Glycerides, C8-10 mono- and di-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

13 Nov - 22 Nov 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates

EC Number:

307-333-3

EC Name:

Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates

Cas Number:

97593-30-1

IUPAC Name:

97593-30-1

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Chemical name: acetylated glyceride-mixture
- Physical state: clear yellowish liquid
- Analytical purity: 100%
- Purity test date: 15 Oct 2007
- Lot/batch No.: CH70920C
- Expiration date of the lot/batch: 20 Sep 2008
- Stability under test conditions: The batch used was analytically examined for identity prior to study initiation and was approved for use for the test period. A stability test in the solvent did not reveal significant degradation of the active ingredient.
- Storage condition of test material: at room temperature

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

Controls 1

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])

Controls 2

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative