Glycerol trimyristate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537,TA 1538, TA 97 TA 100 and TA 98 and in E. coli WP2 uvrA pKM

Link to relevant study records

Reference

Reference 1

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

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

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Apr -21 Apr 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1983

Deviations:

yes

Remarks:

only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft 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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/β-Naphthoflavone

Test concentrations with justification for top dose:

5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 95% ethanol (100 and 50 µL/plate in the plate incorporation and preincubation tests, respectively).
- Justification for choice of solvent/vehicle: in a preliminary test, the solubility of the test compound was determined in a number of solvents suitable for the Ames test, namely water, dimethylsulfoxide, glycerol, dimethyl formamide, formamide, ethanol, acetone, dioxane, tetrahydrofuran and tetrahydrofurfuryl alcohol. In all these solvents, the test compound could not be dissolved in appreciable amounts. Therefore, a suspension of the test material was prepared on the day of testing by mixing the test compound with 95% ethanol, stirring it on a magnetic stirrer and using samples of this stirred suspension for testing. No stability testing or composition analysis was performed on the dosing suspension.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-nitrofluorene (2-NF; 2.5 µg/plate for TA98); sodium azide (SA; 5 and 2.5 µg/plate for TA100 and TA1535, respectively); 9-aminoacridine (9-AA; 40 µg/plate for TA1537; +S9: 2-aminoanthracene (2-AA; 2.5 µg/plate for all strains)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 30 min (second experiment)
- Exposure duration: 72 h (first and second experiment)

NUMBER OF REPLICATIONS: 3 replications in the first and second experiment, respectively.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

Evaluation criteria:

Criteria for determination of a valid test:

The following criteria had to be met for the mutagenicity assay to be considered valid:
- In the solvent control, each tester strain culture exhibited a characteristic mean number of spontaneous revertants.
- To ensure that appropriate numbers of bacteria were plated, overnight culture titers had to be in excess of 1E08 bacteria/mL.
- The mean of each positive control exhibited a significant increase in the number of revertants over the mean value of the respective vehicle control.
- In a standard Ames test, at least four non-toxic dose levels were required to evaluate the assay data. In the current test, due to non-solubility of the test compound, only a limit dose was employed.

Criteria for evaluation of test results:

For a test compound to be considered positive, it had to (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. In a standard Ames test, this increase had to be accompanied by a dose response towards increasing concentrations of the test article. In the current test, a reproducible caused by a treatment with the limit dose would have been sufficient. A test article that did not meet these criteria would be called non-mutagenic in bacteria. Single increases in revertant frequencies, which were not reproducible in two independent tests were considered non-relevant.

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

Remarks:

at 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance is not soluble in water.
- Precipitation: due to non-solubility of the test susbtance in all solvents used routinely in the Ames test, the tester strains were exposed to a suspension of the test compound at a single limit concentration of 5000 µg/plate. Test compound-treated bacterial colonies were counted by hand, as the precipitate formed by the test compound prevented automatic counting.

Table 1. Test Results of Experiment 1 (plate incorporation).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	untreated	110	8	31	12
–	Ethanol	105	7	33	16
–	5000	125P	3P	30P	20P
Positive controls, –S9	Name	SA	SA	2-NF	9-AA
	Concentrations (μg/plate)	5	2.5	2.5	40
	Mean No. of colonies/plate (average of 3)	575	267	123	125
+	untreated	126	11	25	12
+	Ethanol	116	14	33	16
+	5000	136P	8P	29P	10P
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5
	Mean No. of colonies/plate (average of 3)	1394	123	835	120

 

SA = sodium azide

2-NF = 2-nitrofluorene

9-AA = 9-aminoacridine

2-AA = 2-Aminoanthracene

P = Precipitate

 

 

Table 2. Test Results of Experiment 2 (preincubation).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	untreated	142	13	30	14
–	Ethanol	126	9	28	11
–	5000	127P	8P	24P	12P
Positive controls, –S9	Name	SA	SA	2-NF	9-AA
	Concentrations (μg/plate)	5	2.5	2.5	40
	Mean No. of colonies/plate (average of 3)	575	267	123	125
+	untreated	134	7	26	13
+	Ethanol	155	11	27	10
+	5000	128P	10P	31P	13P
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5
	Mean No. of colonies/plate (average of 3)	839	126	657	76

 

SA = sodium azide

2-NF = 2-nitrofluorene

9-AA = 9-aminoacridine

2-AA = 2-Aminoanthracene

P = Precipitate

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 May - 22 Jun 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 3 S. typhimurium strains tested, lack of details on test material

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium, other: TA97, TA98 and TA100

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Toxicity range-finder experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Mutation experiment: 1.6, 8, 40, 200, 1000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethylene glycol dimethyl ether (EGDME)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EGDME

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9 mix: 2-nitrofluorene (50 µg/plate, TA98); sodium azide (2 µg/plate, TA100); 9-aminoacridine (50 µg/plate, TA97); with S9 mix: 2-aminoanthracene (5 µg/plate, all strains)

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: thinning of background layer and/or presence of microcolonies

Evaluation criteria:

At least 2-fold increase of revertant numbers compared to controls and dose-response correlation.

Statistics:

Analysis of variance and regression analysis.

Key result

Species / strain:

S. typhimurium, other: TA97, TA98 and TA100

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
- Water solubility: limited solubility at 1000 µg/plate, but at this dose the precipitated test agent did not interfere with the counting of colonies. Thus 1000 µg/plate was chosen as the highest concentration fur mutation testing.
- Precipitation: yes, at 1000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
Stain TA100 was used for testing up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed in TA100 in the absence or presence of S9 mix. At 5000 µg/plate precipitation occurred.

Table 1: Ames Test Results after treatment with C10-18 triglycerides

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA97	TA98	TA100
-	0 (vehicle)	96.0	31.6	120.0
-	1.6	80.7	21.7	131.3
-	8	103.3	23.7	117.5
-	40	88.3	17.0	141.0
-	200	94.0	23.3	144.3
-	1000	92.7^ST	16.0^P	124.0^P
Positive controls - S9	Name	9AA	2NF	SA
	Concentrations (μg/plate)	50	50	2
	Number of colonies/plate	446.4	920.4	886.8
+	0 (vehicle)	132.2	46.8	138.8
+	1.6	145.0	34.3	152.7
+	8	152.0	36.7	140.3
+	40	165.7	37.0	141.3
+	200	142.3	44.7	143.7
+	1000	142.0^P	52.3^P	139.7^P
Positive controls + S9	Name	2AA	2AA	2AA
	Concentrations (μg/plate)	5	5	5
	Number of colonies/plate	499.8	689.2	888.4

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

SA = sodium azide

9AA = 9-aminoacridine

P= Precipitation observed

ST= signs of toxicity observed (thinning of background layer and/or presence of microcolonies)

 

 

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 4

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]

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Analogue justification

No data on the in vitro genetic toxicity of target substance Glycerol trimyristate (CAS 555-45-3) are available. The genetic toxicity assessment was therefore based on studies conducted with analogue (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 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 read across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 142-18-7

A bacterial gene mutation assay with 2,3-dihydroxypropyl laurate was conducted following a protocol similar to OECD guideline 471 and GLP (WoE, 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.

CAS 91052-13-0

A bacterial gene mutation assay with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was performed according to OECD guideline 471 and GLP (WoE, 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.

CAS 555-43-1

The potential mutagenicity of Glycerol tristearate was assessed in a bacterial mutation assay performed with a protocol similar to OECD guideline 471 and in compliance with GLP (WoE, 1997). S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were exposed to concentrations of 5000 µg/plate with or without S9-mix in two independent experiments. No cytotoxicity and no increase in the mean number of revertants were observed. The solvent and positive control values were within the historical control values. Based on the results of this study, the test substance did not induce mutagenicity in the selected strains of S. typhimurium in the presence and absence of metabolic activation. As only a single concentration was used, this study may not be used for classification purposes.

 

CAS 67701-26-2

The potential mutagenicity of C12-C18 trialkyl glyceride was assessed in a bacterial mutation assay performed following a protocol similar to OECD guideline 471 and under GLP conditions (WoE, 1987). S. typhimurium TA 97, TA 98 and TA 100 were tested in the presence or absence of a liver metabolic activation system (S9-mix). In a preliminary experiment, the Salmonella strain TA 100 was exposed to concentrations of 8-5000 µg/plate. No cytotoxicity occurred up to 5000 µg/plate, but precipitation of the test substance was observed at this test concentration. Although limited solubility was also observed at 1000 µg/plate, precipitation of the test substance at this concentration did not interfere with colony counting. Concentrations ranging from 1.6 to 1000 µg/plate were selected for treatment in the main assay (plate incorporation method), with and without metabolic activation. No increase in the mean number of revertants per plate was observed when compared to controls. The positive and negative controls included for each test strain were shown to be valid. The test substance was considered to be non-mutagenic under the conditions of the study, with and without metabolic activation.

 

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of Glycerol trimyristate (CAS 555-45-3). Analogue read-across from 4 source substances was applied from in vitro studies in bacterial cells and the results of the available in vitro studies were consistently negative. Based on the available data and following the analogue approach, Glycerol trimyristate is not expected to be mutagenic in vitro in bacterial cells.

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 trimyristate (CAS 555-45-3) 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.

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