Reaction mass of 2,2-bis(hydroxymethyl)propane-1,3-diyl bis(2-ethylhexanoate) and 3-[(2-ethylhexanoyl)oxy]-2-{[(2-ethylhexanoyl)oxy]methyl}-2-(hydroxymethyl)propyl 2-ethylhexanoate and 3-[(2-ethylhexanoyl)oxy]-2,2-bis{[(2-ethylhexanoyl)oxy]methyl}propyl 2-ethylhexanoate

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Pentaerythritol tetra(2-ethylhexanoate). 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:

2005

Report date:

2005

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[[(2-ethyl-1-oxohexyl)oxy]methyl]propane-1,3-diyl bis(2-ethylhexanoate)

EC Number:

230-743-8

EC Name:

2,2-bis[[(2-ethyl-1-oxohexyl)oxy]methyl]propane-1,3-diyl bis(2-ethylhexanoate)

Cas Number:

7299-99-2

Molecular formula:

C37H68O8

IUPAC Name:

3-[(2-ethylhexanoyl)oxy]-2,2-bis{[(2-ethylhexanoyl)oxy]methyl}propyl 2-ethylhexanoate

Details on test material:

- Name of test material (as cited in study report): Pentaerythritol tetra(2-ethylhexanoate)
- Analytical purity: 98.4 %
- Physical state: colourless liquid
- Analytical purity: 98.4% (GC)
- Lot/batch No.: TOL-887
- Stability under test conditions: proven by re-analysis
- Storage condition of test material: airtight at room temperature in the dark

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:

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 and 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose-finding study:
-S9 mix: 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
+S9 mix: 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)

Main study:
-S9 mix: 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate (all strains)
+S9 mix: 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate (all strains)

Re-study:
-S9 mix; 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate (TA1535)

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2, for TA100, TA98, WP2 uvrA), sodium azide (NaN3, for TA1535) and 9-aminoacridine hydrochloride (9-AA, for TA1537) +S9 mix; 2-Aminoanthracene (2-AA, for all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background lawn

OTHER:
A re-test with TA1535 without S9 mix was performed to show reproducibility.

Evaluation criteria:

The test substance was considered positive if the number of revertant colonies increased more than double compared to the number of revertant of the vehicle control as well as a dose-dependeny was found.

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:

no cytotoxicity

Remarks:

precipitation was observed at concentrations of ≥ 1250 µg/plate at the end of the exposure period

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitation was observed at concentrations of ≥ 1250 µg/plate at the end of the exposure period

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No increase in revertant colonies was observed in the test with either the non-activation method (-S9 mix) or the activation method (+S9 mix).
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: deposits of oily droplets were oberseved at concentrations including and higher than 1250 µg/plate

RANGE-FINDING/SCREENING STUDIES:
Since contamination was found in strain TA1535 without S9 mix, a retest with this strain was performed. However, no increase in number of revertants was found in any strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Pretest:

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	120 ± 10	-	25 ± 3	22 ± 1	7 ± 3
–	8.19	18 ± 6	-	24 ± 3	22 ± 2	5 ± 1
–	20.5	126 ± 7	-	24 ± 3	23 ± 4	7 ± 3
–	51.2	110 ± 4	-	26 ± 4	30 ± 2	7 ± 1
–	128	117 ± 8	-	30 ± 2	26 ± 3	7 ± 2
–	320	117 ± 9	-	23 ± 2	23 ± 3	5 ± 2
–	800	118 ± 9	-	22 ± 3	25 ± 3	7 ± 3
–	2000 +	113 ± 4	-	28 ± 3	28 ± 4	8 ± 3
–	5000 +	118 ± 3		24 ± 3	25 ± 3	8 ± 2
Positive controls, –S9	Name	AF-2	-	AF-2	AF-2	9-AA
	Concentrations (μg/plate)	0.01	-	0.01	0.1	80
	Mean No. of colonies/plate (average of 3 ± SD)	590 ± 25	-	151 ± 9	706 ± 25	197 ± 25
+	0	121 ± 8	12 ± 2	24 ± 2	28 ± 5	14 ± 1
+	8.19	132 ± 6	10 ± 2	22 ± 2	34 ± 3	12 ± 2
+	20.5	124 ± 7	10 ± 2	28 ± 3	33 v 3	14 ± 3
+	51.2	127 ± 2	12 ± 2	30 ± 4	35 ± 1	12 ± 3
+	128	121 ± 4	12 ± 2	25 ± 3	30 ± 3	11 ± 1
+	320	126 ± 11	12 ± 3	25 ± 4	28 ± 3	12 ± 2
+	800	139 ± 11	11 ± 1	25 ± 2	28 ± 4	12 ± 2
+	2000 +	132 ± 4	11 ± 1	28 ± 5	31 ± 3	13 ± 2
+	5000 +	134 ± 7	9 ± 3	31 ± 2	30 ± 3	14 ± 2
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1044 ± 114	304 ± 24	682 ± 17	359 ± 13	135 ± 12

+: precipitation

AF-2:2 -(2 -furyl)-3 -(5 -nitro-2 -furyl) acrylamide

NaN3: Sodium azide

9 -AA:9 - aminoacridine hydrochloride

2 -AA.2 - aminoanthracene

Main test:

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	119 ± 5	10 ± 1	24 ± 3	20 v 1	7 ± 1
–	78.1	126 ± 12	9 ± 3	27 ± 3	19 ± 3	9 ± 4
–	156	120 ± 11	14 ± 1	24 ± 2	22 ± 2	8 ± 1
–	313	123 ± 7	13 ± 1	23 ± 3	17 ± 3	6 ± 1
–	625	122 ± 16	11 ± 3	23 ± 2	17 ± 2	7 ± 1
–	1250 +	112 ± 3	13 ± 3	26 ± 3	24 ± 6	7 ± 1
–	2500 +	128 ± 9	11 ± 2	27 ± 4	22 ± 3	9 v 3
Positive controls, –S9	Name	AF-2	NaN3	AF-2	AF-2	9-AA
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	80
	Mean No. of colonies/plate (average of 3 ± SD)	741 ± 57	436 ± 25	152 ± 12	689 ± 53	205 ± 6
+	0	131 ± 5	11 ± 2	29 ± 8	30 ± 5	12 ± 2
+	78.1	132 ± 13	12 ± 1	27 ± 9	30 ± 4	13 ± 4
+	156	145 ± 5	12 ± 1	24 ± 4	34 ± 5	10 ± 3
+	313	131 ± 3	13 ± 2	26 ± 6	29 ± 2	16 ± 5
+	625	148 ± 6	12 ± 2	24 ± 3	26 ± 1	13 ± 3
+	1250 +	135 ± 145	10 ± 1	27 ± 4	28 ± 3	11 ± 3
+	2500 +	152 ± 3	10 ± 2	31 ± 4	28 ± 3	14 ± 4
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1047 ± 53	362 ± 6	720 ± 49	343 ± 45	129 ± 13

+: precipitation

AF-2: 2 -(2 -furyl)-3 -(5 -nitro-2 -furyl) acrylamide

NaN3: Sodium azide

9 -AA: 9 - aminoacridine hydrochloride

2 -AA. 2 - aminoanthracene

Re-test:

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type
		TA1535
–	0	9 ± 2
–	78.1	8 ± 1
–	156	13 ± 2
–	313	13 ± 1
–	625	11 ± 3
–	1250	10 ± 1
–	2500	10 ± 3
Positive controls, –S9	Name	NaN3
	Concentrations (μg/plate)	0.5
	Mean No. of colonies/plate (average of 3 ± SD)	523 ± 20

NaN3: Sodium azide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative