Nonanoic acid, mixed esters with heptanoic acid, pentaerythritol and valeric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the polyol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the polyol esters category.

 

Ames test (OECD 471, GLP): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100 and TA 98, and E. coli WP2 uvrA pKM 101

Chromosome aberration (OECD 473): negative in CHO cells with and without metabolic activation

Read-across from structural analogue source substance Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)

Gene mutation in mammalian cells (OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Read-across from structural analogue source substance 2,2-bis[[(1-oxopentyl)oxy]methyl] propane-1,3-diyl divalerate (CAS No. 15834-04-5)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April - 20 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministeriel des Produits Chimiques, France

Type of assay:

bacterial reverse mutation assay

Target gene:

"his operon" (for S. typhimurium strains) and "trp operon" (for E.coli strains)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity study: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
First experiment (direct incorporation): 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment (pre-incubation) experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Remarks:

untreated control plates

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Untreated negative controls:

yes

Remarks:

untreated control plates

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Remarks:

untreated control plates

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Remarks:

untreated control plates

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cis-platinum (II) diammine dichloride

Untreated negative controls:

yes

Remarks:

untreated control plates

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Untreated negative controls:

yes

Remarks:

untreated control plates

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: dimethylbenzanthracene

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn


OTHER: a preliminary toxicity test was carried out in the strains TA100

Evaluation criteria:

The result of the test is considered positive if a concentration dependent relationship is obtained in one or several of the 5 strains, with and/or without metabolic activation. A mutagenic effect is taken into account for a given test concentration if the number of revertant colonies is at least two-fold that of spontaneous revertant colonies for S. typhimurium TA 98, TA 100 and E. coli WP2 uvr A pKM 101 and three-fold for S. typhimurium TA 1535 and TA 1537. All results must be confirmed in an independent experiment.

Statistics:

Mean values and standard deviations were calculated.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

E. coli WP2 uvr A pKM 101

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
- Precipitation: no precipitate was observed at at any dose level tested

RANGE-FINDING/SCREENING STUDIES: The dose range for the main test was determined in a preliminary toxicity assay where the test material was tested at the following doses: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation. The test material was non-toxic to the strains of bacteria used (TA 100) at any dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Furthermore positive control values were at least two times the respective vehicle control value for each strain. The historical control ranges are presented in the table below.

Due to the complexity and the amount of information, all relevant tables summarising results and historical control data are attached as pdf document (Tables_OECD 471.pdf).

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar - 11 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

First experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with metabolic activation (12%, v/v)); 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL (without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

in the absence of S9-mix Migrated to IUCLID6: 15 and 5 µg/mL for 3 and 24 h treatment period

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in the presence of S9-mix Migrated to IUCLID6: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: cells were exposed to the test material for 3 h and 24 h
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

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

Evaluation criteria:

Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.
There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency.

Statistics:

The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well

Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10E005] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100

RSday2= CEday2(test) / CEday2(control) x 100

The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10E005]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count/1.25x10E005]


The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:

MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10E-006

Small and large colony mutation frequencies were calculated in an identical manner.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 100 µg/mL


RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h, respectively, with a number of increasing test substance concentrations. The highest concentration tested was 200 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h incubation. 24 h incubation resulted in 64% relative suspension growth in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls.

Table 1: Experiment 1 - 3 hours with and without S9 mix

Dose (µg/ml)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	mutation frequency x 10^-6
					total
Without metabolic activation, 3 h treatment
SC1	100	94	100	100	89
SC2		108			73
0.03	98	101	100	98	63
0.1	92	99	98	90	83
0.3	111	102	101	112	58
1	107	98	97	104	64
3	110	101	100	110	83
10	98	99	98	96	83
33	98	110	109	106	90
100*	74	94	93	69	97
MMS	70	63	63	44	1022
With 8% (v/v) metabolic activation, 3 h treatment
SC1	100	77	100	100	82
SC2		84			87
0.03	96	90	112	107	71
0.1	92	104	129	119	60
0.3	80	108	135	108	55
1	93	105	131	121	69
3	97	90	112	109	65
10	95	84	104	99	71
33	93	81	101	94	91
100*	42	83	103	43	98
CP	20	37	47	9	1107

 

Table 2: Experiment 2 - 3 hours with and 24 hours without S9 mix

Dose (µg/ml)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	mutation frequency x 10^-6
					total
Without metabolic activation, 24 h treatment
SC1	100	102	100	100	62
SC2		104			57
0.1	97	83	80	78	87
1	94	105	102	96	68
3	102	90	87	89	65
10	104	115	111	115	54
33	105	83	80	84	53
100*	102	98	95	97	55
200*	116	104	101	116	52
250*	112	108	105	118	51
MMS	80	81	79	63	631
With 12% (v/v) metabolic activation, 3 h treatment
SC1	100	77	100	100	60
SC2		91			84
0.03	116	58	69	81	108
0.1	97	80	95	93	86
0.3	94	80	95	90	76
1	99	81	97	96	88
3	102	89	106	108	71
10	104	86	103	106	73
33	119	86	103	122	83
100*	105	77	91	96	72
CP	31	54	64	20	814

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:

Interpretation of results:
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

(in both experiments, cultures without metabolic activation were exposed to the test substance for about 16 h, no positive control for the 40 h time point)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A Medium containing 10% (v/v) fetal bovine serum and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: WBL clone

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

40, 80 and 160 µg/mL with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on results of a solubility test, acetone was selected as the vehicle. The test substance was not soluble in water or dimethyl sulfoxide at any of the concentrations (10, 25, 50% (v/v)) tested. The test substance was soluble as a 50% mixture in acetone.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (v/v) in acetone; + S9: 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL (v/v) in acetone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
+S9: ca. 3 h (± 0.5 h)
- S9: ca. 16 h (± 0.5 h)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 16 h (± 0.5 h); second experiment - ca. 16 and 40 h (± 0.5 h)

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL Colcemid® (10 mg/mL (v/v) in cell culture medium)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 replications (16 h) and 1 replication (40 h), respectively

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

Evaluation criteria:

A test substance was considered positive in the chromosome aberration test if:
1. A statistically significant dose-related increase in the percentage of aberrant cells and in at least one of the treatment groups, the percentage of aberrant cells exceeds 5%. OR
2. A reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
If neither of the above conditions exist, the test substance is considered nonmutagenic or negative for inducing chromosomal aberrations in this system.

Species / strain:

Chinese hamster Ovary (CHO)

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: The test substance is not soluble in water, therefore it was dissolved in acetone.
- Precipitation: Final concentrations of the test substance in medium of 10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL were tested by visual and microscopic methods for precipitation immediately, 30 minutes and 3 h after dosing. Traces of the test substance were observed microscopically at all test concentrations equal to or greater than 78 µg/mL. Therefore, the upper limit of the culture medium solubility of the test substance was considered to be between 39 and 78 µg/mL. Based on these results, the study director selected the following concentrations for the toxicity pretest: 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
In the main experiments, slight precipitation was observed in the second experiment after 16 h at 160 µg/mL without metabolic activation. Precipitation was not noted at any other harvest of a 160 µg/mL culture.

RANGE-FINDING/SCREENING STUDIES: To determine a concentration selection for the aberration assay, a toxicity pretest was conducted with concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL of the test substance with and without metabolic activation. The concentrations tested were based on the results of a culture medium solubility test. The cultures with metabolic activation were treated for 3 h (± 0.5 h). The cultures without metabolic activation were treated until 2-3 h prior to harvest. All cultures were harvested about 16 h from the beginning of treatment. After harvest, the number of cells that survived treatment were counted using a hemacytometer to evaluate cytotoxicity and the mitotic indices (number of mitotic cells per 1000 total cells) were determined to evaluate cell cycle suppression. The selected concentrations for the aberration assay were based on the results of the cell count data and mitotic index data. The highest reduction in cell survival was observed at 160 µg/mL without metabolic acvtivation, where reduction in viability of 37% was noted. Other less notable reductions in cell survival were noted (see table 3), but were not indicative of a concentration-related trend. Based on these results, the concentrations selected for the aberration assay were 40, 80 and 160 µg/mL.

Table 1. Test results of experiment 1

Test item	Concentration	Mitotic Index	Aberrant cells	Aberration frequency
	in µg/mL	in %	in %	in %
Exposure period 16 h, fixation time 16 h, without S9 mix
vehicle	0.5% (v/v)	6.8	0.5	0.5
MNNG	0.6	6.2	22.5**	24.5
Test substance	40	5.6	0.5	0.5
	80	6.5	0.5	0.5
	160	7.2	1.0	1.0
Exposure period 3 h, fixation time 16 h, with S9 mix
Acetone	0.5% (v/v)	5.5	1.0	1.0
DMBA	10	2.6	33.5**	42.0
Test substance	40	5.3	1.5	1.5
	80	6.3	0.5	0.5
	160	4.6	2.0	2.0

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

 

Table 2. Test results of experiment 2

Test item	Concentration	Mitotic Index	Aberrant cells	Aberration frequency
	in µg/mL	in %	in %	in %
Exposure period 16 h, fixation time 16 h, without S9 mix
vehicle	0.5% (v/v)	7.2	1.0	1.0
MNNG	0.6	5.4	17.5**	17.5
Test substance	40	7.1	1.0	1.0
	80	5.4	0.5	0.5
	160	7.1	2.5	2.5
Exposure period 3 h, fixation time 16 h, with S9 mix
Acetone	0.5% (v/v)	2.2	0.0	0.0
DMBA	10	4.5	33.0**	43.0
Test substance	40	2.2	1.0	1.0
	80	2.4	2.0	2.0
	160	2.0	1.5	1.5
Exposure period 16 h, fixation time 40 h, without S9 mix
Acetone	0.5% (v/v)	3.4	2.5	2.0
MNNG #	0.6	---	---	---
Test substance	40	3.0	4.0	4.5
	80	2.2	3.0	3.0
	160	3.4	2.0	2.0
Exposure period 3 h, fixation time 40 h, with S9 mix
Acetone	0.5% (v/v)	4.8	2.5	2.5
DMBA #	10	---	---	---
Test substance	40	5.4	2.0	2.0
	80	4.8	2.0	2.0
	160	5.0	0.5	0.5

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

# According to the study report, positive controls were not required for the 40 h harvest.

Table 3. Toxicity pretest results

Treatment Group in µg/mL	Cell Survival in %*
	+ S9	- S9
non-treated	107	102
vehicle	100	100
0.625	122	114
1.25	95	72
2.5	121	88
5	108	68
10	111	108
20	89	102
40	99	93
80	78	93
160	120	63

  * % cell survival as compared to vehicle

Conclusions:

Interpretation of results:
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the polyol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the polyol esters category.

 

Mammalian erythrocyte micronucleus formation in vivo (OECD 474): negative after intraperitoneal and oral application

Read-across from structural analogue source substances Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS No. 68424-31-7) and Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the polyol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

In vitro gene mutation in bacteria

The potential to induce gene mutations in bacteria has been investigated using Monopentaerythritol tetraesters and dipentaerythritol hexaesters of valeric, heptanoic and nonanoic acids.

The mutagenic potential of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of valeric, heptanoic and nonanoic acids was tested in a bacterial reverse mutation study according to OECD Guideline 471 and under GLP conditions (LEMI, 2017). Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvr A pKM 101 were tested. Two independent experiments were carried out. In the first experiment the plate incorporation method was applied, while in the repeat experiment the pre-incubation procedure was used. In both experiments, the tester strains were exposed to solutions of the test substance in ethanol at concentrations of 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation. Metabolic activation was simulated by a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Aroclor 1254. Appropriate vehicle, negative and positive controls were also included in the study design. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls indicating the satisfactory performance of the test and the activity of the metabolising system. There was no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed at any concentration either with or without metabolic activation. Therefore, the test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

In vitro cytogenicity / chromosome aberration in mammalian cells

A relevant key study investigating the potential to induce chromosome aberration in mammalian cells performed with a suitable structural analogue source substance is available.

An in vitro mammalian chromosome aberration test was performed with Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD Guideline 473 and under GLP conditions (Exxon, 1995e). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were exposed to the test substance for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. The test substance was dissolved in acetone and used at concentrations of 40, 80 and 160 µg/mL. In the second experiment cells were again exposed for 3 h and for 16 h followed by 16 h expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 h followed by 40 h expression time with and without metabolic activation, respectively. The same substance concentrations as in first experiment were used. The test substance did not induce cytotoxicity but a precipitate was visible in the second experiment at 160 µg/mL after 16 h incubation without metabolic activation. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. N-Methyl-N-Nitro-N-Nitrosoguanidine and 7,12-Dimethylbenz[a]anthracene were used as positive control materials inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

In vitro gene mutation in mammalian cells

There is one relevant key study investigating the potential to induce gene mutation in mammalian cells available. The study has been performed with a suitable structural analogue source substance.

An in vitro mammalian cell gene mutation assay according to OECD Guideline 476 and GLP was performed with 2,2-bis[[(1-oxopentyl)oxy]methyl] propane-1,3-diyl divalerate (CAS No. 15834-04-5) in mouse lymphoma L5178Y cells (Notox, 2010). In the first experiment, the cells were treated for 3 h with 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL in the presence or absence of S9-mix (8% (v/v)). In the second experiment, concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL were applied with metabolic activation (12%, v/v) for 3 h and 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL without metabolic activation for 24 h. The test substance was tested up to precipitating concentration (100 µg/mL and above). Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Two relevant studies with structural analogue source substances and investigating the potential to induce micronuclei in erythrocytes in vivo are available. Both studies are taken into account in a Weight-of-Evidence approach.

Fatty acids, C5-10, esters with pentraerythritol (CAS No. 68424-31-7) was found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application (Zeneca, 1992). A single intraperitoneal injection was given to groups of 5 male and 5 female mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 hours after dosing. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times. Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is considered not to be biologically significant compared to the concurrent control values. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Fatty acids, C8-10 mixed esters with dipenaterythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS No. 189200-42-8) was tested for its clastogenic potential in an in vivo micronucleus assay (Exxon, 1995f). Male and female CD-1 mice (5 per sex and dose) were administered 500, 1000, and 2000 mg/kg bw by oral gavage for three times. The period between the oral applications was approx. 24 h. Cyclophosphamide (20 mg/kg bw) as positive control substance and concurrent vehicle controls were also part of the study design. No toxicity was observed up to the highest dose tested. Bone marrow samples were taken 24 h after the last application of the test substance. No cytotoxicity was observed, since there were no statistically significant decreases in the percentage of polychromatic erythrocytes of the treated animals in comparison with the vehicle control. Treatment of the animals with the test substance did not lead to a dose-dependent and statistically significant increase in micronuclei formation. The sensitivity of the test system and the validity of the assay were demonstrated by a statistically significant increase in micronucleated polychromatic erythrocytes induced by the positive control, when compared to the vehicle control. In summary, the test substance was found not to be genotoxic in the in vivo micronucleus assay.

Conclusion on genetic toxicity

Several reliable studies performed with either the target substance or analogue source substances are available investigating their genotoxic potential. Genotoxic effects considered include gene mutation in bacteria and mammalian cells as well as clastogenicity both in vitro and in vivo. The available data demonstrate the lack of genotoxic effects since all tests performed were negative. Thus, no hazard regarding genotoxicity is identified for the target substance Monopentaerythritol tetraesters and dipentaerythritol hexaesters of valeric, heptanoic and nonanoic acids.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 information on intrinsic properties of substances may be generated by means other than tests, e.g. using information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI 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 grouping concept is applied to the target substance Monopentaerythritol tetraesters and dipentaerythritol hexaesters of valeric, heptanoic and nonanoic acids, data gaps can be filled by interpolation, as part of a read across approach from representative structural analogue source substances to avoid unnecessary animal testing.

The grouping concept is also used to derive the C&L of the target substance taking the properties of the source substances into account. Based on the grouping concept, all available data on genotoxicity both in vitro and in vivo do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.