Fatty acids, C9, hexaesters with dipentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

01 Feb - 21 Apr 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No data on analytical purity. Read across study.

Justification for type of information:

Based on its composition and physico chemical properties, 3,5,5-trimethylhexanoic acid mixed tetraesters with pentaerythritol and valeric acid is considered to be a suitable read across substance to address the genotoxicity endpoint of the test substance, fatty acids, C9, hexaesters with dipentaerythritol.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No details on the purity of the test substance

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No details on the purity of the test substance

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced S9-Mix from male Spraque-Dawley rats

Test concentrations with justification for top dose:

1. Experiment:
4(20) h without S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL
4(20) h with S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL

2. Experiment
24 h without S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL
4(20) h with S9: 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

in absence of S9-Mix Migrated to IUCLID6: 750 µg/mL

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in presence of S9-Mix Migrated to IUCLID6: 25 µg/mL

Details on test system and experimental conditions:

Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
METHOD OF APPLICATION: in medium (Eagle´s minimal essential medium with HEPES buffer supplemented with L-glutamine, Pen/Strep, amphotericin B and 15% FCS

DURATION
- Exposure duration: Experiment I: 4 h with or without S9 mix .
- Experiment II: Either 4 h with S9 mix or 24 h without S9 mix.
- Expression time (cells in growth medium): 20 h after 4 h exposure to the test substance and 20 h expression time


SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 min

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Statistics:

The frequency of cells with aberrations and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at and above 1250 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at and above 1250 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: Medium was "cloudy" from 156.25 µg/mL and a precipitate was seen from 1250 µg/mL without effects on the toxicity responsefects:

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, data were in range with the historical control data.

Remarks on result:

other: strain/cell type: lymphocytes

Remarks:

Migrated from field 'Test system'.

Table 1: Experiment 1: 4 h treatment without metabolic activation

Treatment group (µg/ml)	Number of cells scored	Total gaps	Chromatid		Chromosome		others	Total abberations		Abberant cells
			Breaks	Exchanges	Breaks	Exchanges	x	(+Gaps)	(+Gaps)	(+Gaps)	(+Gaps)
Vehicle control	200	5	0	1	0	1	0	7	2	7	2
1250	200	5	0	0	3	0	0	8	3	8	3
2500	200	2	2	0	0	0	0	4	2	4	2
5000	200	5	4	0	0	1	0	10	5	9	5
EMS 750	200	24	9	5	5	0	0	43	19	35	17

Table 2: Experiment 1: 4 h treatment with metabolic activation

Treatment group (µg/ml)	Number of cells scored	Total gaps	Chromatid		Chromosome		others	Total abberations		Abberant cells
			Breaks	Exchanges	Breaks	Exchanges	x	(+Gaps)	(+Gaps)	(+Gaps)	(+Gaps)
Vehicle control	200	4	1	0	0	0	0	5	1	4	1
1250	200	6	4	0	0	0	0	10	4	9	4
2500	200	4	2	0	0	0	0	6	2	5	2
5000	200	2	4	0	1	0	0	7	5	7	5
CP 25	200	13	12	3	2	0	0	30	17	24	15

Table 3: Experiment 2: 20 h treatment without metabolic activation

Treatment group (µg/ml)	Number of cells scored	Total gaps	Chromatid		Chromosome		others	Total abberations		Abberant cells
			Breaks	Exchanges	Breaks	Exchanges	x	(+Gaps)	(+Gaps)	(+Gaps)	(+Gaps)
Vehicle control	200	2	2	1	0	0	0	5	3	5	3
1250	200	7	0	0	1	0	0	8	1	8	1
2500	200	0	1	0	0	0	0	1	1	1	1
5000	200	6	0	0	0	0	0	6	0	6	0
EMS 500	200	53	41	13	2	0	0	109	56	74	48

Table 4: Experiment 2: 4 h treatment with metabolic activation

Treatment group (µg/ml)	Number of cells scored	Total gaps	Chromatid		Chromosome		others	Total abberations		Abberant cells
			Breaks	Exchanges	Breaks	Exchanges	x	(+Gaps)	(+Gaps)	(+Gaps)	(+Gaps)
Vehicle control	200	0	0	0	2	0	0	2	2	2	2
1250	200	4	2	2	3	0	0	9	5	7	3
2500	200	5	2	0	1	0	0	8	3	8	3
5000	200	3	2	0	2	0	0	7	4	6	3
CP 25	200	10	10	2	0	0	0	22	12	15	11

The test substance was non-clastogenic to human lymphocytes in vitro.

Conclusions:

Under the study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

An in vitro mammalian chromosome aberration test was performed with the read across substance, 3,5,5-trimethylhexanoic acid mixed tetraesters with pentaerythritol and valeric acid, in primary human lymphocytes according to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9 mix). In the first experiment test substance concentrations of 39.06, 78.13, 156.25, 312.5; 625; 1250; 2500 and 5000 µg/mL in acetone were used for 4 h of exposure with and without metabolic activation. In the second experiment 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/mL were used for 24 h exposure without S9 and 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 4 h with S9. Ethylmethanesulphate and cyclophosphamide were used as positive control substances. Evaluation of 200 cells from each culture for chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level in comparison to the negative controls. The test substance demonstrated showed no cytotoxicity. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro (Wright, 1999).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

26 Mar - 11 May 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read across study

Justification for type of information:

Based on its composition and physico chemical properties, pentaerythritol tetravalerate is considered to be a suitable read across substance to address the genotoxicity endpoint of the test substance, fatty acids, C9, hexaesters with dipentaerythritol.

Reason / purpose for cross-reference:

read-across source

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:

mammalian cell gene mutation assay

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.

Key result

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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:

The test substance was not mutagenic in the mouse lymphoma L5178Y test system under the study conditions.

Executive summary:

An in vitro mammalian cell gene mutation assay was performed with the read across substance, pentaerythritol tetravalerate, in L5178Y mouse lymphoma cells according to OECD Guideline 476, in compliance with GLP. In the first experiment, the test item was tested up to concentrations of 100 μg/mL in the absence and presence of 8% (v/v) S9 mix, respectively. The incubation time was 3 h. In the second experiment, the substance was tested up to cytotoxic levels of 100 µg/mL and 250 µg/L in the presence and absence of 12% (v/v) S9 mix respectively. The incubation times were 24 h and 3 h for incubations in the absence and presence of S9 mix, respectively. In the test, no cytotoxicty was noticed. However, the test item was test up at or above the precipitating dose level of 100 µg/ml. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 11-fold for methylmethansulfonate (MMS) in the absence of S9 mix, and by 13- and 11-fold for cyclophosphamide (CP) in the presence of S9 mix. In the absence and presence of S9 mix, the test substance did not induce a significant increase in the mutation frequency. In the absence presence of S9 mix, the substance did not induce a significant increase in the mutation frequency in the second experiment. It is concluded that the test substance was not mutagenic in the mouse lymphoma L5178Y test system under the study conditions (Verspeek, 2010).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read across study

Justification for type of information:

Based on its composition and physico chemical properties, fatty acids, C5-9, hexaesters with dipentaerythritol is considered to be a suitable read across substance to address the genotoxicity endpoint of the test substance, fatty acids, C9, hexaesters with dipentaerythritol.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

S9 mix

Test concentrations with justification for top dose:

the doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 microg per plate, one plate per dose, both in the presence and absence of S9 mix.
Since no cytotoxicity was observed on the dose range finding study, the highest dose level of test article used in the mutagenicity assay was the same as the hihest dose tested in the range finding

The doses tested were 33.3, 100, 333, 1000, 3330, and 5000 microg per plate.

Vehicle / solvent:

ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Positive control substance:

other: ICR-191

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Conclusions:

Under the study conditions of the Ames assay, the test substance was not mutagenic with or without metabolic activation.

Executive summary:

A study was conducted to evaluate the mutagenicity of the read across substance, fatty acids, C5-9, hexaesters with dipentaerythritol,according to OECD 471, in compliance with GLP. The tester strains used were Salmonella typhimirium TA98, TA100, TA1535 and TA1537, as well as Escherichia coli WP2uvra. The assay was conducted with 6 concentrations of the substance (33.3, 100, 333, 1000, 3330 and 5000 µg/plate) in both the presence and absence of Arochlor-induced rat liver S9 mix, along with concurrent vehicle and positive controls, using three plates per dose. The results of the initial mutagenicity assay were confirmed in an independent experiment. Under the study conditions, the test substance did not cause a positive increase in the mean number of revertants per plate in any of the tester strains, either in the presence or the absence of S9 mix, and was therefore considered to be non-mutagenic (Mecchi, 2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

13 May - 08 July 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Based on its composition and physico chemical properties, fatty acids, C5-10, esters with pentaerythritol is considered to be a suitable read across substance to address the genotoxicity endpoint of the test substance, fatty acids, C9, hexaesters with dipentaerythritol.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

The study consisted in two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethalities or severe toxicity observed over a four-day observation period following a single intraperitoneal injection.
In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

24 h and 48 h

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg bw in physiological saline

Tissues and cell types examined:

Monochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg bw. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing


DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin


METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.

Evaluation criteria:

Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point.
Percentage of polychromatic erythrocytes.

Statistics:

The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine wether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times.
Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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, however, considered not to be statistically significant compared to the concurrent control values.
The positive control induced stastistically 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.

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

 

Table 1: Males

Group	Compound	Dose	Mean Incidence
			24 h	48 h
11	Vehicle control (corn oil)	10 mL/kg	0.8 ± 0.8	1.0 ± 1.2
12	Cyclophosphamide	65 mg/kg	24.4 ± 6.0**
13	Test substance	5000 mg/kg	0.6 ± 0.6	0.4 ± 0.6

 

Table 2: Females

Group	Compound	Dose	Mean Incidence
			24 h	48 h
11	Vehicle control (corn oil)	10 ml/kg	0.2 ± 0.5	1.4 ± 1.1
12	Cyclophosphamide	65 mg/kg	18.4 ± 7.3**
13	Test substance	5000 mg/kg	0.4 ± 0.9	0.4 ± 0.9

 

 

Mean percentage of polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

 

Table 3: Males

Group	Compound	Dose	Mean Incidence
			24 h	48 h
11	Vehicle control (corn oil)	10 ml/kg	48.0 ± 5.6	44.3 ± 7.5
12	Cyclophosphamide	65 mg/kg	41.4 ± 4.4*
13	Test substance	5000 mg/kg	42.2 ± 7.0*	43.3 ± 1.9

 

Table 4: Female

Group	Compound	Dose	Mean Incidence
			24 h	48 h
11	Vehicle control (corn oil)	10 ml/kg	41.9 ± 4.8	41.9 ± 1.7
12	Cyclophosphamide	65 mg/kg	45.9 ± 3.49
13	Test substance	5000 mg/kg	46.5 ± 5.8	48.0 ± 5.2

Conclusions:

Under the conditions of the in vivo mouse micronucleus assay, the test substance was not clastogenic.

Executive summary:

An in vivo study was conducted to determine the genotoxic potential of the read across substance, fatty acids, C5-10, esters with pentaerythritol, according to OECD Guideline 474 (mouse micronucleus test), in compliance with GLP. A single intraperitoneal injection was given to groups of 5 male and 5 female CD-1 mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 h after dosing. The positive control (cyclophosphamide) 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. 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, however, considered not to be biologically significant compared to the concurrent control values. Therefore, 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. Under the conditions of the in vivo mouse micronucleus assay, the test substance was not clastogenic (Griffiths, 1992).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Study 1:

A study was conducted to evaluate the mutagenicity of the read across substance, fatty acids, C5-9, hexaesters with dipentaerythritol, according to OECD 471, in compliance with GLP. The tester strains used were Salmonella typhimirium TA98, TA100, TA1535 and TA1537, as well as Escherichia coli WP2uvra. The assay was conducted with 6 concentrations of the substance (33.3, 100, 333, 1000, 3330 and 5000 µg/plate) in both the presence and absence of Arochlor-induced rat liver S9 mix, along with concurrent vehicle and positive controls, using three plates per dose. The results of the initial mutagenicity assay were confirmed in an independent experiment. Under the study conditions, the test substance did not cause a positive increase in the mean number of revertants per plate in any of the tester strains, either in the presence or the absence of S9 mix, and was therefore considered to be non-mutagenic (Mecchi, 2008).

Study 2:

An in vitro mammalian chromosome aberration test was performed with the read across substance, 3,5,5-trimethylhexanoic acid mixed tetraesters with pentaerythritol and valeric acid, in primary human lymphocytes according to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9 mix). In the first experiment test substance concentrations of 39.06, 78.13, 156.25, 312.5; 625; 1250; 2500 and 5000 µg/mL in acetone were used for 4 h of exposure with and without metabolic activation. In the second experiment 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/mL were used for 24 h exposure without S9 and 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 4 h with S9. Ethylmethanesulphate and cyclophosphamide were used as positive control substances. Evaluation of 200 cells from each culture for chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level in comparison to the negative controls. The test substance demonstrated showed no cytotoxicity. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro (Wright, 1999).

 

Study 3:

An in vitro mammalian cell gene mutation assay was performed with the read across substance, pentaerythritol tetravalerate, in L5178Y mouse lymphoma cells according to OECD Guideline 476, in compliance with GLP. In the first experiment, the test item was tested up to concentrations of 100 μg/mL in the absence and presence of 8% (v/v) S9 mix, respectively. The incubation time was 3 h. In the second experiment, the substance was tested up to cytotoxic levels of 100 µg/mL and 250 µg/L in the presence and absence of 12% (v/v) S9 mix respectively. The incubation times were 24 h and 3 h for incubations in the absence and presence of S9 mix, respectively. In the test, no cytotoxicty was noticed. However, the test item was test up at or above the precipitating dose level of 100 µg/ml. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 11-fold for methylmethansulfonate (MMS) in the absence of S9 mix, and by 13- and 11-fold for cyclophosphamide (CP) in the presence of S9 mix. In the absence and presence of S9 mix, the test substance did not induce a significant increase in the mutation frequency. In the absence presence of S9 mix, the substance did not induce a significant increase in the mutation frequency in the second experiment. It is concluded that the test substance was not mutagenic in the mouse lymphoma L5178Y test system under the study conditions (Verspeek, 2010).

In vivo

An in vivo study was conducted to determine the genotoxic potential ofthe read across substance,fatty acids, C5-10, esters with pentaerythritol,according to OECD Guideline 474 (mouse micronucleus test), in compliance with GLP. A single intraperitoneal injection was given to groups of 5 male and 5 female CD-1 mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 h after dosing. The positive control (cyclophosphamide) 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. 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, however, considered not to be biologically significant compared to the concurrent control values. Therefore, 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. Under the conditions of the in vivo mouse micronucleus assay, the test substance was not clastogenic (Griffiths, 1992).

Justification for classification or non-classification

Based on the results of in vitro studies with appropriate read across substances, the test substance does not require classification for genotoxicity according to EU CLP (EC 1272/2008) criteria.