Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Bacterial Mutagenicity (Ames test, OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and TA 102 with and without metabolic activation

Mammalian cytogenicity (CA, OECD 473): negative in cultured peripheral human lymphocytes with and without metabolic activation

Mammalian mutagenicity (MLA, OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar - 08 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1997

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:

TK 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 rats pretreated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment I:
- 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (8%, v/v))
Experiment II:
- 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (12%, v/v))

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

+ S9: cyclophosphamide, 15 and 5 µg/mL for 3 and 24 h treatment, respectively; - S9: methylmethanesulfonate, 7.5 µg/mL

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Pre-incubation period: No
- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively.
- Expression time (cells in growth medium): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. 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: relative total growth

Evaluation criteria:

Several criteria including a concentration-related, or a reproducible increase in mutation frequencies determined a positive result.

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

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 333 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 750 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 or 24 h incubation.

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

Table 1: Results of experiment 1

Dose (µg/ml)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	mutation frequency x 10E6
					total
Without metabolic activation, 3 h treatment
SC1	100	104	100	100	74
SC2		85			97
0.3	99	98	104	102	74
1	101	102	108	109	71
3	100	101	107	107	94
10	93	98	104	97	67
33	120	94	100	120	63
100	113	101	107	121	61
333*	104	113	120	124	64
750*	405	101	107	112	74
MMS	71	68	72	51	835
With 8% (v/v) metabolic activation, 3 h treatment
SC1	100	70	100	100	65
SC2		69			64
0.3	96	60	86	83	74
1	115	68	98	113	60
3	109	40	57	62	84
10	127	72	104	132	52
33	114	46	66	75	84
100	122	76	108	133	63
333*	115	62	89	102	72
750*	104	58	84	87	53
CP	50	32	45	22	1617

 

Table 2: Results of experiment 2

Dose (µg/ml)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	mutation frequency x 10E6
					total
Without metabolic activation, 24 h treatment
SC1	100	66	100	100	90
SC2		79			75
0.3	112	77	106	119	88
1	116	80	110	128	82
3	117	72	100	117	79
10	120	85	117	140	66
33	114	74	101	116	83
100	121	69	95	115	83
333*	116	70	97	112	70
750*	116	66	91	106	71
MMS	101	49	67	68	1502
With 12% (v/v) metabolic activation, 3 h treatment
SC1	100	93	100	100	80
SC2		93			76
0.3	103	84	90	93	74
1	113	83	89	101	81
3	107	97	104	112	60
10	105	94	101	107	80
33	103	93	100	103	67
100	102	105	114	116	57
333*	106	91	99	104	74
750*	103	93	100	103	73
CP	72	75	81	58	1082

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:

negative

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jan - 07 Feb 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP -guideline study with acceptable restrictions (no analytical purity reported)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no analytical purity reported

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2000

Deviations:

yes

Remarks:

no analytical purity reported

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/beta-naphthoflavone

Test concentrations with justification for top dose:

Experiment I+II:
- 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: N-ethyl-N´-nitro-N-nitrosoguanidine (ENNG, 3 or 5 µg/plate) for TA 100 and TA 1535; 9-Aminoacridine (9AA, 80 µg/plate) for TA 1537; Mitomycin C (MMC, 0.5 µg/plate) for TA 102; 4-Nitroquinoline-1-oxide (4NQO, 0.2 µg/plate) for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: 2-Aminoanthracene (2AA, 1 or 2 µg/plate) for TA 100, TA 1535 and TA 1537; Benzo(a)pyrene (BP, 5 µg/plate) for TA 98; 1,8-Dihydroxyanthraquinone (DAN, 10 µg/plate) for TA 102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

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

Evaluation criteria:

The test material may be considered positive mutagenic in this test system if the following criteria are met: the test item should induce a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	80 ± 5.7	24 ± 6.1	294 ± 18.1	24 ± 4.6	18 ± 2.5
–	50	81 ± 6.1	22 ± 5.9	297 ± 19.3	23 ± 1.0	14 ± 2.1
–	150	86 ± 3.1	23 ± 3.2	310 ± 18.2	21 ± 4.6	15 ± 4.7
–	500	82 ± 11.0	23 ± 2.1	330 ± 31.5	22 ± 4.2	19 ± 4.4
–	1500	89 ± 1.5	23 ± 4.0	311 ± 14.0	29 ± 5.7	16 ± 3.2
–	5000	85 ± 5.9 P	30 ± 2.6 P	321 ± 34.6 P	28 ± 4.2 P	16 ± 4.6 P
Positive controls, –S9	Name	ENNG	ENNG	MMC	4NQO	9AA
	Concentrations (μg/plate)	3	5	0.5	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	439 ± 33.5	464 ± 29.4	740 ± 113.4	96 ± 5.9	1510 ± 150.1
+	0	92 ± 10.4	25 ± 4.7	339 ± 24.3	40 ± 3.2	23 ± 0.6
+	50	109 ± 11.5	23 ± 7.2	375 ± 14.6	39 ± 9.2	18 ± 2.0
+	150	99 ± 18.0	19 ± 1.5	359 ± 22.4	45 ± 1.2	24 ± 3.1
+	500	94 ± 17.7	15 ± 2.6	358 ± 23.7	45 ± 4.6	26 ± 1.5
+	1500	102 ± 9.6	23 ± 5.6	358 ± 32.1	35 ± 2.0	21 ± 5.2
+	5000	102 ± 6.8	22 ± 4.7	369 ± 72.2	39 ± 4.6	25 ± 2.1
Positive controls, +S9	Name	2AA	2AA	DAN	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	2112 ± 97.7	269 ± 37.8	706 ± 32.6	232 ± 19.2	217 ± 12.4

Table 2. Test results of experiment 2 (plate incorporation). 

ith or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	82 ± 8.1	21 ± 4.0	249 ± 20.7	18 ± 0.6	10 ± 2.6
–	50	70 ± 13.1	15 ± 2.6	269 ± 9.8	24 ± 1.5	12 ± 5.5
–	150	79 ± 15.5	18 ± 1.5	279 ± 34.8	24 ± 4.5	6 ± 2.3
–	500	81 ± 7.8	19 ± 6.4	268 ± 2.3	22 ± 6.1	7 ± 1.0
–	1500	88 ± 8.0	22 ± 6.1	275 ± 8.3	27 ± 7.0	13 ± 3.5
–	5000	75 ± 12.1 P	21 ± 3.6 P	290 ± 50.2 P	24 ± 2.9 P	10 ± 6.4 P
Positive controls, –S9	Name	ENNG	ENNG	MMC	4NQO	9AA
	Concentrations (μg/plate)	3	5	0.5	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	679 ± 29.4	933 ± 340.6	1167 ± 40.7	188 ± 11.7	2357 ± 179.5
+	0	87 ± 21.1	10 ± 4.4	330 ± 21.2	40 ± 2.1	19 ± 3.8
+	50	86 ± 7.2	13 ± 4.0	369 ± 18.9	33 ± 4.6	17 ± 2.6
+	150	105 ± 10.4	11 ± 4.4	349 ± 27.5	42 ± 1.0	14 ± 7.1
+	500	80 ± 8.0	15 ± 6.1	364 ± 8.3	35 ± 2.5	18 ± 3.6
+	1500	85 ± 6.7	12 ± 3.0	369 ± 34.9	38 ± 2.0	18 ± 1.5
+	5000	88 ± 12.9 P	11 ± 2.5 P	365 ± 15.0 P	36 ± 8.2 P	19 ± 4.4 P
Positive controls, +S9	Name	2AA	2AA	DAN	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	3159 ± 145.2	313 ± 14.5	1021 ± 104.5	312 ± 32.4	222 ± 10.2

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

MMC = mitomycin C

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = benzo(a)pyrene

DAN = 1,8 -dihydroxanthraquinone

P = Precipitate 

Conclusions:

negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06. Oct. - 16. Feb. 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study. (Purity of test substance not given, evaluation criteria not given.)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

purity of test substance is not given (responsibility of the sponsor)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagles essential medium with HEPES buffer (MEM), supplemented with:
L-glutamine, penicillin/streptomycin, amphotericin B, 15% foetal calf serum
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats pretreated with phenobarbitone (80 mg/kg) and ß-naphtoflavone (100 mg/kg)

Test concentrations with justification for top dose:

Experiment I:
- 40, 80, 160, 240*, 320*, 400* µg/mL (with and without metabolic activation)
Experiment II:
- 40, 80, 160, 240*, 320*, 400* µg/mL (with and without metabolic activation)
* Dose levels (plus control dose) selected for metaphase analysis

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC; 0.2 and 0.4 µg/mL; -S9), Cyclophosphamide (CP; 10 µg/mL; +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24 h (without)
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20 h; 24 h treatment: 0 h

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

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

no data

Statistics:

The frequency of cells with aberrations excluding gaps 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: cultured peripheral human lymphocytes

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
- Precipitation: cloudy precipitates were observed at and above 40 and 80 µg/mL in the 24-hour continuous and 4-hour pulse treatment groups, respectively

RANGE-FINDING/SCREENING STUDIES:
The dose range tested was 10-320 µg/mL. The test material produced some weak toxicity in the 4-hour treatment group but not the 24-hour treatment group. Toxicity could not be reproduced in the main experiment (scorable metaphases at every dose level).
COMPARISON WITH HISTORICAL CONTROL DATA:
The results are in range with historical control data.

Table 3 + 4: Test results of experiment I.

Test item	Concentration	Mitotic Index	Aberrant cells in %
Experiment I	in µg/mL	in %	with gaps	without gaps
Exposure period 4h, fixation time 20h, without S9 mix
control	0	100	1	0
MMC	0.4	35	53	37
Test substance	240	98P	0.5	0
	320	110P	0	0
	400	91P	0.5	0.5
Exposure period 4h, fixation time 20h, with S9 mix
control	0	100	1	0.5
CP	10	20	35.5	28.5
Test substance	240	89P	1.5	0
	320	89P	0.5	0
	400	108P	3.5	1

Test item	Concentration	Mitotic Index	Aberrant cells in %
Experiment II	in µg/mL	in %	with gaps	without gaps
Exposure period + fixation time 24h, without S9 mix
control	0	100	1.5	0
MMC	0.4	41	70	67
Test substance	240	61	0.5	0.5
	320	53	0.5	0
	400	83	0	0
Exposure period 4h, fixation time 20h, with S9 mix
control	0	100	0.5	0
CP	10	30	67	56
Test substance	240	103	1	0
	320	76	1	0
	400	110	1.5	0.5

Table 5 +6: Mean Frequency of Polyploid Cells (%)

Experiment I dose level µg/mL	harvest time 24 hours
	4 hours without S9	4 hours with S9
0	0.0	0.0
240	0.0	0.0
320	0.0	0.0
400	0.0	0.0
MMC 0.4	0.0	NA
CP 10	NA	0.0

dose level µg/mL	harvest time 24 hours
	24 hours without S9	4 hours with S9
0	0.5	0.0
240	0.0	0.0
320	0.0	0.0
400	0.0	0.5
MMC 0.4	0.0	NA
CP 10	NA	0.0

CP: Cyclophosphamide

MMC: Mitomycin C

Conclusions:

negative

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July - 27 July 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (Strain S. typhimurium TA102 or E.coli WP2 were not tested).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strain S. typhimurium TA102 or E.coli WP2 not tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa-, uvR-, Strains 98 and 100 also R+

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: rfa-, uvR-

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Experiment I+II:
- 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 µg/plate; TA100 and TA1535 (-S9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate; TA1537 (-S9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

40 µg/plate; TA98 and TA1538 (- S9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

all strains with metabolic activation; 2.5 µg/plate: TA1535, TA1537; 5 µg/plate: TA100, TA1538, TA98

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was seen at concentrations of 1000 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Table 1: Mutagenicity of the test item on bacteria - experiment I

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA1538	TA98	TA1537
-	Buffer	116	9	10	22	7
	Vehicle	112	11	12	34	10
-	8	114	10	7	29	10
-	40	111	10	8	34	7
-	200	114	8	9	29	8
-	1000	120	12	7	25	6
-	5000	132	6	9	29	6
Positive controls - S9	Name	SA	SA	4NP	4NP	9AA
	Concentrations (μg/plate)	2.0	2.0	40	40	80
	Number of colonies/plate	818	621	1792	1571	1025
+	Buffer	116	11	15	38	6
+	Vehicle	103	12	15	35	10
+	8	115	12	16	34	8
+	40	120	9	11	39	8
+	200	113	12	13	38	10
+	1000	126	11	14	39	7
+	5000	130	13	12	3934	7
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5.0	2.5	5.0	5.0	2.5
	Number of colonies/plate	1594	210	1769	1572	66

4NP= 4-nitro-o-phenylendiamine

SA = sodium azide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

Table 2: Mutagenicity of the test item on bacteria - experiment II

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA1538	TA98	TA1537
-	Buffer	122	11	13	34	6
	Vehicle	127	13	9	39	13
-	8	116	12	11	30	10
-	40	110	14	12	31	8
-	200	121	13	14	39	5
-	1000	111	17	18	29	7
-	5000	122	16	13	32	10
Positive controls - S9	Name	SA	SA	4NP	4NP	9AA
	Concentrations (μg/plate)	2.0	2.0	40	40	80
	Number of colonies/plate	1018	824	1929	1643	1017
+	Buffer	127	15	19	40	9
+	Vehicle	116	21	20	43	10
+	8	128	11	19	46	10
+	40	126	17	19	49	6
+	200	115	13	19	45	9
+	1000	121	19	16	49	10
+	5000	127	21	14	40	7
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5.0	2.5	5.0	5.0	2.5
	Number of colonies/plate	1535	327	1384	1515	64

4NP= 4-nitro-o-phenylendiamine

SA = sodium azide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

 

Conclusions:

negative

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Aug - 09 Sep 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (tester strains with AT reversion site missing)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1983

Deviations:

yes

Remarks:

tester strains with AT reversion site missing (S. typhimurium TA102 or E. coli WP2 were not tested)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (intraperitoneally) and beta-Naphthoflavone (orally)

Test concentrations with justification for top dose:

Experiment I+II:
- 8, 40, 200, 1000 and 5000 µg/plate (with and withoutmetabolic activation)

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: sodium azide (2 µg/plate, TA100 and TA1535); 9-aminoacridine (80µg/plate, TA537); 4-nitro-o-phenylendiamine (40 µg/plate, TA98 and TA1538); +S9: 2-aminoanthracene (2.5 or 5 µg/plate, all strains)

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylendiamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: growth reduction of background bacteria

Evaluation criteria:

The test material was considered positive if the following criteria were met: The plate background of non-converted bacteria did not show any growth reduction versus the respective negative controls. The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range. The positive controls showed mutation rates exceeding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains by the factor 3.0. At more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with negative controls in the tester strain TA 100, for the other tester strains, an increase in the mutation rates of more than 3.0 above the corresponding negative controls.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Table 1: Results of experiment 1:

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 100	TA1535	TA1537	TA1538	TA98
-	Medium	113	11	11	8	20
-	Vehicle	78	8	7	6	18
-	8	78	8	5	6	16
-	40	80	8	4	6	18
-	200	80	10	5	6	28
-	1000	90	11	8	5	23
-	5000	90	6	5	5	22
Positive controls - S9	Name	SA	SA	9AA	4NP	4NP
	Concentrations (μg/plate)	2.0	2.0	80	40	40
	Number of colonies/plate	745	518	773	1875	1531
+	Medium	119	16	8	13	21
+	Vehicle	91	11	6	7	18
+	8	118	17	9	6	21
+	40	110	10	6	10	18
+	200	109	14	5	9	23
+	1000	118	12	4	11	17
+	5000	110	10	9	8	23
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5.0	2.5	2.5	5.0	5.0
	Number of colonies/plate	318	264	257	2155	1808

SA: sodium azide

9AA : 9-aminoacridine

4NP : 4-nitro-o-phenylenediamine

2-AA: 2 -aminoanthracene

 

Table 2: Results of experiment 2:

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 100	TA1535	TA1537	TA1538	TA98
-	Medium	100	10	7	8	20
-	Vehicle	98	9	9	10	9
-	8	85	13	11	4	9
-	400	92	10	5	9	11
-	200	84	7	6	11	12
-	1000	77	13	6	8	13
-	5000	99	10	2	9	11
Positive controls - S9	Name	SA	SA	9AA	4NP	4NP
	Concentrations (μg/plate)	2.0	2.0	80	40	40
	Number of colonies/plate	790	583	922	2031	1631
+	Medium	99	15	10	13	22
+	Vehicle	80	15	8	17	31
+	8	88	14	8	11	28
+	40	91	16	13	18	27
+	200	110	12	9	16	26
+	1000	83	12	14	17	27
+	5000	103	16	12	15	30
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5.0	2.5	2.5	5.0	5.0
	Number of colonies/plate	1339	84	215	1192	1069

SA: sodium azide

9AA : 9 -aminoacridine

4NP : 4 -nitro-o-phenylenediamine

2-AA: 2 -aminoanthracene 

Conclusions:

negative

Reference 6

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

Justification for type of information:

Please refer to the Additional Information field in the endpoint study summary

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and 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:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

Remarks on result:

other: BASF, 1991

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to the Additional Information field in the endpoint study summary

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

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

Remarks on result:

other: Arizona, 2004

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to the Additional Information field in the endpoint study summary

Reason / purpose for cross-reference:

read-across source

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Croda, 2010

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across

There are only limited data available addressing the genetic toxicity of Fatty acids, C16-18 (even numbered) and C18-unsatd., branched and linear, tri- and tetraesters with pentaerythritol (CAS 85186-72-7). In accordance with Article 13 (1) of Regulation (EC) No. 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

 

Having regard to the general rules for the read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a comparable pattern as a result of structural similarity, the substances Fatty acids, C16-18, esters with pentaerythritol (CAS 85116-93-4), Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (CAS 85186-89-6) and Fatty acids, C16-18 and C18uns., branched and linear ester with trimethylolpropane (CAS 403507-18-6) are selected as source substances.

 

In vitro mutagenicity in bacteria

CAS 85116-93-4

The mutagenic potential of Fatty acids, C16-18 (even numbered), esters with pentaerythritol (CAS 85116-93-4) was tested in a reverse mutation assay comparable to OECD guideline 471 and under GLP conditions (BASF, 1991). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were used. Tester strains were incubated with test material dissolved in Tween 80 at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9-mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C16-18, esters with pentaerythritol did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

 

CAS 85186-89-6

The mutagenic potential of Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (Formerly CAS 85186-89-6) was examined in a reverse mutation assay comparable to OECD guideline 471 and under GLP conditions (BASF, 1999). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were used. Tester strains were incubated with test material dissolved in acetone at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) with and without the addition of a metabolic activation system (phenobarbital and beta-naphthoflavone induced rat liver S9-mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed but the test substance was tested up to limit concentrations. Thus, Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

 

CAS 403507-18-6

The mutagenic potential of Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane (CAS 403507-18-6) was tested in a reverse mutation assay comparable to OECD guideline 471 and under GLP conditions (Arizona, 2002). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Tester strains were incubated with test material dissolved in acetone at concentrations of 50, 150, 500, 1500 and 5000 µg/plate with and without the addition of a metabolic activation system (phenobarbital and beta-naphthoflavone induced rat liver S9-mix). Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent vehicle controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed, but the test substance was tested up to precipitating concentrations. Thus, Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

 

In vitro cytogenicity in mammalian cells

CAS 403507-18-6

An in vitro mammalian chromosome aberration test was performed with Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane (CAS 403507-18-6) in cultured peripheral human lymphocytes comparable to OECD guideline 473 and under GLP conditions (Arizona, 2004). 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 cells were exposed for 4 hours to the test substance dissolved in acetone at concentrations of 240, 320, 400 µg/mL with and without metabolic activation. In the second experiment cells were exposed for 4 hours to 240, 320, 400 µg/mL with metabolic activation and for 24 hours to 240, 320, 400 µg/mL followed by 24 hours expression time without metabolic activation. The test substance did not induce cytotoxicity but a cloudy precipitate was already visible at 40 µg/mL. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Mitomycin C and Cyclophosphamide 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 200 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 human lymphocytes in vitro.

 

In vitro mutagenicity in mammalian cells

CAS 85186-89-6

An in vitro Mammalian Cell Gene Mutation Assay according to OECD guideline 476 and GLP was performed with Fatty acids, C8-10(even), C14-18(even) and C16-18(even)-unsatd., triesters with trimethylolpropane (CAS 85186-89-6) in mouse lymphoma L5178Y cells (Croda, 2010). The cells were treated for 3 and 24 hours with 8% (v/v) and without S9-mix in the first experiment, respectively, and with 12% (v/v) with and without S9-mix in the second experiment, respectively. In the first experiment the test substance was tested at 0.3, 1, 3, 10, 33, 100, 333 and 750 μg/mL up to precipitation with 8% (v/v) and without S9-mix for 3 h. In the second experiment the test substance was tested at 0.3, 1, 3, 10, 33, 100, 333 and 750 μg/mL up to precipitation with 12% (v/v) for 3 hours and without S9-mix for 24 hours. 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 Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

Conclusion for genetic toxicity

There is no study available addressing the in vitro genetic toxicity of the target substance Fatty acids, C16-18 (even numbered) and C18-unsatd., branched and linear, tri- and tetraesters with pentaerythritol (CAS 85186-72-7). Therefore, analogue read-across from source substances was applied from in vitro studies on bacterial and mammalian cells, using three source substances. The results of the available in vitro studies on source substances did not provide evidence indicative for mutagenic and clastogenic potential. Based on the available data, and following the analogue approach Fatty acids, C16-18 (even numbered) and C18-unsatd., branched and linear, tri- and tetraesters with pentaerythritol (CAS 85186-72-7) is not expected to be mutagenic and clastogenic in vitro. 

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” 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 analogue concept is applied to Fatty acids, C16-18 (even numbered) and C18-unsatd., branched and linear, tri- and tetraesters with pentaerythritol (CAS 85186-72-7), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.