2 µg/plate) for TA100 and TA1535; 9-aminoacridine (80 µg/plate) for TA1537; 4-nitro-o-phenylendiamine (40 µg/plate) for TA98 and TA1538; with S9: 2 aminoanthracene (2.5 or 5 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn

Evaluation criteria:

A combination of the following criteria was considered as a positive result:
- The plate background of non-reverted bacteria did not show any growth reduction versus the respective negative controls.
- The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1).
- As a rule, the positive control showed mutation rates exceeding the control values of TA100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0.
- At more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with the negative controls in the tester strain TA100. For the other tester strains, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive.

Reproducibility
If the test substance induces reverse mutations in only one test, and if this effect cannot be reproduced in one or several repeated tests, the initially positive test data will lose their significance.
The criteria for interpretation specified here do not apply absolutely. Other aspects in connection with this in vitro test system may as well be taken into account for the final assessment of the test substance.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 40 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 40 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitations were not noted.

COMPARISON WITH HISTORICAL CONTROL DATA: number of revertants in the negative and vehicle controls (with and without S9) were all within the characteristic spontaneous mutation ranges of the test batches presented in Table 1.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first experiment, the background lawn was reduced or completely cleared in:
TA100 from 40 µg/plate (-S9) and from 200 µg/plate (+S9)
TA1535 from 40 µg/plate (-S9) and from 200 µg/plate (+S9)
TA1537 from 40 µg/plate (-S9 and +S9)
TA1538 from 40 µg/plate (-S9) and from 200 µg/plate (+S9)
TA98 from 200 µg/plate (-S9 and +S9)

- In the second experiment, the background lawn was reduced or completely cleared in:
TA100 from 50 µg/plate (-S9) and from 100 µg/plate (+S9)
TA1535 from 100 µg/plate (-S9 and +S9)
TA1537 from 50 µg/plate (-S9 and +S9)
TA1538 from 50 µg/plate (-S9 and +S9)
TA98 from 100 µg/plate (-S9 and +S9)

Table 2. Group mean values of revertant colonies per treatment group (first experiment).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 100	TA1535	TA1537	TA1538	TA98
–	Untreated	168	20	13	27	28
–	Vehicle	134	17	14	28	32
–	8	119	16	9	29	28
–	40	87	10	4	T	24
–	200	T	T	T	T	20
–	1000	T	T	T	T	T
–	5000	T	T	T	T	T
Positive controls, –S9	Name	Sodium azide	Sodium azide	9-AA	4-NOPD	4-NOPD
	Concentrations (μg/plate)	2	2	80	40	40
	Mean No. of colonies/plate (average of 3)	474	455	604	1675	1359
+	Untreated	152	22	17	23	51
+	Vehicle	159	18	13	31	38
+	8	126	18	12	23	32
+	40	132	13	12	25	30
+	200	96	15	5	23	27
+	1000	45	T	T	8	10
+	5000	20	T	T	T	T
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	5	2.5	2.5	5	5
	Mean No. of colonies/plate (average of 3)	1562	183	87	1339	940

 

T = toxic

9-AA = 9-aminoacridine

4-NOPD = 4-nitro-o-phenylendiamine

2-AA = 2-aminoanthracene

 

 

Table 3. Group mean values of revertant colonies per treatment group (second experiment).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 100	TA1535	TA1537	TA1538	TA98
–	Untreated	121	13	12	28	24
–	Vehicle	141	13	9	24	31
–	6.25	135	20	14	26	26
–	12.5	137	19	11	34	26
–	25	112	12	7	30	29
–	50	79	13	8	T	28
–	100	T	T	T	T	20
Positive controls, –S9	Name	Sodium azide	Sodium azide	9-AA	4-NOPD	4-NOPD
	Concentrations (μg/plate)	2	2	80	40	40
	Mean No. of colonies/plate (average of 3)	612	518	457	1741	1449
+	Untreated	128	14	12	26	38
+	Vehicle	129	16	17	27	26
+	6.25	135	14	14	27	42
+	12.5	114	13	12	30	27
+	25	129	17	13	28	26
+	50	119	15	8	21	28
+	100	111	15	12	T	23
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	5	2.5	2.5	5	5
	Mean No. of colonies/plate (average of 3)	1591	166	143	1499	780

 

T = toxic

9-AA = 9-aminoacridine

4-NOPD = 4-nitro-o-phenylendiamine

2-AA = 2-aminoanthracene

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 Apr - 24 Apr 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only low test concentrations used due to growth inhibition in Salmonella, but not in E.coli strains.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further 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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)

Test concentrations with justification for top dose:

0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metablic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.

Statistics:

The results at each concentration were demonstrated with the mean and the standard deviation.

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

1st experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	14 ±2	399 ±25	12±2.5 [39]
TA 100	103±5.0	825 ± 34.6	113 ± 9.7 [4.9]
TA 1537	11±1.5	1989± 52.0	13±0.6 [10]
TA 98	19 ±1.7	561 ± 19	18 ±4.4 [1.2]
WP2uvrA	25 ±4.0	243 ±24.7	30 ±2.6 [5000]
with S9-mix
TA 1535	11± 0.6	399 ±25	12 ±2.5 [20]
TA 100	111 ± 6.1	825 ± 34.6	126 ±7.5 [39]
TA 1537	17 ±4.7	1989± 52.0	20±3.8 [20]
TA 98	29±1.5	561 ± 19	28 ±5.0 [39]
WP2uvrA	28 ±0.6	243 ±24.7	33±7.0 [5000]
2nd experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	10±2.3	388± 16.4	8 ±0.6 [10]
TA 100	121 ± 6.0	780 ±7.1	114 ±7.8 [20]
TA 1537	11 ±1.0	2057±200.9	15 ±0 [4.9]
TA 98	17 ±1.2	577 ±32.8	20±1.5  [1.2]
WP2uvrA	20 ±2.0	219 ±28:1	27 ±4.6 [1250]
with S9-mix
TA 1535	9 ±2.1	388 ±16.4	11 ±3.2 [78]
TA 100	114 ±12.7	780 ±7.1	117±9.3 [10]
TA 1537	23 ±4.7	2057 ±200:9	21±2.3 [10]
TA 98	25±3.8	577 ±32.8	29 ±2.6 [39]
WP2uvrA	25± 3.2	219 ±28:1	35 ±2.1 [1250]

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Nov - 22 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Düsseldorf, Germany

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

50, 158, 500, 1581 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9 (µg/plate, strain): sodium azide (10, TA1535); Nitrofurantoin (0.2, TA100); 4-nitro-1,2-phenylene diamine (0.5 and 10, TA98 and TA1537, respectively); Mitomycin C (0.2, TA102 [plate incorporation]); cumene hydroperoxide (50, TA102 [preincubation])

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9 (µg/plate, strain): 2-aminoanthracene (3, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 in each experiment (plate incorporation and preincubation)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and/or reduction in background growth

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1535, TA98 and TA100: at 1581 µg/plate and above (with S9); TA1537: at 500 µg/plate and above (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1581 µg/plate and above (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1581 µg/plate, the substance started to precipitate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls (DMSO) were within the expected range and the positive controls showed sufficient effects when compared to the testing laboratory's historical negative and positive control data presented in the study report for the preceding 10 years.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as assessed by a reduction in the number of revertants per plate and/or reduction in background lawn, were observed in the plate incorporation assay in:

TA1535 at 5000 µg/plate with metabolic activation
TA100 at 1581 µg/plate and above with metabolic activation
TA1537 at 1581 µg/plate and above with metabolic activation

In the preincubation assay, cytotoxic effects were observed in:

TA1535 at 1581 µg/plate and above with metabolic activation
TA100 at 1581 µg/plate and above with metabolic activation
TA1537 at 500 µg/plate and above with metabolic activation, and at 5000 µg/plate without metabolic activation
TA98 at 1581 µg/plate and above with metabolic activation
TA102 at 1581 µg/plate and above with metabolic activation

Table 1. Test results of first experiment (plate incorporation).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	DMSO	130 ± 19	11 ± 3	263 ± 5	28 ± 6	7 ± 2
–	50	147 ± 12	8 ± 2	255 ± 6	21 ± 6	9 ± 1
–	158	159 ± 14	11 ± 4	215 ± 35	19 ± 5	7 ± 2
–	500	141 ± 15	10 ± 2	235 ± 9	26 ± 2	6 ± 1
–	1581	142 ± 14P	11 ± 1P	253 ± 28P	24 ± 7P	5 ± 2P
–	5000	143 ± 20P	9 ± 3P	231 ± 16P	23 ± 9P	5 ± 2P
Positive controls, –S9	Name	NF	Na-azide	MMC	4-NPDA	4-NPDA
	Concentrations (μg/plate)	0.2	10	0.2	0.5	10
	Mean No. of colonies/plate (average of 3 ± SD)	387 ± 16	527 ± 24	517 ± 16	137 ± 5	88 ± 8
+	DMSO	184 ± 4	12 ± 2	340 ± 39	35 ± 8	12 ± 3
+	50	187 ± 6	12 ± 4	333 ± 13	31 ± 6	12 ± 4
+	158	198 ± 20	11 ± 3	348 ± 8	33 ± 7	10 ± 1
+	500	176 ± 8	13 ± 5	311 ± 8	30 ± 6	10 ± 1
+	1581	156 ± 12P*	10 ± 5P	322 ± 45P	24 ± 5P	6 ± 2P*
+	5000	151 ± 8P*	7 ± 3P*	300 ± 27P	27 ± 11P	4 ± 1P*
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	3	3	3	3	3
	Mean No. of colonies/plate (average of 3 ± SD)	2434 ± 260	149 ± 26	847 ± 80	1340 ± 110	446 ± 46

 

NF = nitrofurantoin

Na-azide = sodium azide

MMC = mitomycin C

4-NPDA = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

P = precipitate

* = cytotoxicity

 

 

Table 2. Test results of second experiment (preincubation).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	DMSO	131 ± 5	12 ± 2	266 ± 4	16 ± 3	8 ± 1
–	50	112 ± 16	9 ± 1	268 ± 6	17 ± 2	6 ± 1
–	158	113 ± 17	9 ± 1	266 ± 13	13 ± 4	6 ± 1
–	500	111 ± 12	8 ± 1	263 ± 12	15 ± 4	7 ± 3
–	1581	104 ± 14	8 ± 2	222 ± 28	16 ± 7	6 ± 2
–	5000	99 ± 3P	7 ± 1P	254 ± 16P	12 ± 5P	0 ± 0BP
Positive controls, –S9	Name	NF	Na-azide	Cumene	4-NPDA	4-NPDA
	Concentrations (μg/plate)	0.2	10	0.2	0.5	10
	Mean No. of colonies/plate (average of 3 ± SD)	560 ± 17	701 ± 27	503 ± 27	137 ± 5	102 ± 6
+	DMSO	199 ± 6	12 ± 0	354 ± 16	27 ± 0	8 ± 2
+	50	211 ± 9	11 ± 3	320 ± 22	25 ± 5	10 ± 1
+	158	197 ± 33	12 ± 3	334 ± 31	22 ± 4	6 ± 1
+	500	100 ± 14	9 ± 2	267 ± 21	16 ± 3	5 ± 2
+	1581	73 ± 5B	7 ± 2	178 ± 5B	10 ± 4B	1 ± 1BP
+	5000	71 ± 14BP	3 ± 2BP	156 ± 22BP	1 ± 2BP	0 ± 0BP
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	3	3	3	3	3
	Mean No. of colonies/plate (average of 3 ± SD)	2021 ± 235	176 ± 19	594 ± 47	1719 ± 168	178 ± 36

 

NF = nitrofurantoin

Na-azide = sodium azide

Cumene = cumene hydroperoxide

4-NPDA = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

B = background lawn reduced

P = precipitate

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read-across

Data on the genetic toxicity of Glycerol monomyristate (CAS 27214 -38 -6) are not available. The assessment of genetic toxicity was therefore based on data from source substances 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).

Bacterial mutagenicity

A bacterial gene mutation assay with Glycerol tristearate (CAS 97593 -30 -1) was performed in compliance with OECD guideline 471 and GLP (Lanxess, 2007). In two independent experiments, S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to concentrations of 50-5000 µg/plate in the presence or absence of metabolic activation system (S9-mix). In the first experiment cytotoxic effects were evident in the presence of S9-mix at ≥ 1581 (TA 100 and TA 1537) and at 5000 µg/plate (TA 1535). In the second experiment, cytotoxicity was observed in all strains at ≥ 1581 µg/plate (with S9-mix), except for TA 1537 which showed cytotoxicity already at ≥ 500 µg/plate in the presence of S9-mix and at 5000 µg/plate in the absence of metabolic activation. Precipitation of the test substance was noted at ≥ 1581 in the first experiment and at 5000 µg/plate in the second experiment, respectively. No increase in the mean number of revertants was observed in any tester strain in the two independent experiments independent of the metabolic activation. The negative (solvent) controls were within the historical range and the positive controls showed the expected results. Based on the results of both experiments, the test substance did not induce mutagenic effects in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

A bacterial gene mutation assay with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052 -13 -0) was performed according to OECD guideline 471 and GLP (Riken, 2010). Two independent experiments were performed both in the presence or absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E. coli WP2 uvrA. In the preliminary toxicity screening, growth inhibitory effects were observed at ≥ 20 µg/plate in S. typhimurium TA 98 and TA 1537 (without metabolic activation), at ≥ 78 µg/plate in S. typhimurium TA 100 and TA 1535 (without metabolic activation), and at ≥ 313 µg/plate in all S. typhimurium strains with metabolic activation. Based on these results, concentrations ranging from 0.61 to 78 µg/plate were used for the tester strains TA 100, TA 1535, TA98 and TA 1537 in the absence of metabolic activation, whereas concentrations ranging from 10 to 313 µg/plate were applied for treatment of the tester strains TA 100, TA 1535, TA 98 and TA 1537 in the presence of metabolic activation. Since no cytotoxicity was seen in E. coli WP2 uvrA, the maximum test concentration of 5000 µg/plate and concentrations of 2500, 1250, 625 and313 µg/plate were selected for treatment in the main assay. Precipitation of the test substance was observed on the plates with E. coli WP2 uvrA at test concentrations ≥ 1250 µg/plate without metabolic activation and at ≥ 2500 µg/plate with metabolic activation in both experiments. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance in the presence or absence of metabolic activation. The positive and negative controls revealed the expected results. Under the conditions of this assay, the test substance did not induce gene mutations in the selected strains of S. typhimurium and in E. coli WP2 uvrA in the absence and presence of metabolic activation, respectively.

A bacterial gene mutation assay with 2,3-dihydroxypropyl laurate (CAS 142 -18 -7) was conducted in compliance with OECD guideline 471 and GLP (BASF, 1995). Two independent experiments were conducted both in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were treated with concentrations ranging from 8 to 5000 µg/plate. In the second experiment, concentrations ranging from 6.25 to 100 µg/plate were tested. In both experiments, cytotoxicity was observed at concentrations ≥ 40 µg/plate in the bacterial strains. The mean number of revertants was not increased at any concentrations tested. The positive and negative controls included revealed the expected results. Under the experimental conditions reported, the test substance did not induce mutations in the selected strains of S. typhimurium with or without metabolic activation.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

2,3-Dihydroxypropyl oleate (CAS 111 -03 -5) was assessed in an in vitro mammalian chromosome aberration test in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 and under GLP conditions (MHLW, 2005). In a preliminary cytotoxicity test, cells were exposed to concentrations ranging from 6.96 to 3565 µg/mL for a continuous 24-h exposure period with metabolic activation (S9-mix) or a short-term 6 h exposure with and without S9-mix. The test substance was not cytotoxic after short-term exposure at any concentration in the presence of S9 mix. Without metabolic activation, relative growth was reduced to about 71% of the control value after a period of 6 h. Continuous exposure for 24 h without S9-mix caused no cytotoxicity up to concentrations of 1783 µg/mL. At 3565 µg/mL, relative growth was decreased to ca. 13 % of the control value. The concentration leading to 50% cytotoxicity was calculated to be 2738 µg/mL. Based on these results, concentrations of 891, 1783 and 3565 µg/mL (with and without S9 mix) were selected for chromosome analysis after short-term exposure, whereas concentrations of 55.7, 111, 223 and 446 µg/mL (without S9-mix) were chosen for chromosome analysis after continuous exposure. No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. No cytotoxic effects were observed after short-term exposure, but the test substance was cytotoxic at 446 µg/mL after 24-h continuous treatment. Visible precipitation of the test substance was observed at concentrations ≥ 223 µg/mL, which however did not interfere with chromosomal analysis. The positive controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU) in the presence and absence of metabolic activation.

The clastogenic potential of Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052 -13 -0) was assessed in a GLP-study in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 (Riken, 2010). In a preliminary cell growth inhibition test with concentrations ranging from 9.8 to 5000 µg/plate, no significant inhibition of cell growth was observed after 4 h exposure in the presence and absence of metabolic activation (S9 mix). However, a moderate reduction of cell growth of ca. 30-40% compared to controls was observed at concentrations ranging from 156-1250 µg/mL after 24 and 48 h continuous exposure. Based on the results of this study, concentrations of 20, 39, 78 and 156 µg/mL (± S9 mix) were used for the analysis of chromosomal aberrations after short-term exposure (6 h), whereas concentrations of 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL (± S9 mix) were chosen for analysis chromosomal aberrations after continuous exposure (24 and 48 h). No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. No cytotoxic effects were observed in any of the experiments performed. An oily precipitation of the test substance was observed at concentrations ≥ 78 µg/mL, but did not interfere with chromosomal analysis of the cells. The positive controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU) in the presence and absence of metabolic activation

Genetic toxicity (mutagenicity) in mammalian cells in vitro

The SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols was assessed using a gene mutation assay in cultured mammalian cells (Chinese hamster ovary (CHO-K1) cells) similarly to OECD guideline 476 (Hayes et al., 1994). Gene mutations at the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/mL. The highest concentration was limited by the low solubility of the fats in the assay medium. No significant cytotoxicity was reported. An increase in mutant frequency was not observed at any concentration tested whether with or without metabolic activation. The positive controls significantly increased the mutant frequency. Therefore, it was concluded that under the conditions of the study, the test material was not mutagenic at the HPRT locus of Chinese hamster ovary cells in the absence and presence of metabolic activation.

Overall conclusion for genetic toxicity

There are no studies on the genetic toxicity of Glycerol monomyristate (CAS 27214 -38 -6) in bacterial and mammalian cells. Analogue read-across from source substances was applied from in vitro studies in bacterial cells, from in vitro studies on cytogenicity and from gene mutation in mammalian cells. The results of the available in vitro studies were all negative. Based on the available data and following the analogue approach, Glycerol monomyristate 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 Glycerol monomyristate, data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

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.