Reaction product of 3,5,5-trimethyl-hexanoic acid and 2-methylpropanoic acid and pentaerythritol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Spetember 2011 - 02 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau

Version / remarks:

25 November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 2-methylpropanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2-{[(2-methylpropanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

EC Number:

813-120-0

Cas Number:

1262967-45-2

Molecular formula:

C21H36O8 C26H46O8 C31H56O8 C36H66O8 C41H76O8

IUPAC Name:

3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 2-methylpropanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2-{[(2-methylpropanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

Test material form:

liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Diluted in acetone at a top concentration of 50 mg/mL (used to prepare plates at 5000 µg/plate)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Diluted in acetone.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: No

Method

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver

Test concentrations with justification for top dose:

Seven concentrations up to 5000 µg/plate i.e. 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: test item was not soluble in DMSO and acetone was preferred over ethanol.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Not reported

DURATION
- Preincubation period: N/A
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: thinning/ absence of bacteria lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response was reproducible under the same treatment conditions.

The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

The statistical significance of results was analysed using Dunnett’s test to aid in the evaluation of potential positive responses.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1       Test 1 (in the presence of S9 mix)

 

Strain	Concentration (µg/plate)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	Solvent control	37.3	3.1		38, 40, 34
	5	44.3	2.1	1.2	45, 46, 42
	15	33.0	5.3	0.9	35, 27, 37
	50	38.7	0.6	1.0	38, 39, 39
	150	42.3	8.3	1.1	45, 49, 33
	500	37.3	6.5	1.0	37, 31, 44
	1500	42.0	3.6	1.1	43, 38, 45
	5000	40.0	3.0	1.1	43, 40, 37
TA100	Solvent control	154.0	20.0		137, 149, 176
	5	138.0	12.5	0.9	134, 152, 128
	15	149.0	8.7	1.0	159, 144, 144
	50	141.0	15.1	0.9	143, 125, 155
	150	153.7	27.2	1.0	123, 163, 175
	500	155.0	9.2	1.0	153, 165, 147
	1500	151.3	7.8	1.0	145, 160, 149
	5000	138.3	5.9	0.9	145, 136, 134
TA1535	Solvent control	21.7	1.5		23, 23, 22
	5	18.7	1.2	0.9	20, 18, 18
	15	19.3	1.2	0.9	20, 20, 18
	50	25.0	1.7	1.2	24, 24, 27
	150	23.3	1.2	1.1	22, 24, 24
	500	20.3	4.9	0.9	26, 18, 17
	1500	21.3	4.2	1.0	18, 20, 26
	5000	18.7	2.3	0.9	20, 20, 16
TA1537	Solvent control	12.0	1.0		12, 13, 11
	5	11.7	1.5	1.0	13, 10, 12
	15	11.3	1.5	0.9	11, 13, 10
	50	9.7	2.5	0.8	10, 7, 12
	150	9.7	2.3	0.8	11, 11, 7
	500	13.0	2.0	1.1	13, 15, 11
	1500	12.7	2.9	1.1	11, 16, 11
	5000	7.7	2.1	0.6	6, 10, 7
WP2 uvrA (pKM101)	Solvent control	139.0	7.9		142, 130, 145
	5	157.7	21.9	1.1	145, 145, 183
	15	156.7	9.1	1.1	147, 165, 158
	50	134.7	31.0	1.0	165, 136, 103
	150	158.7	10.0	1.1	149, 169, 158
	500	157.7	16.3	1.1	165, 139, 169
	1500	146.0	15.6	1.1	154, 128, 156
	5000	128.0	15.6	0.9	110, 138, 136
TA98	2^a	313.0	31.7	8.4	280, 343, 317
TA100	2^b	902.0	138.7	5.9	787, 1056, 863
TA1535	2^b	509.3	22.2	23.5	535, 497, 496
TA1537	50^c	648.3	69.9	54.0	725, 632, 588
WP2 uvrA (pKM101)	2^d	2259.0	200.3	16.3	2280, 2448, 2049

^a2-nitrofluorene

^bsodium azide

^c9-aminoacridine

^d4-nitroquinoline-1-oxide

 

Table 2       Test 1 (in the absence of S9 mix)

 

Strain	Concentration (µg/plate)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	Solvent control	52.3	1.2		53, 51, 53
	5	58.3	8.7	1.1	68, 51, 56
	15	52.3	3.1	1.0	53, 49, 55
	50	45.7	6.4	0.9	42, 53, 42
	150	47.3	2.3	0.9	46, 50, 46
	500	49.7	4.2	0.9	53, 45, 51
	1500	41.0	7.5	0.8	40, 49, 34
	5000	41.0	6.1	0.8	44, 34, 45
TA100	Solvent control	164.7	10.1		170, 153, 171
	5	162.0	17.6	1.0	155, 182, 149
	15	154.3	5.0	0.9	149, 155, 159
	50	156.0	13.0	0.9	171, 148, 149
	150	159.3	18.5	1.0	138, 171, 169
	500	148.7	8.1	0.9	145, 158, 143
	1500	146.3	8.4	0.9	156, 141, 142
	5000	148.3	12.7	0.9	163, 141, 141
TA1535	Solvent control	19.3	2.3		18, 18, 22
	5	22.0	3.5	1.1	20, 26, 20
	15	22.7	0.6	1.2	23, 22, 23
	50	17.3	1.2	0.9	18, 18, 16
	150	14.0	1.7	0.7	13, 13, 16
	500	17.7	2.9	0.9	21, 16, 16
	1500	17.7	2.1	0.9	16, 20, 17
	5000	15.0	2.0	0.8	17, 13, 15
TA1537	Solvent control	38.0	4.6		42, 39, 33
	5	33.0	6.0	0.9	27, 39, 33
	15	32.0	7.2	0.8	38, 24, 34
	50	31.7	5.5	0.8	26, 32, 37
	150	29.7	3.2	0.8	26, 32, 31
	500	22.7	2.3	0.6	24, 24, 20
	1500	24.0	1.7	0.6	23, 26, 23
	5000	27.0	3.5	0.7	29, 29, 23
WP2 uvrA (pKM101)	Solvent control	159.7	10.5		160, 149, 170
	5	154.7	19.1	1.0	175, 137, 152
	15	146.3	14.0	0.9	160, 132, 147
	50	158.0	11.3	1.0	145, 165, 164
	150	142.3	19.6	0.9	137, 164, 126
	500	139.3	11.9	0.9	143, 149, 126
	1500	142.7	15.1	0.9	160, 136, 132
	5000	133.0	5.0	0.8	138, 128, 133
TA98	5^a	219.7	29.2	4.2	236, 186, 237
TA100	5^b	2259.7	405.0	13.7	1858, 2668, 2253
TA1535	5^b	302.0	4.6	15.6	297, 306, 303
TA1537	5^a	241.7	17.0	6.4	241, 259, 225
WP2 uvrA (pKM101)	10^b	1332.7	61.1	8.3	1398, 1323, 1277

^abenzo(a)pyrene

^b2-aminoanthracene

 

Table 3       Test 2 (in the presence of S9 mix)

 

Strain	Concentration (µg/plate)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	Solvent control	36.7	4.6		42, 34, 34
	50	35.7	2.9	1.0	39, 34, 34
	150	36.7	8.5	1.0	45, 37, 28
	500	38.3	10.8	1.0	46, 43, 26
	1500	41.3	5.7	1.1	46, 43, 35
	5000	38.7	5.0	1.1	44, 38, 34
TA100	Solvent control	123.3	16.3		120, 109, 141
	50	138.7	16.2	1.1	120, 148, 148
	150	129.0	6.1	1.0	136, 125, 126
	500	117.7	8.1	1.0	109, 119, 125
	1500	118.7	6.5	1.0	112, 125, 120
	5000	122.7	3.1	1.0	126, 122, 120
TA1535	Solvent control	22.7	0.6		22, 23, 23
	50	26.0	2.0	1.1	24, 26, 28
	150	23.0	1.0	1.0	23, 22, 24
	500	22.3	7.0	1.0	15, 23, 29
	1500	20.7	1.2	0.9	20, 22, 20
	5000	20.7	1.2	0.9	20, 20, 20
TA1537	Solvent control	16.7	5.0		22, 16, 12
	50	15.3	2.1	0.9	16, 13, 17
	150	19.7	1.5	1.2	18, 21, 20
	500	14.0	1.7	0.8	13, 16, 13
	1500	18.3	1.5	1.1	17, 18, 20
	5000	14.7	1.5	0.9	15, 16, 13
WP2 uvrA (pKM101)	Solvent control	145.7	16.4		152, 127, 158
	50	156.3	21.6	1.1	141, 181, 147
	150	152.7	9.1	1.0	161, 154, 143
	500	152.0	6.2	1.0	159, 150, 147
	1500	140.0	8.7	1.0	145, 145, 130
	5000	145.3	13.6	1.0	150, 156, 130
TA98	2^a	404.7	29.7	11.0	438, 395, 381
TA100	2^b	1015.3	103.6	8.2	1125, 1002, 919
TA1535	2^b	701.3	24.0	30.9	729, 689, 686
TA1537	50^c	501.7	20.6	30.1	500, 523, 482
WP2 uvrA (pKM101)	2^d	1816.7	181.5	12.5	1782, 2013, 1655

^a2-nitrofluorene

^bsodium azide

^c9-aminoacridine

^d4-nitroquinoline-1-oxide

 

Table 4       Test 2 (in the absence of S9 mix)

 

Strain	Concentration (µg/plate)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	Solvent control	47.0	6.2		42, 54, 45
	50	51.0	9.5	1.1	62, 46, 45
	150	43.7	1.5	0.9	45, 44, 42
	500	51.7	11.2	1.1	64, 42, 49
	1500	46.3	10.0	1.0	50, 35, 54
	5000	44.7	4.6	1.0	50, 42, 42
TA100	Solvent control	133.7	21.0		150, 110, 141
	50	136.0	4.4	1.0	131, 139, 138
	150	141.7	12.3	1.1	152, 145, 128
	500	148.3	9.9	1.1	155, 137, 153
	1500	140.7	13.6	1.1	128, 139, 155
	5000	133.7	6.8	1.0	139, 136, 126
TA1535	Solvent control	25.0	7.2		17, 31, 29
	50	20.7	5.0	0.8	26, 20, 16
	150	21.3	4.6	0.9	16, 24, 24
	500	22.0	5.3	0.9	28, 18, 20
	1500	19.0	2.6	0.8	17, 22, 18
	5000	19.3	2.9	0.8	21, 21, 16
TA1537	Solvent control	30.0	6.9		26, 26, 38
	50	28.3	4.0	0.9	26, 33, 26
	150	28.3	2.3	0.9	27, 27, 31
	500	28.3	3.2	0.9	32, 27, 26
	1500	31.3	6.8	1.0	39, 29, 26
	5000	27.0	1.7	0.9	26, 29, 26
WP2 uvrA (pKM101)	Solvent control	156.0	14.4		172, 152, 144
	50	165.0	11.3	1.1	159, 158, 178
	150	158.3	15.5	1.0	158, 174, 143
	500	154.7	4.5	1.0	155, 159, 150
	1500	157.7	4.6	1.0	155, 163, 155
	5000	152.7	4.2	1.0	156, 154, 148
TA98	5^a	380.7	36.9	8.1	409, 394, 339
TA100	5^b	2087.7	206.8	15.6	1864, 2272, 2127
TA1535	5^b	243.7	68.6	9.7	169, 304, 258
TA1537	5^a	236.0	14.5	7.9	222, 251, 235
WP2 uvrA (pKM101)	10^b	1179.3	60.6	7.6	1117, 1238, 1183

^abenzo(a)pyrene

^b2-aminoanthracene

 

 

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 (S. typhimurium) and WP2uvrA (pKM101) (Escherichia coli) were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in acetone at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in a screening experiment followed by concentrations of 50, 150, 1500 and 5000 µg/plate in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix. The S9 mix content was increased from 10 % v/v to 20 % v/v in the secondary test.

 

The test item was tested up to the standard limit concentration recommended in the regulatory guidelines i.e. 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains.

 

There was no evidence of cytotoxicity or induced mutant colonies (over background levels) in any of the test item treated colonies in either of the two tests.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.