Decanoic acid, mixed esters with dipentaerythritol, heptanoic acid and octanoic acid.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance, CAS: 70851-04-6; EC: 453-480 -2 is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. Based on the available information to read across to, the substance is not expected to be mutagenic.

Hatcol 5127 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

Remarks:

Type of genotoxicity: gene mutation in bacteria / mammilian cell lines and chromosome abberiation

Type of information:

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

Remarks:

4 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the justification provided in section 13

Reason / purpose for cross-reference:

read-across source

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:

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:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

other: Unacceptable vehicle control values for the tester strain TA1537 and TA 98 were repeated.

Untreated negative controls validity:

not specified

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:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Key result

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2)

Conclusions:

The substance, CAS: 70851-04-6; EC: 453-480-2; is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. Based on the available information to read across to, the substance is not expected to be mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May - 29 Aug 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

September 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

September 1995

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium strains)
trp operon ( E. coli strains)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from livers of male rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0, 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

- S9: sodium azide (1 μg/plate, TA1535 and TA100); 9-aminoacridine (75 µg/plate, TA 1537); 2-nitrofluorene (1 µg/plate, TA98 and TA 1538); methylmethanesulfonate (1000 µg/plate, WP2 uvrA); +S9: 2-aminoanthracene (1 μg/pate, all strains)

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h
- Expression time (cells in growth medium): 48 to 72 h

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn wit a dissecting microscope

Evaluation criteria:

Revertant colonies were counted and the mean and standard deviation were calculated and compared to the controls.
All Salmonella tester strains must demonstrate the presence of the deep rough mutation and the deletion of the uvrA gene. Cultures of the TA98 and TA100 strains must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion of the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. Tester strain titers must be above 30.000.000 cells/ml. The mean of each positive control must be at least three-fold increased to the controlls. A minimum of three non-toxic dose levels are recquired to evaluate assay data.

Statistics:

Mean 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:

other: Unacceptable vehicle control values for the tester strain TA1537 and TA 98 were repeated.

Untreated negative controls validity:

not specified

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:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: >100 µg/plate

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:

other: all strains/cell types tested

Table 1: Test 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)
		Base-pair substitution type			Frameshift type
		TA 1535	TA1537	TA98	TA100	TA 1538	WP2uvrA
-	Vehicle	12	4	17	111	5	21
-	10	3	4	15	115	6	16
-	33	5	5	19	113	6	11
-	100	9	4	19	98	7	12
-	333	7	5	11	116	4	10
-	1000	8	6	21	120	7	13
Positive controls - S9	Name	SA	9AA	2NF	SA	2NF	MMS
	Concentrations (μg/plate)	1.0	75	1.0	1.0	1.0	1000
	Number of colonies/plate	241	40	105	377	179	143
+	Vehicle	14	5	18	133	7	11
+	10	9	5	27	114	12	16
+	33	9	3	21	113	8	13
+	100	8	7	26	108	9	13
+	333	9	4	27	115	6	8
+	1000	10	5	17	111	9	13
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1.0	1.0	1.0	1.0	1.0	10
	Number of colonies/plate	72	127	888	904	783	57

SA: sodium azide

9AA : 9-aminoacridine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

2NF: 2-nitrofluorene

 

 

Table 2: Test results of experiment 2/3:

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 1535	TA1537	TA98	TA100	TA 1538	WP2uvrA
-	Vehicle	14	10	23	126	11	27
-	10	7	13	16	117	7	27
-	33	9	15	23	124	8	19
-	100	5	13	22	120	6	18
-	333	12	9	17	110	11	16
-	1000	8	14	17	125	5	21
Positive controls - S9	Name	SA	9AA	2NF	SA	2NF	MMS
	Concentrations (μg/plate)	1.0	75	1.0	1.0	1.0	1000
	Number of colonies/plate	429	757	125	601	221	195
+	Vehicle	8	5	19	147	14	24
+	10	9	7	17	142	16	30
+	33	10	5	19	136	18	25
+	100	10	5	20	132	13	31
+	333	10	4	17	138	12	19
+	1000	10	7	19	125	12	19
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1.0	1.0	1.0	1.0	1.0	10
	Number of colonies/plate	85	97	530	647	1041	88

SA: sodium azide

9AA : 9 -aminoacridine

MMS: methylmethanesulfonate

2-AA: 2 -aminoanthracene

2NF: 2 -nitrofluorene

Conclusions:

Interpretation of results:
negative

Reference 3

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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

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 4

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:

other: Comparable to guideline study with acceptable restrictions (shorter exposure period. Lack of data on test substance, no positive controls for 40 h time point)

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.

Key result

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.

Remarks on result:

other: all strains/cell types tested

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

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no details on analytical purity given)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no analytical purity reported

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium) and trp operon (E. coli)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254

Test concentrations with justification for top dose:

Range-finding toxicity study (in TA 100 and WP2 uvrA): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Main study (all strains): 33.3, 100, 333, 1000, 3330 and 5000 µg/plate, with and without metabolic activation

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: 2-NF (1 µg/plate, TA 98); SA (2 µg/plate, TA 100 and TA 1535); ICR-191 (2 µg/plate, TA 1537); 4-NQO (1 µg/plate, WP2 uvrA); +S9: BP (2.5 µg/plate, TA 98); 2-AA (2.5-25 µg/plate, TA 100, TA 1535, TA 1537 and WP2 uvrA)

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene; ICR-191

Remarks:

2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn

Evaluation criteria:

The results of the test were considered positive, if the following criteria were met:
- tester strains TA 98, TA 100 and WP2 uvrA: for a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
- tester strains TA 1535 and TA 1537: for a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.

Statistics:

Mean values and standard deviations of revertants per plate 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, slight precipitation of the test substance was observed in all experiments at concentrations ≥ 100 µg/plate. In the absence of S9 mix, slight precipitates were noted at ≥ 333 µg/plate in the preliminary cytotoxicity study and at ≥ 1000 µg/plate in the main study.

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity study, the tester strains TA 100 and WP2 uvrA were treated with the test substance at concentrations ranging from 6.67 to 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). No cytotoxicity was observed in these strains up to the limit dose of 5000 µg/plate, neither with nor without addition of S9 mix.

Remarks on result:

other: all strains/cell types tested

Table 1. Test results of experiment (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)
EXPERIMENT
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC	25 ± 2	80 ± 9	14 ± 8	3 ± 1	26 ± 3
Test material
33.3 µg	19 ± 4	88 ± 5	12 ± 5	4 ± 4	19 ± 2
100 µg	21 ± 7	96 ± 12	11 ± 2	5 ± 4	19 ± 4
333 µg	22 ± 2	92 ± 9	13 ± 2	5 ± 5	23 ± 10
1000 µg	25 ± 6	97 ± 11	25 ± 2	5 ± 3	30 ± 3
3330 µg	22 ± 1	101 ± 3	10 ± 1	3 ± 2	32 ± 2
5000 µg	20 ± 6	85 ± 6	15 ± 3	4 ± 2	27 ± 3
PC
2-NF	206 ± 42	-	-	-	-
SA	-	549 ± 71	480 ± 3	-	-
ICR-191	-	-	-	277 ± 33	-
4-NQO	-	-	-	-	260 ± 43
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC	36 ± 2	102 ± 2	19 ± 1	7 ± 1	25 ± 5
Test material
33.3 µg	36 ± 7	93 ± 9	15 ± 2	8 ± 4	22 ± 5
100 µg	34 ± 7	97 ± 17	16 ± 4	9 ± 2	22 ± 6
333 µg	34 ± 6	87 ± 6	17 ± 2	9 ± 4	27 ± 3
1000 µg	39 ± 9	94 ± 17	17 ± 3	8 ± 2	26 ± 6
3330 µg	38 ± 8	92 ± 6	16 ± 4	6 ± 3	29 ± 11
5000 µg	34 ± 5	139 ± 5	20 ± 3	3 ± 2	24 ± 4
PC
BP	471 ± 14	-	-	-	-
2-AA	-	918 ± 296	124 ± 6	171 ± 32	278 ± 24
SC = Solvent control; PC = Positive control substances; SD = standard deviation; 2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Conclusions:

Interpretation of results:
negative

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 February 2004 to 27 February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine & trytophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation system
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.

Preparation of S9-fraction
The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were injected intraperitoneally with a solution (20% (w/v)) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).
Before use, all S9 batches were characterized with the metabolic activation requiring positive control; benzo(a)pyrene (Sigma) in tester strain TA98 at the concentration of 5 μg/plate.

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox) and 15.2 mg glucose--phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCI2 solution; 1 ml 0.33 M KCI solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction)
to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment. The S9-batch used was no. 03-11.

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with strain TA 100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Hatcol 5127 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Vehicle / solvent:

The test substance was dissolved in ethanol (Lichrosolv, Merck Eurolab B.V.). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle of the test article, being ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

methylmethanesulfonate

other: 2-aminoanthracene (2M)

Details on test system and experimental conditions:

Test System
Test System: Salmonella typhimurium bacteria and Escherichia coli bacteria
Source: Salmonella typhimurium strains: Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. TA98 received on 21-02-1991, used batch: TA98.120203; TA1535 received on 30-07-2 001, used batch: TA1535.120203; TA1537 received on 30-07-2001, used batch: TA1537.120203.
Xenometrc, Boulder, Co, U.S.A. (obtained from N.V. Organon). TA100 received on 19-09-2002, used batch: TA100.140B03. Prof. Dr. B.A. Bridges, University of Sussex, Brighton, U.K.
Escherichia coli strain: WP2uvrA received on 23-10-1987, used batch: EC.11 0703
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor – plasmid pKM101 (increase error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa: deep rough (defective lipopolysaccharide cellcoat)
gal: mutation in the galactose metabolism
chl: mutation in nitrate reductase
bio: defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicilin resistance (TA9B and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coliWP2uvrA strain detects base-pair substitutions. The strain lacks an
excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by
permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the
number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196 °C).

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking
incubator (37 °C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were
used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter:
18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Merck Eurolab B.V.). N.B.
The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck Eurolab B.V.) and 15 μg/plate histidine (Merck Eurolab B.V.) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) Sodium Chloride (Merck Eurolab B.V.) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3 °C.

Environmental conditions
All Incubations were carried out in the dark at 37 ± 1 °C. The temperature was monitored during the experiment.

Study design
Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix.
Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. This dose range finding test was reported as a part
of the first experiment of the mutation assay. The highest concentration of Hatcol 5150 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh
bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation
assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube
was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 h. After this
period revertant colonies (histidine independent (His +) for Salmonella typhimurium bacteria and tryptophan independent (Trp +) for Escherichia coli) were
counted.

Colony counting
The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain- at any concentration is not greater than two
times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the
number induced by the solvent control in any of the tester strains, either with or without metabolic
activation. However. any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final
evaluation decision.

Statistics:

No formal hypothesis testing was done.

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:

valid

True 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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test
Hatcol 5127 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33,100,333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Precipitate
The test substance precipitated in the top agar at concentrations of 100 µg/plate and upwards. Precipitation of Hatcol 5127 on the plates was observed at the start and at the end of the incubation period at concentrations of 333 µg/plate and upwards.
Toxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity
No increase in the number of revertants was observed upon treatment with HATCOL 5127 under all conditions test.

Mutation assay
Based on the results of the dose range finding test, Hatcol 5127 was tested up to concentrations of 333 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second µexperiment was performed with the strains TA 1535, TA 1537, TA98, TA 100 and WP2uvrA.
Precipitate
Hatcol 5127 precipitated in the top agar at concentrations of 100 and 333 µg/plate. Precipitation of Hatcol 5127 on the plates was observed at the start and at the end of the incubation period at the concentration 333 µg/plate.
Toxicity
ln both mutation assays, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity
No increase in the number of revertants was observed upon treatment with Hatcol 5127 under allconditions tested.

Experiment 1: Mutagenic response of Hatcol 5127 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (+/- S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	837 +/- 49	689 +/- 148	280 +/- 36	1103 +/- 58	608 +/- 101
Solvent control	10 +/- 4	8 +/- 2	23 +/- 2	129 +/- 13	10 +/- 1
3	12 +/- 3	8 +/- 4	22 +/- 3	133 +/- 8	8 +/- 3
10	13 +/-2	4 +/- 2	22 +/- 5	135 +/- 11	6 +/- 1
33	11 +/- 1	5 +/- 2	22 +/- 1	125 +/- 16	6 +/- 0
100	9 +/- 1	7 +/- 6	25 +/-	121 +/- 11	8 +/- 0
333^SP	12 +/- 5	7+/- 4	23 +/- 5	115 +/- 7	7 +/- 0
1000^SP				138 +/- 6	7 +/- 1
3330^SP				110 +/- 7	7 +/- 2
5000^SP				125 +/- 10	6 +/- 2
With S9-mix^1
Positive control	139 +/- 12	190 +/-13	688 +/- 45	756 +/- 59	214 +/- 14
Solvent control	13 +/- 1	8 +/- 4	33 +/- 1	121 +/- 3	10 +/- 2
3	11 +/- 5	6 +/- 4	26 +/- 9	125 +/- 2	11 +/- 2
10	13 +/- 5	8 +/- 2	30 +/- 2	116 +/- 17	6 +/- 3
33	13 +/- 4	6 +/- 2	28 +/- 4	125 +/- 5	9 +/- 2
100	14 +/- 5	6 +/- 2	33 +/- 3	115 +/- 2	10 +/- 3
333^SP	12 +/- 4	8 +/- 1	28 +/- 5	120 +/- 11	9 +/- 3
1000^SP				104 +/- 6	11 +/- 2
3330^SP				53 +/- 1	9 +/- 3
5000^SP				103 +/- 16	8 +/- 1

Solvent control: 0.1 ml ethanol

1: S9 mix contained 5% (v/v) S9 fraction

SP: Slight precipitate

 

Experiment 2: Mutagenic response of Hatcol 5127 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (+/- S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	981 +/- 22	435 +/-23	336 +/- 31	1221 +/-47	892 +/-23
Solvent control	8 +/- 1	5 +/-2	16 +/- 2	133 +/- 13	9 +/- 2
3	7 +/- 2	5 +/-2	17 +/-5	135 +/- 14	8 +/- 3
10	7 +/-1	6 +/-1	16 +/- 4	142 +/- 19	9 +/- 1
33	7 +/- 2	5 +/-2	18 +/- 5	138 +/- 11	9 +/- 3
100	10 +/- 1	4 +/- 0	16 +/- 4	137 +/- 21	7 +/- 3
333^SP	5 +/-1	4 +/- 1	15 +/- 3	128 +/-10	5 +/- 2
With S9-mix^1
Positive control	86 +/- 10	217 +/-24	585 +/- 44	721 +/- 48	95 +/-8
Solvent control	8 +/-2	5 +/- 2	22 +/- 4	108 +/- 6	7 +/- 1
3	6 +/- 2	6 +/- 1	22 +/- 3	112 +/- 6	10 +/- 3
10	11 +/-1	4 +/-1	19 +/- 2	118 +/- 12	9 +/- 5
33	8 +/- 2	4 +/-1	21 +/- 2	108 +/-13	6 +/- 3
100	6 +/- 2	4 +/-1	20 +/- 3	123 +/- 6	6 +/- 3
333^SP	7 +/- 2	7 +/-3	19 +/- 2	108 +/- 5	10 +/- 3

Solvent control: 0.1 ml ethanol

1: S9 mix contained 10% (v/v) S9 fraction

SP: Slight precipitate

 

Conclusions:

All bacterial strains showed negative responses aver the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertant in two independently repeated experiments . The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Hatcol 5127 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of Hatcol 5127 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

 

Hatcol 5127 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Araclor-1254 induced rat liver S9-mix).

 

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals Guideline no. 471 “ Genetic Toxicology: Bacterial Reverse Mutation Test” Adopted July 21, 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity ; B.13/14: "Mutagenicity: Reverse Mutation Test using bacteria". EEC Publication Commission Directive (Pub1ished June 8, 2000).

 

Batch D19628 of Hatcol 5127 was a light yellow liquid. The test substance was dissolved in ethanol.

 

In the dose range finding test, Hatco1 5127 was tested up to concentrations of 5000µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 5127 precipitated on the plates at dose levels of 333µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Based on the results of the dose range finding test, Hatcol 5127 was tested in the first mutation assay at a concentration range of 3 to 333µg/p1ate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA90. ln the second mutation assay, Hatcol 5127 was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Hatcol 5127 precipitated on the plates at the top dose of 333µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

 

Hatcol 5127 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific control values were within our laboratory historical control values indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Hatcol 5127 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative results obtained in an in vivo chromosome aberration study with Fatty acids, C5-10, esters with pentaerythritol (CAS 68424-31-7)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Evaluation of the mutagenic activity of Hatcol 5127 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

Hatcol 5127 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Araclor-1254 induced rat liver S9-mix).

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals Guideline no. 471 “ Genetic Toxicology: Bacterial Reverse Mutation Test” Adopted July 21, 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity ; B.13/14: "Mutagenicity: Reverse Mutation Test using bacteria". EEC Publication Commission Directive (Pub1ished June 8, 2000).

Batch D19628 of Hatcol 5127 was a light yellow liquid. The test substance was dissolved in ethanol.

In the dose range finding test, Hatcol 5127 was tested up to concentrations of 5000µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 5127 precipitated on the plates at dose levels of 333µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, Hatcol 5127 was tested in the first mutation assay at a concentration range of 3 to 333µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. ln the second mutation assay, Hatcol 5127 was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Hatcol 5127 precipitated on the plates at the top dose of 333µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

Hatcol 5127 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific control values were within our laboratory historical control values indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Hatcol 5127 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Read-across summaries:

CAS 11138-60-6

In an Ames test conducted with the Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol (CAS 11138-60-6), Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli WP2 uvr A were treated according to OECD Guideline 471 (Bailey, 1996). The test substance was diluted in ethanol and test substance concentrations of 0, 10, 33, 100, 333 and 1000 µg/plate were tested in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). Precipitation of the test substance was observed at and above 100 µg/plate. The test material caused no cytotoxicity up to the highest, precipitating dose. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

CAS 67762-53-2

The mutagenic potential of Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) was tested in a reverse mutation assay according to OECD Guideline 471 and under GLP conditions (Mecchi, 1999). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used. Tester strains were incubated with test material dissolved in ethanol at concentrations of 33.3, 100, 333, 1000, 3330 and 5000 µg/plate with and without the addition of a metabolic activation system (Aroclor 1254 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 negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C5-9, tetraesters with pentaerythritol did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

CAS 189200-42-8

The mutagenic potential of Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8) was tested in a reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Przygoda, 1995). The following Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 were used. Tester strains were incubated with the test material dissolved in acetone at concentrations of 0.5, 5, 50, 500, 5000 µg/plate in the first experiment and 50, 100, 500, 1000 and 5000 µg/plate in the repeat experiment with and without the addition of a metabolic activation system (Arochlor 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 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 but beading of the test substance occured in the initial assay and repeat assay at 500 µg/plate and above with and without metabolic activation in all strains. Thus, Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritoland tripentaerythritoldid 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 11138-60-6

An in vitro mammalian chromosome aberration test was performed with Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol (CAS 11138-60-6) in Chinese Hamster Ovary cells (CHO) according to OECD Guideline 473 and under GLP conditions (Gudi, 1996). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (Arochlor 1254 induced rat liver S9-mix). Cells were exposed for 4 and 20 hours without and for 4 hours with metabolic activation. The test substance was dissolved in ethanol and used at concentrations of 625, 1250, 2500, 5000 µg/mL. Cytotoxicity was observed at the highest dose tested regardless of metabolic activation. Mitomycin C and cyclophosphamide was used as positive control without and with metabolic activation respectively. Vehicle (solvent) controls induced aberration frequencies within the range expected. Positive control material induced 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 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.

CAS 189200-42-8

An in vitro mammalian chromosome aberration test was performed with Fatty acids C8-10, mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol (CAS 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD Guideline 473 and under GLP conditions (Przygody, 1995). 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 hours and for 16 hours followed by 16 hours 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 hours and for 16 hours followed by 16 hours expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 hours followed by 40 hours 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 hours 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

CAS 15834-04-5

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 15834-04-5) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). In the first experiment, the cells were treated for 3 hours 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, test 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 hours. The test substance was tested up to precipitating concentrations (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 2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

 

Genotoxicity in vivo

Fatty acids, C5-10, esters with pentraerythritol (CAS No. 68424-31-7) were found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application. 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, however, 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 (Griffiths, 1992).

Justification for classification or non-classification

Based on the information available for the substance and the information obtained from read-across, the substance is not expected to be mutagenic according to the criteria laid out by Regulation 1272/2008.