Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Test with the registered substance as well as RA from source substances (CAS 63705-03-3 and CAS 130905-60-1)

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

RA from source substances (CAS 49553-7-6)

Chromosome aberration (OECD 473): negative in V79 cells with and without metabolic activation

RA from source substances (CAS 63705-03-3 and CAS 130905-60-1)

Chromosome aberration (OECD 473): negative in human peripheral blood lymphocytes with and without metabolic activation

RA from source substance (CAS 49553-76-6)

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

RA from source substance (CAS 49553-76-6)

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

RA from source substance (CAS 63705-03-3)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May - 10 June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983 (2)

Deviations:

yes

Remarks:

S. typhimurium TA 1538 instead of E. coli or S. typhimurium TA102 tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated December 29, 1992

Deviations:

yes

Remarks:

S. typhimurium TA 1538 instead of E. coli or S. typhimurium TA102 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:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital or Naphthoflavone.

Test concentrations with justification for top dose:

1st experiment : 8; 40; 200; 1000 and 5000 µg/plate
2nd experiment: 20, 60, 200, 600 and 1800 µg/plate

Vehicle / solvent:

The test item was dissolved in DMSO and diluted with DMSO to the desired concentrations just before the start of the test. The solution medium was chosen according to the solubility properties tested preliminary before start of the study.

Untreated negative controls:

yes

Remarks:

Solution medium DMSO and the untreated fresh cell suspensions served as negative controls.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: sodium azide in strains TA 100 and TA 1535, 9-aminoacridine in strain TA 1537, 4-nitro-o-phenylendiamine in strains TA 98 and TA 1538 +S9: 2-aminoanthracene in 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: triplicate plates in both experiments

DETERMINATION OF CYTOTOXICITY
- Method: mean number of revertant colonies, inspection of bacterial background lawn

Evaluation criteria:

A combination of the following criteria was considered as a positive result:
- 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.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slightly toxic effects were noted at the concentration of 1000 µg/plate or higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: the mean revertant number of the vehicle and positive controls was within their respective historical ranges.

Table 1: Experiment 1:

single data of revertant rates without and with metabolic activation

without S9-Mix

strain			TA 100			TA	1535			TA 1537			TA 1538			TA 98
	µg/plate	1	2	3	1		2	3	1	2	3	1	2	3	1	2	3
neg. control		121	115	103	15	8		13	11	13	11	21	12	11	14	23	23
solvent		94	97	90	21	19		14	12	19	8	4	18	10	21	18	22
cone. 1	8	107	101	106	18	12		15	22	12	13	13	16	8	32	31	19
cone. 2	40	77	112	93	8	12		8	13	19	13	12	6	7	19	19	20
cone. 3	200	99	124	89	15	13		12	8	5	15	8	8	6	32	16	30
cone. 4	1000	74	89	110	13	16		8	7	12	8	2	1	4	27	23	19
cone. 5	5000	48	66	32	8	14		11	0	0	0	0	0	0	25	10	13
pos. control	A	887	919	910	585	692		708	858	1407	849	2442	2297	2468	1870	1572	1520
pos. control	B	118	92	158	19	16		15	8	11	15	22	21	16	37	32	21

 

with S9-Mix

strain			TA 100			TA	1535			TA	1537			TA 1538			TA 98
	µg/plate	1	2	3	1		2	3	1		2	3	1	2	3	1	2	3
neg. control		106	119	116	11	8		16	12	18		4	27	18	22	36	30	27
solvent		127	120	83	25	8		14	15	13		13	20	16	22	40	29	29
cone. 1	8	143	108	126	11	15		13	16	13		8	10	16	22	30	33	41
cone. 2	40	120	120	120	17	12		10	15	13		10	14	12	15	35	23	37
cone. 3	200	103	106	105	14	11		13	6	6		8	19	13	15	36	33	33
cone. 4	1000	103	73	108	17	7		10	15	10		14	13	12	8	37	27	35
cone. 5	5000	65	60	69	11	10		16	6	6		1	7	17	11	24	25	35
pos. control	A	232	260	268	207	157		191	472	643		712	2729	2610	2571	2347	2101	2075
pos. control	B	1811	1690	1811	244	274		292	387	321		324	1293	1365	1359	4031	3965	3962

Slightly toxic effects were noted at the concentration of 1000 µg/plate orhigher.

Table 2: Experiment 2:

single data of revertant rates without and with metabolic activation

without S9-Mix

strain			TA 100			TA	1535			TA 1537			TA 1538			TA 98
	µg/plate	1	2	3	1		2	3	1	2	3	1	2	3	1	2	3
neg. control		116	111	120	14	11		5	13	17	8	17	11	13	14	20	24
solvent		108	121	110	13	16		20	15	14	12	12	11	15	24	26	36
cone. 1	20	91	95	102	16	21		16	17	13	19	28	21	20	19	15	25
cone. 2	60	103	107	82	12	14		10	10	8	14	12	10	12	14	19	27
cone. 3	200	126	90	93	19	11		28	15	6	10	6	8	8	27	25	20
cone. 4	600	105	119	81	6	19		18	12	4	16	8	13	6	17	29	14
cone. 5	1800	65	66	67	10	10		15	2	4	5	3	5	3	22	23	22
pos. control	A	858	870	866	671	686		669	1100	1004	1805	1937	1874	1847	1492	1537	1340
pos. control	B	140	130	100	28	15		13	22	12	28	24	31	16	28	27	24

 

with S9-Mix

strain			TA 100			TA	1535			TA	1537			TA 1538			TA 98
	µg/plate	1	2	3	1		2	3	1		2	3	1	2	3	1	2	3
neg. control		122	122	115	8	8		17	10	17		14	12	14	15	19	28	39
solvent		125	98	106	8	21		18	11	22		11	8	23	15	31	37	32
cone. 1	20	125	122	111	16	12		11	10	11		19	27	14	16	35	35	28
cone. 2	60	99	107	113	16	12		20	17	7		15	11	17	18	33	33	37
cone. 3	200	110	121	88	10	12		4	13	22		6	15	14	12	40	32	21
cone. 4	600	97	97	87	17	12		12	7	7		17	5	13	8	30	24	38
cone. 5	1800	82	67	75	14	7		17	10	6		10	7	11	11	25	35	28
pos. control	A	285	330	297	239	276		236	509	855		865	2323	2515	2037	2104	1698	1598
pos. control	B	1534	1368	1644	299	338		326	375	323		344	1435	1501	1527	3782	3720	4105

 

Table 3: Summarizing table of experiment 1 and 2 (mean values and standard deviation (SO))

experiment 1

treatment	strains:
groups      µg/pl.	TA 100 -  (SO) +     (SO)	-	TA 1535 (SO)     +		(SO)	-	TA 1537 (SO)     +		(SO)	-	TA (SO)	1538 +	(SO)	-	TA (SO)	98 +	(SO)
medium	113  9  113     7	12	3	12	4	11	1	11	7	14	6	22	4	20	6	31	5
solvent	94  3  110    24	18	4	16	9	13	5	13	1	11	7	19	3	21	2	33	7
substance 8	105  3  126     18	15	3	13	2	16	6	12	4	12	4	16	6	27	7	35	6
40	94  18 120    0	10	2	13	4	15	4	12	3	8	3	13	2	19	1	32	7
200	104 18 105    2	13	2	12	2	10	5	7	1	8	1	16	3	26	9	34	2
1000	91  18  95    19	12	4	11	5	9	2	13	3	2	2	11	2	23	4	33	6
5000	48  17  65    4	11	3	12	3	0	0	5	3	0	0	12	5	16	8	28	6
positive          A	905 17 253    19	662 17	67 2	185 270	25 24	1038 11	320 3	609 344	123 37	2402 20	92 3	2636 1339	82 40	1654 30	189 8	2174 3986	150 39
controls         B   123 33 1771  70

 

experiment 2

treatment groups            µg/pl.	strains: TA 100 -  (SO)  +     (SO)	-	TA 1535 (SO)     +		(SO)	-	TA 1537 (SO) +		(SO)	-	TA (SO)	1538 +	(SO)	-	TA (SO)	98 +	(SO)
medium solvent	116  4     119     4 113  7     110     14	10 16	4 4	11 16	5 7	13 13	4 2	13 14	4 6	13 12	3 2	13 16	2 7	19 29	5 6	29 33	10 3
substance 20	96  5    119    7	18	3	13	3	16	3	13	5	23	4	19	7	20	5	33	4
60	97  14    106    7	12	2	16	4	11	3	13	5	11	1	15	4	20	7	34	2
200	103 20    106    17	19	8	8	4	10	4	14	8	8	1	13	2	24	3	31	10
600	101 19    93    6	15	7	13	3	11	6	11	6	9	3	9	4	20	8	31	7
1800	66  1    75    7	11	3	13	5	4	2	8	2	4	1	10	2	23	1	29	5
positive           A	865  6     304     23	676 18	9 8	250 321	22 20	1303 21	437 8	743 347	203 26	1886 24	46 7	2292 1487	241 47	1456 26	103 2	1800 3869	268 207
controls         B 123 21 1515 139

Conclusions:

Interpretation of results:
negative

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 - 12 Aug 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

S. typhimurium TA 1538 instead of E. coli or S. typhimurium TA102 tested; lack of data on test substance

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

S. typhimurium TA 1538 instead of E. coli or S. typhimurium TA102 tested; lack of data on test substance

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor or Luminal

Test concentrations with justification for top dose:

As none of the solvent specified for the Ames Test could be used, the product was tested directly and undiluted. Thus, it was not possible to indicate concentrations as µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: nitrofluorene (2.5 µg/plate) for TA 98 and 1538; sodium azide (2.5 µg/plate) for TA 100 and 1535; aminoacridine (50 µg/plate) for 1537; with S9: aminoanthracene (10 µg/plate) and endoxan (100 µg/plate) for TA 100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (spot-test)

DURATION
- Exposure duration: 96 h

NUMBER OF REPLICATIONS: 3 replications in 1 experiment

Statistics:

Mean values and standard deviation were calculated

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

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test item is insoluble

Strain: S. typhimurium TA 98

	Revertants per plate						Quotient
Dose/plate [µg/plate]	-S9	M	SD	+S9	M	SD	-S9	+S9
Water	33	34	6	62	46	14	1.0	1.0
	28			36
	40			39
Spot-Test	27	24	4	56	55	3	0.7	1.2
	20			52
	26			57
Nitrofluorene 2.5	192	217	22	-	-	-	6.4	-
	224			-
	235			-

-: not tested; M: mean; SD: standard-deviation; Reference: water; -S9: without S9 mix; +S9: with S9 mix; Conditions: arochlor induced +S9 mix

 

Strain: S. typhimurium TA 100

	Revertants per plate						Quotient
Dose/plate [µg/plate]	-S9	M	SD	+S9	M	SD	-S9	+S9
Water	136	142	13	203	185	17	1.0	1.0
	156			169
	133			183
Spot-Test	134	130	6	157	167	11	0.9	0.9
	132			179
	123			165
Na azide 2.5	515	524	11	-	-	-	3.7	-
	522			-
	536			-
Aminoanthracen 10	137	159	25	2812	2862	129	1.1	15.5
	154			2765
	187			3008

-: not tested; M: mean; SD: standard-deviation; Reference: water; -S9: without S9 mix; +S9: with S9 mix; Conditions: arochlor induced +S9 mix

 

Strain: S. typhimurium TA 1535

	Revertants per plate						Quotient
Dose/plate [µg/plate]	-S9	M	SD	+S9	M	SD	-S9	+S9
Water	23	21	5	13	15	2	1.0	1.0
	15			14
	25			17
Spot-Test	27	20	8	20	18	5	1.0	1.2
	22			12
	11			21
Na azide 2.5	476	473	11	-	-	-	22.5	-
	482			-
	460			-

-: not tested; M: mean; SD: standard-deviation; Reference: water; -S9: without S9 mix; +S9: with S9 mix; Conditions: arochlor induced +S9 mix

 

Strain: S. typhimurium TA 1537

	Revertants per plate						Quotient
Dose/plate [µg/plate]	-S9	M	SD	+S9	M	SD	-S9	+S9
Water	10	12	1	20	19	2	1.0	1.0
	12			20
	13			17
Spot-Test	12	9	3	29	28	8	0.7	1.5
	6			35
	8			19
Na azide 2.5	62	72	26	-	-	-	6.1	-
	101			-
	52			-

-: not tested; M: mean; SD: standard-deviation; Reference: water; -S9: without S9 mix; +S9: with S9 mix; Conditions: arochlor induced +S9 mix

 

Strain: S. typhimurium TA 1538

	Revertants per plate						Quotient
Dose/plate [µg/plate]	-S9	M	SD	+S9	M	SD	-S9	+S9
Water	29	27	4	46	47	1	1.0	1.0
	30			47
	23			47
Spot-Test	33	31	8	46	52	6	1.1	1.1
	38			55
	22			56
Nitrofluorene 2.5	139	136	6	-	-	-	5.0	-
	129			-
	140			-

-: not tested; M: mean; SD: standard-deviation; Reference: water; -S9: without S9 mix; +S9: with S9 mix; Conditions: arochlor induced +S9 mix

Conclusions:

Interpretation of results:
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb - May 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

(less metaphases scored (200 per concentration instead of 300))

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in May 1983

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in July 2016

Deviations:

yes

Remarks:

(less metaphases scored (200 per concentration instead of 300))

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in Dec 1992

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Engelhardt, BASF, Ludwigshafen, Germany
- Cell cycle length: approx. 13 h
- Methods for maintenance in cell culture if applicable: cultured at 37°C, 5% CO2, approx. 95% rel. humidity, subcultured 4 and 7 days after seeding the first time.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM3 (minimal essential medium) (Lot No. 22Q2655, Life Technologies, Germany), with 3% of fetal calf serum, 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin
- Properly maintained: yes

Cytokinesis block (if used):

yes, Colcemid treatment (0.2 µg/mL) added to the cultures 2 h prior to cell preparation

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 1: 40, 200, 400 µg/mL
Experiment 2: after 18 h: 40, 200, 400 µg/mL, after 28 h: 400 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not provided in the study report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM3 + 1% Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1x10^5 (18 h exposure); 5x10^4 (28 h exposure)

DURATION
- Exposure duration: 3 h (with S9 Mix, experiment 1+2), 16 h and 26 h (without S9 Mix, experiment 1+2)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h (experiment 1); 18 and 28 h (experiment 2)

SPINDLE INHIBITOR (cytogenetic assays): colcemide (0.2 µg/mL)

STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: duplicate cultures in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After fixation steps, cells were dropped onto microscopic slides and dried for approx. 24 h and stained with a giemsa solution.

NUMBER OF CELLS EVALUATED: at least 2000 (1000/slide)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): where possible 100 metaphases/culture (= 200 metaphases/experimental point)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:

The chosen cell line (V79) provides a convenient in vitro mammalian model which has been proposed in the literature and is well established in in vitro cytogenetics.

Evaluation criteria:

The assay was considered valid if the following criteria were met:

• The numbers of cells with chromosomal aberrations (excl. gaps) in the negative control had to be within the normal range (< 5%). The percentage of polyploid and endoreduplicated cells should be < 10%).
• Approx. 200 cells per treatment group (approx. 100 for each positive control) are analysable for chromosomal abberations.
• Treatment with positive controls (MMC and CP) in at least one concentration tested leads to clear increases in the frequency of cells with structurally altered chromosomes (>> 5% excl. gaps).
• There is acceptable homogeneity between replicate cultures as demonstrated by the binomial dispersion test (this criterion might not be met if treatment with a test chemical results in a positive response).

The test substance was considered as clastogenic if the following criteria were met:
• A statistically significant increase in the number of cells with chromosomal aberrations (excl. gaps) in one or more concentrations.
• The number of aberrant cells (excl. gaps) exceeded the normal range of the test system (>> 5%).
• The positive results could be varified in an independent experiment.

Increases in the proportion of cells with gaps or increases in the number of cells with structural abberations not exceeding the normal range are considered on a case by case basis. The possible influence of pH, S9 mix or osmolality on the occurance of chromosomal aberrations will also be considered.
As this assay is not designed to detect numerical aberrations, polyploidy and endoreduplications are reported when seen, but these data are not used for any kind of interpretation.

Statistics:

The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The possible influence of pH or osmolality on the occurance of chromosomal aberrations will also be considered.
- Water solubility: < 0.001 g/L
- Definition of acceptable cells for analysis: only intact cells with good choromosome morphology and without overlapping nuclei or debris and cells with 20-24 centromers were scored.

RANGE-FINDING/SCREENING STUDIES: The doses/concentrations were selected based on the results of a preliminary range finding test where 10 different concentrations (40 - 400 µg/mL) were tested in presence and absence of exogenous metabolic activation.

HISTORICAL CONTROL DATA: No historical control data were provided in the report.

Table 1: Summary of data obtained in chromosomal aberration test #1

	Dose [µg/mL]	Time [h]	S9 Mix	Mitrotic index [% mean]	Total # of mitotic cells scored	Proportion of cells with exchanges [%]	Proportion of cells with aberrations incl. gaps [%]	Proportion of cells with aberrations excl. gaps [%]	Significance
Negative control	0	20	-	9.0	200	0	1.5	0	no
Test substance	40			8.7		0.5	4.5	2.5	no
	200			10.8		1	3.5	2.5	no
	400			11.5		1	4	1	no
Positive control (MMC)	0.03			5.6		6.5	20	16.5	yes
Negative control	0	20	+	7.1	200	0.5	4.5	2	no
Test substance	40			8.6		0	2	0	no
	200			7.3		0	2.5	0.5	no
	400			6.8		0	1	0	no
Positive control (CP)	3			3.2		19.5	31	23.5	yes

Table 2: Summary of data obtained in chromosomal aberration test #2

	Dose [µg/mL]	Time [h]	S9 Mix	Mitrotic index [% mean]	Total # of mitotic cells scored	Proportion of cells with exchanges [%]	Proportion of cells with aberrations incl. gaps [%]	Proportion of cells with aberrations excl. gaps [%]	Significance
Negative control	0	20	-	10.7	200	0.5	1.5	0.5	no
Test substance	40			13.2		0.5	3.5	1.5	no
	200			11.3		0.5	2.5	0.5	no
	400			11		0	2.5	0.5	no
Positive control (MMC)	0.03			6.6		7.5	14.5	12.5	yes
Negative control	0	20	+	7.9	200	1.5	5	2.5	no
Test substance	40			8.3		0	2	0	no
	200			8.5		0.5	3	1	no
	400			8.4		1.5	4	2	no
Positive control (CP)	3			4.9		15.5	26.5	25	yes
Negative control	0	43	-	8.6	200	0	0.5	0	no
Test substance	400			9.3		0	1	0	no
Positive control (CP)	0.03			5.8		14	31.5	26.5	yes
Negative control	0	43	+	7.7	200	1	3.5	1.5	no
Test substance	400			7.7		0.5	2	0.5	no
Positive control (CP)	3			7.6		15.5	27	22.5	yes

MMC = mitomycin C

CP = cyclophosphamide

Conclusions:

In conclusion, the test item did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, it is considered not clastogenic in this test system.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 - 24 Sep 2012

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium) and trp operon (for 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:

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

Test concentrations with justification for top dose:

Preliminary cytotoxicity test (Experiment I): 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate with and without metabolic activation
Main assay (Experiment II): 333, 667, 1000, 3333, and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

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: BaP (2.5 µg/plate, TA 98); 2-AA (2.5 µg/plate, TA 100, TA 1535 and TA 1537; 25 µg/plate, WP2 uvrA)

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: acridine mutagen ICR-191 and 2-aminoanthracene

Remarks:

BaP: benzo[a]pyrene; 4-NQO: 4-nitroquinoline N-oxide; acridine mutagen ICR-191: ICR-191; SA: sodium azide; 2-aminoanthracene: 2-AA; 2-NF: 2-nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: duplicate plates in the preliminary cytotoxicity test and triplicate plates in the main assay

DETERMINATION OF CYTOTOXICITY
- Method: mean number of revertant colonies, inspection of bacterial background lawn

Evaluation criteria:

Criteria for a positive response:
- Strains TA1535 and TA1537: data were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate had to be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
- Strains TA98, TA100 and WP2uvrA: data sets were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate has to be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Species / strain:

S. typhimurium, other: TA 1535, TA1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate: TA 1537 (-S9) and TA 1535 (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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, test substance precipitation was observed starting at 3333 μg/plate for all tester strains with the exception of WP2 uvrA where precipitation was observed only at 5000 μg/plate. In the presence of S9 mix, precipitation was observed starting at 3333 μg/plate for all tester strains with the exception of TA 100 and WP2uvrA. Precipitation was not observed with WP2 uvrA in the activated condition and was only observed at 5000 μg/plate with tester strain TA 100. However, none of the precipitates prevented accurate colony counting.

RANGE-FINDING/SCREENING STUDIES: in the preliminary cytotoxicity test, a 50% reduction in revertant colonies was observed with TA 1537 in the presence of metabolic activation starting at 3333 µg/plate. No cytotoxicity was observed in any of the other tester strains after treatment with concentrations ranging from 33.3 to 5000 µg/plate in the presence and absence of metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: the mean revertant number of the vehicle and positive controls was within their respective historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: a >50% reduction in revertant colonies was observed at 5000 μg/plate for both TA1537 in the absence of S9 activation, as well as for TA1535 in the presence of S9 activation.

Table1. Test results of first experiment – preliminary cytotoxicity test

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=2 ± SD)
EXPERIMENT I (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (DMSO)	17 ± 1	111 ± 4	16 ± 0	5 ± 1	31 ± 1
Test item
33.3 µg	20 ± 4	98 ± 6	20 ± 2	14 ± 1	38 ± 7
66.7 µg	24 ± 6	110 ± 11	11 ± 3	10 ± 1	33 ± 8
100 µg	23 ± 6	101 ± 16	14 ± 7	3 ± 3	33 ± 4
333 µg	25 ± 5	91 ± 0	14 ± 4	5 ± 0	35 ± 4
667 µg	21 ± 4	84 ± 18	18 ± 4	4 ± 1	39 ± 6
1000 µg	28 ± 1	100 ± 6	9 ± 6	7 ± 0	38 ± 14
3333 µg	22 ± 4P	80 ± 4P	9 ± 6P	10 ± 7P	39 ± 8
5000 µg	23 ± 7P	89 ± 1P	18 ± 0P	6 ± 6P	26 ± 4P
PC
2-NF (1.0 µg)	143 ± 0	-	-	-	-
SA (2.0 µg)	-	1295 ± 50	1115 ± 115	-	-
ICR-191 (2.0 µg)	-	-	-	1440 ± 53	-
4-NQO (1.0 µg)	-	-	-	-	540 ± 13
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (acetone)	33 ± 8	139 ± 1	12 ± 2	15 ± 4	56 ± 2
Test item
33.3 µg	41 ± 9	132 ± 4	17 ± 1	11 ± 1	61 ± 4
66.7 µg	39 ± 6	135 ± 8	11 ± 3	10 ± 1	49 ± 5
100 µg	27 ± 4	140 ± 11	11 ± 1	13 ± 0	54 ± 8
333 µg	33 ± 4	116 ± 7	11 ± 1	12 ± 3	55 ± 8
667 µg	32 ± 4	73 ± 7	12 ± 2	11 ± 4	45 ± 4
1000 µg	34 ± 0	87 ± 20	11 ± 1	12 ± 6	49 ± 1
3333 µg	31 ± 6P	80 ± 9	10 ± 4P	5 ± 6P	47 ± 4
5000 µg	25 ± 1P	94 ± 16P	8 ± 2P	7 ± 1P	51 ± 8
PC
BP (2.5 µg)	485 ± 87	-	-	-	-
2-AA (2.5 µg)	-	2501 ± 137	196 ± 1	181 ± 45	-
2-AA (25 µg)	-	-	-	-	257 ± 8
SC = Solvent control; PC = Positive control substances; SD = standard deviation; BaP: benzo[a]pyrene; 4-NQO: 4-nitroquinoline N-oxide; acridine mutagen ICR-191: ICR-191; SA: sodium azide; 2-aminoanthracene: 2-AA; 2-NF: 2-nitrofluorene P = precipitate; M = manual counting necessary

Table 2. Test results of second experiment – Main assay

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± SD)
EXPERIMENT II (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (DMSO)	18 ± 2	86 ± 7	11 ± 8	8 ± 2	25 ± 3
Test item
333 µg	18 ± 3	74 ± 2	6 ± 4	5 ± 1	25 ± 5
667 µg	14 ± 3	74 ± 3	9 ± 3	4 ± 1	28 ± 6
1000 µg	16 ± 1	69 ± 2	8 ± 3	5 ± 1	24 ± 2
3333 µg	18 ± 5P	78 ± 16P	9 ± 3P	6 ± 3P	26 ± 4
5000 µg	19 ± 3P	74 ± 3P	6 ± 4P	3 ± 1P, M	26 ± 3P
PC
2-NF (1.0 µg)	150 ± 3	-	-	-	-
SA (2.0 µg)	-	1031 ± 19	956 ± 37	-	-
ICR-191 (2.0 µg)	-	-	-	1649 ± 123	-
4-NQO (1.0 µg)	-	-	-	-	456 ± 90
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (acetone)	28 ± 6	99 ± 11	15 ± 2	5 ± 1	33 ± 1
Test item
333 µg	27 ± 1	94 ± 24	8 ± 2	9 ± 2	32 ± 5
667 µg	23 ± 4	72 ± 5	7 ± 3	6 ± 1	29 ± 3
1000 µg	20 ± 4P	72 ± 13	8 ± 2	5 ± 4	29 ± 4
3333 µg	19 ± 2P	63 ± 6P	8 ± 3P	5 ± 3P	35 ± 9P
5000 µg	22 ± 6P	63 ± 7P	6 ± 4P	4 ± 2P	30 ± 10P
PC
BP (2.5 µg)	284 ± 12	-	-	-	-
2-AA (2.5 µg)	-	2337 ± 174	155 ± 5	119 ± 22	-
2-AA (25 µg)	-	-	-	-	200 ± 19
SC = Solvent control; PC = Positive control substances; SD = standard deviation; BaP: benzo[a]pyrene; 4-NQO: 4-nitroquinoline N-oxide; acridine mutagen ICR-191: ICR-191; SA: sodium azide; 2-aminoanthracene: 2-AA; 2-NF: 2-nitrofluorene P = precipitate

Conclusions:

Interpretation of results:
negative

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Aug - 12 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Target gene:

not applicable

Species / strain / cell type:

other: human peripheral blood lymphocytes (from healthy donor(s) less than 50 years old without previous chemotherapy or radiotherapy; and without recent (within the last 6 months) viral disease or X-ray exposure)

Details on mammalian cell type (if applicable):

- Type and identity of media: complete medium (RPMI 1640 medium containing approximately 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units penicillin/mL, and 100 μg streptomycin/mL) supplemented with 1-2% phytohemagglutinin-M (PHA-M)
- 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 treated with Aroclor 1254

Test concentrations with justification for top dose:

Without S9-mix, 4-h exposure: 25, 50, 100, 150, 200 µg/mL
With S9-mix, 4-h exposure: 50, 100, 200, 300, 400 µg/mL
Without S9-mix, 22-h exposure: 10, 25, 50, 75, 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The vehicle of choice for this study was DMSO, which permitted preparation of the highest workable/soluble stock concentration. Under the conditions of this test system, the final concentration of DMSO in the treatment medium did not exceed 1% of the treatment medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

mitomycin C (-S9): 0.2 µg/mL (4 and 22 h exposure period), cyclophosphamide (+S9): 10 µg/mL for the 4 h exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 µg/mL medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 100 from each duplicate culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

An assay was considered acceptable for evaluation of test results only if all of the following criteria were satisfied.
Negative Controls: The frequency of cells with structural chromosome aberrations was within the negative historical control range.
Positive Controls: The percentage of cells with structural chromosome aberrations were statistically significantly greater (p < 0.05, Fisher’s exact test) than the vehicle control response.

The following conditions were used as a guide to determine a positive response:
• A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response.
• The observed increased frequencies were accompanied by a concentration-related increase.
• A statistically significant increase was observed at the highest dose only.
• Note: Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.

The following condition was used as a guide to determine an equivocal response:
• Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.

The test substance was judged negative if the following condition was met:
• There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.

Statistics:

Data were evaluated using scientific judgment. Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Statistical analysis consisted of a Fisher’s exact test (with Bonferroni-Holm Adjustment) to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only on values within a test condition only if statistically significant values, based on the Fisher’s exact test, are found. At the discretion of the study director, statistical analyses may be conducted on the percentage of cells with numerical aberrations as well.

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 150 μg/mL in the 4-hour nonactivated test condition, at 100 μg/mL in the 22-hour non-activated test and in the 4-hour S9-activated test at 200 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: In the preliminary toxicity assays, osmolality and pH measurements were taken from 2 test substance concentrations (2500 and 5000 μg/mL) and the vehicle control media. The pH of the non-activated test system was 8.02 in the vehicle compared with 7.89 at 2500 μg/mL and 7.87 at 5000 μg/mL. The pH of the S9-activated test system was 7.50 in the vehicle compared with 7.37 at 2500 μg/mL and 7.34 at 5000 μg/mL. The osmolality of the nonactivated test system was 443 in the vehicle compared with 339 at 2500 μg/mL and 358 at 5000 μg/mL. The osmolality of the S9-activated test system was 462 in the vehicle compared with 418 at 2500 μg/mL and 384 at 5000 μg/mL. There were no observed increases in osmolality that were ≥ 20%, and therefore, not considered significant.
- Precipitation: Precipitation was observed in the 4-hour S9-activated test condition at the end of treatment only at 400 μg/mL.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the cells were treated for 4 and 22 hours in the non-activated test condition and for 4 hours in the S9-activated test condition. All cells were harvested 22 hours after treatment initiation. The cells were exposed to 9 concentrations of the test substance ranging from 25 to 5000 μg/mL, as well as a vehicle control. Precipitation was observed in the treatment media at ≥ 2500 μg/mL at the beginning of treatment in each test condition. In the 4-hour test conditions precipitation was observed at ≥ 500 μg/mL at the end of treatment while no precipitation was observed in the 22-hour test condition at the end of treatment. Substantial toxicity (greater than 50% reduction in mitotic index relative to the vehicle control) was observed at 250 μg/mL in the 4-hour non-activated test condition, at 500 μg/ml in the 4-hour S9-activated test condition and at 100 μg/mL in the 22-hour non-activated test condition. Based on the findings from the preliminary toxicity assay, the highest concentration chosen for the chromosome aberration assay was based on test substance induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical control data were given in the study report for the negative and positive controls of studies conducted from 2006 to 2011. The results for the positive and negative controls were within the historical control ranges given.

Table 1: Cytogenic analysis of cells treated with the test substance in the presence or absence of exogenous metabolic acitvation

				Cell scored		Aberrations per Cell		Cells with Aberrations#
Treatment (µg/mL)	S9 activation	Treatment time	Mitotic index (%)	Numerical	Structural	Mean	SD	Numerical (%)	Structural (%)
Vehicle	-	4	14.0	200	200	0.005	0.007	0.0	0.5
25	-	4	12.3	200	200	0.000	0.000	0.0	0.0
50	-	4	11.7	200	200	0.000	0.000	0.0	0.0
100	-	4	8.6	200	200	0.030	0.028	1.0	2.0
150	-	4	3.6	200	200	0.015	0.007	0.0	1.5
MMC 0.2	-	4	7.6	200	200	0.080	0.042	0.0	8.0*
Vehicle	+	4	11.6	200	200	0.010	0.014	0.0	1.0
50	+	4	11.0	200	200	0.005	0.007	1.5	0.5
100	+	4	10.0	200	200	0.010	0.014	0.0	1.0
200	+	4	6.3	200	200	0.020	0.014	0.0	2.0
CP 10	+	4	7.0	200	200	0.125	0.035	0.0	10.5*
Vehicle	-	22	8.0	200	200	0.025	0.007	0.0	2.5
25	-	22	6.5	200	200	0.000	0.000	0.0	0.0
50	-	22	5.2	200	200	0.005	0.007	0.0	0.5
100	-	22	2.6	200	200	0.010	0.014	0.0	1.0
MMC 0.2	-	22	5.1	200	200	0.135	0.007	0.0	12.5*

*: Statistically significant difference from control at p < 0.05 by Fisher's test.

#: Excluding cells with only gaps

Conclusions:

Interpretation of results:
negative

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 Aug - 08 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Office of Food Additive Safety, Redbook 2000

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: treatment medium: Fischer's Medium for Leukemic Cells of Mice with 0.1% Pluronics (F0P); restrictive medium for cleansing; washing medium: F0P supplemented with 10% horse serum, 2 mM L-glutamine, 100 U penicillin/mL and 100 μg streptomycin/mL (F10P); cloning medium: cloning medium (C.M.) containing 0.24% dissolved Noble agar in F0P plus 20% 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 Aroclor 1254

Test concentrations with justification for top dose:

Preliminary experiment: 0.5, 1.5, 5, 15, 50, 150, 1500 and 5000 µg/mL
- 4 h treatment: with and without metabolic activation
- 24 treatment: without metabolic activation

Main assay - concentrations used for treatment:
- Experiment I - 4 h treatment: 10 to 150 µg/mL with and without metabolic activation
- Experiment II -24 h treatment: 5 to 75 µg/mL without metabolic activation

Main assay - concentrations used for cloning:
- Experiment I - 4 h treatment: 10, 20, 30, 40 and 50 µg/mL with and without metabolic activation
- Experiment II -24 h treatment: 5, 10, 20, 30 and 40 µg/mL without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: methylmethanesulfonate, 15 and 20 µg/mL (first experiment) and 5 and 7.5 µg/mL (second experiment); +S9: 7,12-Dimethyl-benz(a)anthracene, 1.25 and 1.5 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 h (± S9); Experiment II: 24 h (-S9)
- Expression time (cells in growth medium): for expression of the mutant phenotype in the 4-hour exposure, the cultures were counted using an electronic cell counter and adjusted to 3E+5 cells/mL one and two days after treatment in 20 and 10 mL total volume, respectively. For the 24-hour exposure, cultures were adjusted to 3E+5 cells/mL in 20 mL immediately after test article removal, then two and three days after treatment in 20 and 10 mL total volume, respectively. For expression of the TK-/- cells, cells were placed in cloning medium (C.M.) containing 0.24% dissolved Noble agar in F0P plus 20% horse serum. Two flasks per culture to be cloned were labelled with the test article concentration, activation condition, and either TFT (trifluorothymidine, the selective agent) or VC (viable count). Each flask was filled with 100 mL C.M. and placed in an incubator shaker at 37 ± 1 °C until used. The cells were centrifuged at approximately 200-300 g for 10 minutes and the supernatant was decanted. The cells were then diluted in C.M. to concentrations of 3E+6 cells/100 mL C.M. for the TFT flask and 600 cells/100 mL C.M. for the VC flask. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 μg/mL) and both this flask and the VC flask were placed on the shaker at 125 rpm and 37 ± 1 °C. After 15 minutes, the flasks were removed and the cell suspension was dispensed equally into each of three appropriately labelled Petri dishes. To accelerate the gelling process, the plates were placed in cold storage (2-8 °C) for approximately 30 minutes. The plates were then incubated at 37 ± 1 °C in a humidified 5 ± 1% CO2 atmosphere for 10-14 days.
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-16 days

SELECTION AGENT (mutation assays): 3 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: suspension growth, relative suspension growth

OTHER EXAMINATIONS:
- Other: small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

OTHER: DETERMINATION OF OSMOLALITY: the osmolality of the solvent control and the highest soluble concentration in treatment medium were determined.

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency that occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data (Moore et al., 2006):
- a result was considered positive if a concentration-related increase in induced mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of ≥ 90 mutants per 1E+6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average solvent control mutant frequency was > 90 mutants per 1E+6 clonable cells, a doubling of mutant frequency over the background will also be required (Mitchell et al., 1997).
- a result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants per 1E+6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
- there are some situations in which a chemical would be considered negative when there was no culture showing between 10-20% survival: 1) There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 106) in a series of data points within 100% to 20% survival and there was at least one negative data point between 20% and 25% survival. 2) There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 1E+6) in a series of data points between 100% to 25% survival and there was also a negative data point between 10% and 1% survival (Office of Food Additive Safety, 2001). In this case it would be acceptable to count the TFT colonies of cultures exhibiting < 10% total growth.

Statistics:

The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 1E+6 surviving cells) for each treatment condition was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2 x 10-4) then multiplying by 1E+6. For simplicity, this was described as: (Average # TFT colonies / average # VC colonies) x 200 in the tables. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the solvent control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥ 90 mutants per 10E+6 clonable cells at the Aberdeen meeting in 2003, published in Moore et al., 2006.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9 mix: ≥ 30 µg/mL; +S9: ≥ 50 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: in the preliminary experiment, visible precipitate was present in the treatment medium at concentrations ≥ 500 μg/mL at the beginning of treatment, at concentrations ≥ 1500 μg/mL at the end of the 4-hour treatment period and at a concentration of 5000 μg/mL at the end of the 24-hour treatment period.

RANGE-FINDING/SCREENING STUDIES: in the preliminary range-finding test, overt cytotoxicity (relative suspension growth ≤ 10%) was observed after 4 h at ≥ 50 µg/mL with and without metabolic activation. The suspension growth relative to the solvent control was 0% at concentrations ≥ 150 μg/mL in the presence and absence of S9 activation after the 4-hour treatment and at concentrations ≥ 50 μg/mL in the absence of S9 activation after the 24-hour treatment. Based on the results of the toxicity test, the test article concentrations selected for the initial mutagenesis assay with a 4-hour treatment ranged from 10 to 150 μg/mL for the non-activated and S9-activated cultures. The test article concentrations in the extended treatment assay with a 24-hour treatment ranged from 5 to 75 μg/mL for the non-activated cultures.

COMPARISON WITH HISTORICAL CONTROL DATA: mutant frequencies of the negative and positive controls were within their respective historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the 4-h experiment, significant cytotoxicity was evident at ≥ 30 µg/mL in the absence of S9 mix and at 50 µg/mL in the presence of S9 mix. Exposure to the test substance for 24 h in the absence of S9 mix resulted in marked cytotoxicity at 40 µg/mL. Only little toxicity was noted at the other test concentrations with and without metabolic activation at the different exposure durations.

Table 1. Experiment I - 4 h exposure - With Metabolic Activation

Concentration [µg/mL]	Suspension growth [%]	Relative total growth [%]	Mutant frequency per 1E+06 surviving cells	IMF per 1E+06 surviving cells*
SC	100	100	42	N/A
SC			59
10	107	95	61	11
10	101	93	50	0
20	106	109	40	-10
20	102	94	55	4
30	107	96	59	9
30	95	99	44	-6
40	79	78	58	7
40	70	76	57	7
50	19	18	65	14
50	23	18	71	21
DMBA, 1.5	13	7	560	510
DMBA, 1.25	12	7	532	482

DMBA = 7,12-dimethylbenzanthracene; SC = solvent control (DMSO); IMF = Induced mutant frequency

* compared to a mean solvent mutant frequency of 50 per 10E+06 cells

Table 2. Experiment I - 3 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Suspension growth [%]	Relative total growth [%]	Mutant frequency per 1E+06 surviving cells	IMF per 1E+06 surviving cells*
SC	100	100	65	N/A
SC			50
10	103	89	72	14
10	104	93	49	-9
20	103	93	53	-5
20	96	88	44	-14
30	86	78	47	-11
30	57	48	57	-1
40	55	54	58	0
40	58	51	50	-8
50	20	18	45	-13
50	16	14	68	10
MMS, 20	43	7	1179	1122
MMS, 15	53	20	669	611

MMS = methylmethanesulfonate; SC = solvent control (DMSO); IMF = Induced mutant frequency

* compared to a mean solvent mutant frequency of 58 per 10E+06 cells

Table 3. Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Suspension growth [%]	Relative total growth [%]	Mutant frequency per 1E+06 surviving cells	IMF per 1E+06 surviving cells*
SC	100	100	43	N/A
SC			47
5	107	83	31	-4
5	100	72	31	-2
10	104	77	45	15
10	101	81	39	4
20	103	85	40	3
20	98	82	39	1
30	97	83	34	-5
30	97	79	38	1
40	30	27	49	9
40	33	28	59	21
MMS, 7.5	42	14	268	696
MMS, 5	52	27	256	394

MMS = methylmethanesulfonate; SC = solvent control (DMSO); IMF = Induced mutant frequency

* compared to a mean solvent mutant frequency of 40 per 10E+06 cells

RESULTS OF COLONY SIZING

The size of the colonies was not determined for test substance-treated cultures, since no positive results for mutagenicity were obtained. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Conclusions:

Interpretation of results:
negative

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no E. coli strain or equivalent S. typhimurium strain was tested; lack of data on test substance

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

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:

8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (2 µg/plate; TA 1535, TA 100); 9-Aminoacridine (80 µg/plate; TA 1537); 4-Nitro-o-phenylenediamine (40 µg/plate; TA 1538, TA 98); +S9: 2-Aminoanthracene (2.5 µg/plate for TA 1535, TA 1537; 5 µg/plate for TA 98, TA 100, TA 1538)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
For the test item to be considered mutagenic, two-fold (TA 100) or three-fold (all other strains) increases in mean revertant numbers must be observed at more than one dose level. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: The results show that no cytotoxicity was caused by the test substance.

Table 1: Test result of experiment 1

Bacterial Reverse Mutation Assay, mean revertants colonies/plate
EXPERIMENT 1
S9-Mix	Without
Test item (µg/plate)	TA98	TA 100	TA 1535	TA1537	TA 1538
Negative control	18.0 ± 3.5	102.3 ± 15.3	9.5 ± 2.4	5.0 ± 1.2	11.8 ± 3.2
8	13.6 ± 5.0	106.6 ± 13.0	7.3 ± 1.5	6.0 ± 2.0	9.6 ± 0.57
40	16.0 ± 3.0	99.0 ± 2.0	7.6 ± 3.5	7.0 ± 2.0	9.0 ± 1.0
200	21.0 ± 2.0	100.6 ± 8.0	10.3 ± 5.0	7.6 ± 3.5	11.6 ± 0.57
1000	20.6 ± 3.5	92.3 ± 3.2	7.6 ± 1.1	7.0 ± 1.7	8.0 ± 2.0
5000	20.0 ± 5.2	104.0 ± 11.0	8.6 ± 1.5	6.3 ± 2.5	10.6 ± 0.57
Sodium azide	---	492.0 ± 9.8	576.6 ± 31.6	---	---
9-Aminoacridine	---	---	---	372.0 ± 43.3	---
4-Nitro-o-phenylenediamine	807.0 ± 92.6	---	---	---	1970.6 ± 92.0
S9-Mix	With
Test item (µg/plate)	TA98	TA 100	TA 1535	TA1537	TA 1538
Negative control	25.0 ± 6.2	121.5 ± 11.6	8.5 ± 1.6	7.0 ± 2.0	19.8 ± 4.0
8	22.0 ± 1.7	118.3 ± 13.6	10.0 ± 3.6	6.0 ± 2.6	17.0 ± 4.0
40	19.3 ± 2.5	115.0 ± 3.0	10.3 ± 0.57	5.0 ± 2.6	15.6 ± 3.2
200	30.0 ± 0.0	122.6 ± 3.7	8.0 ± 0.0	5.3 ± 2.3	19.0 ± 8.0
1000	23.3 ± 2.0	117.3 ± 7.0	9.6 ± 2.3	10.0 ± 1.0	10.3 ± 2.0
5000	31.3 ± 8.0	126.3 ± 1.5	11.6 ± 3.7	6.0 ± 1.0	15.6 ± 1.5
2-Aminoanthracene	790.0 ± 2.6	1074.0 ± 39.8	80.3 ± 12.7	57.6 ± 3.2	1097.6 ± 82.0

Table 2: Test result of experiment 2

Bacterial Reverse Mutation Assay, mean revertants colonies/plate
EXPERIMENT 2
S9-Mix	Without
Test item (µg/plate)	TA98	TA 100	TA 1535	TA1537	TA 1538
Negative control	27.6 ± 2.5	97.1 ± 7.3	8.8 ± 2.6	8.6 ± 1.7	12.5 ± 3.7
8	21.3 ± 4.1	98.6 ± 3.0	6.3 ± 2.0	8.6 ± 0.57	11.0 ± 0.0
40	24.6 ± 12.0	111.0 ± 14.4	7.3 ± 0.57	9.0 ± 2.0	7.3 ± 3.2
200	22.6 ± 0.57	99.6 ± 0.57	8.3 ± 5.7	7.3 ± 5.0	9.3 ± 4.1
1000	26.3 ± 6.8	99.6 ± 15.9	8.6 ± 3.5	6.6 ± 0.57	10.3 ± 2.0
5000	17.3 ± 1.1	90.6 ± 4.0	4.6 ± 1.1	5.6 ± 0.57	10.6 ± 0.57
Sodium azide	---	537.6 ± 25.5	591.3 ± 32.3	---	---
9-Aminoacridine	---	---	---	578.0 ± 61.0	---
4-Nitro-o-phenylenediamine	1242.6 ± 30.2	---	---	---	2475.3 ± 75.0
S9-Mix	With
Test item (µg/plate)	TA98	TA 100	TA 1535	TA1537	TA 1538
Negative control	30.1 ± 4.1	104.0 ± 8.1	9.1 ± 3.0	6.0 ± 1.8	15.3 ± 1.8
8	32.3 ± 3.5	113.0 ± 1.7	7.6 ± 0.57	6.3 ± 3.0	18.0 ± 2.6
40	31.3 ± 7.5	103.6 ± 4.0	8.6 ± 2.3	11.0 ± 2.6	18.3 ± 4.0
200	30.3 ± 0.57	115.3 ± 10.4	7.6 ± 2.3	6.6 ± 3.2	13.0 ± 3.4
1000	31.0 ± 1.0	101.6 ± 2.5	8.0 ± 1.7	8.6 ± 2.0	15.3 ± 1.1
5000	26.6 ± 1.5	108.0 ± 5.5	6.3 ± 1.1	9.3 ± 3.0	14.0 ± 5.0
2-Aminoanthracene	755.0 ± 205.6	878.0 ± 81.2	101.0 ± 2.0	52.0 ± 14.1	1343.0 ± 80.7

Conclusions:

Interpretation of results:
negative

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Str. 7, 55116 Mainz

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 µg/mL) and 1% (v/v) amphotericin B (250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

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 1: 3.13, 6.25, 12.5, 25, 50, 100, 200 µg/mL
Experiment 2 and 3: 12.5, 25, 50, 100, 200, 400, 800 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and ethanol, dimethyl sulfoxide (DMSO) was selected as most suitable vehicle, which has been demonstrated to be suitable in in vitro genotoxicity studies and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: ethylmethanesulfonate (500 µg/mL), +S9: cyclophosphamide (0.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 h

SPINDLE INHIBITOR (cytogenetic assays): colcemide (100 µL; stock solution: 10 µg/mL)
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol

NUMBER OF REPLICATIONS: duplicate cultures in three independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphases (Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The V79 in vitro cytogenetic assay was considered valid if the following criteria were met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
• The numbers of cells with structural/numerical aberrations in the negative control had to be within the range of the historical negative control data.
• The positive control substances both with and without S9 mix had to induce a distinct increase of structural chromosome aberrations.

The test substance was considered as “positive” if the following criteria were met:
• A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeded both the concurrent negative/vehicle control value and the historical negative control data range.

A test substance generally was considered as “negative” if the following criteria were met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups was not statistically significant increased above the concurrent negative/vehicle control value and was within the historical negative control data range.

Statistics:

The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 2nd Experiment at 800 μg/mL and in the 3rd Experiment from 200 μg/mL onward at 18 hours sampling time and from 400 μg/mL onward at 28 hours sampling time

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the vehicle control with and without S9 mix. The pH values were not influenced by test substance treatment.
- Effects of osmolality: Osmolarity was measured, at least for the top dose and for the vehicle control with and without S9 mix. Osmolarity was not influenced by test substance treatment.
- Water solubility: The test substance was found to be insoluble in water.
- Precipitation: Precipitation in culture medium (macroscopic) at the end of exposure period was observed from about 50 μg/mL onward.

RANGE-FINDING/SCREENING STUDIES: The doses/concentrations were selected based on the results of a preliminary range finding test of an in vitro Gene Mutation Test in CHO cells (see BASF project No. 50M0401/12M232).

COMPARISON WITH HISTORICAL CONTROL DATA: The structural chromosome aberration rates of the vehicle and positive control groups were within the historical control data range and, thus, fulfilled the acceptance criteria of this study.

Table 1: Summary table - experimental parts without S9 mix

					Genotoxicity				Cytotoxicity
					Aberrant cells [%]
Exp.	Exposure/ preparation period	Test groups [µg/mL]	S9 mix	P	incl. gaps#	ecl. gaps#	with exchanges	Polyploid cells [%]	Cell number [%]	Mitotic index [%]
2	4/18 hrs	Vehicle control²	-	-	5.0	2.5	1.5	0.0	100.0	100.0
		12.50	-	-	n.d.	n.d.	n.d.	n.d.	94.4	n.d.
		25.00	-	-	n.d.	n.d.	n.d.	n.d.	103.9	n.d.
		50.00	-	-	4.0	1.5	0.5	0.0	111.2	97.4
		100.00	-	+	6.0	2.0	1.0	0.0	114.8	97.4
		200.00	-	+	n.d.	n.d.	n.d.	n.d.	112.0	n.d.
		400.00	-	+	6.0	3.0	2.5	0.0	92.4	111.5
		800.00	-	+	n.s.	n.s.	n.s.	n.s.	80.4	n.s.
		Positive control³~	-	-	28.0*	21.0*	13.0*	0.0	n.t.	93.7
3	18/18 hrs	Vehicle control²	-	-	3.5	1.5	1.0	0.0	100.0	100.0
		12.50	-	-	n.d.	n.d.	n.d.	n.d.	98.0	n.d.
		25.00	-	-	2.0	1.0	1.0	0.0	96.9	89.8
		50.00	-	-	5.0	2.0	0.5	0.0	95.8	108.9
		100.00	-	+	4.0	2.0	0.5	0.0	100.4	70.7
		200.00	-	+	n.s.	n.s.	n.s.	n.s.	97.8	n.s.
		400.00	-	+	n.s.	n.s.	n.s.	n.s.	53.4	n.s.
		800.00	-	+	n.s.	n.s.	n.s.	n.s.	27.0	n.s.
		Positive control³	-	-	27.0*	26.0*	23.0*	0.0	n.t.	77.3
3	18/28 hrs	Vehicle control²~	-	-	3.5	1.5	0.5	0.0	100.0	100.0
		12.50	-	-	n.d.	n.d.	n.d.	n.d.	94.9	n.d.
		25.00	-	-	n.d.	n.d.	n.d.	n.d.	93.5	n.d.
		50.00	-	-	n.d.	n.d.	n.d.	n.d.	91.2	n.d.
		100.00	-	-	n.d.	n.d.	n.d.	n.d.	76.2	n.d.
		200.00	-	-	3.5	2.0	1.0	0.0	66.1	74.0
		400.00	-	+	n.s.	n.s.	n.s.	n.s.	24.2	n.s.
		800.00	-	+	n.s.	n.s.	n.s.	n.s.	7.0	n.s.
		Positive control³~	-	-	31.0*	30.0*	27.0*	0.0	n.t.	71.0

#: Inclusive cells carrying exchanges

²: DMSO 1% (v/v)

³: EMS 500 μg/mL

~: Evaluation of a sample of 100 metaphase only due to strong clastogenicity

*: Aberration frequency statistically significant higher than corresponding control values

n.d.: Not determined

n.s.: Not scorable due to poor metaphase quality / strong cytotoxicity

n.t.: Not tested

Table 2: Summary table - experimental parts with S9 mix

					Genotoxicity				Cytotoxicity
					Aberrant cells [%]
Exp.	Exposure/ preparation period	Test groups [µg/mL]	S9 mix	P	incl. gaps#	ecl. gaps#	with exchanges	Polyploid cells [%]	Cell number [%]	Mitotic index [%]
1	4/18 hrs	Vehicle control²	+	-	4.5	3.0	1.5	0.0	100.0	100.0
		3.13	+	-	n.d.	n.d.	n.d.	n.d.	110.3	n.d.
		6.25	+	-	n.d.	n.d.	n.d.	n.d.	119.9	n.d.
		12.50	+	-	n.d.	n.d.	n.d.	n.d.	111.2	n.d.
		25.00	+	-	4.0	1.0	0.0	0.0	121.3	69.1
		50.00	+	+	5.5	2.0	1.0	0.0	117.2	95.7
		100.00	+	+	4.0	2.5	2.0	0.0	98.4	102.3
		200.00	+	+	n.s.	n.s.	n.s.	n.s.	80.4	n.s.
		Positive control³~	+	-	22.0*	21.0*	16.0*	0.0	n.t.	78.1
3	4/28 hrs	Vehicle control²	+	-	2.5	0.5	0.5	0.0	100.0	100.0
		12.50	+	-	3.5	1.0	0.5	0.0	104.5	123.4
		25.00	+	-	2.5	0.0	0.0	0.0	91.1	70.5
		50.00	+	-	8.0	3.5	2.0	0.0	103.3	71.3
		100.00	+	+	n.s.	n.s.	n.s.	n.s.	56.3	n.s.
		200.00	+	+	n.s.	n.s.	n.s.	n.s.	17.0	n.s.
		400.00	+	+	n.s.	n.s.	n.s.	n.s.	2.6	n.s.
		800.00	+	+	n.s.	n.s.	n.s.	n.s.	3.0	n.s.
		Positive control³	+	-	20.0*	19.0*	6.0*	0.0	n.t.	124.2

#: Inclusive cells carrying exchanges

²: DMSO 1% (v/v)

³: EMS 500 μg/mL

~: Evaluation of a sample of 100 metaphase only due to strong clastogenicity

*: Aberration frequency statistically significant higher than corresponding control values

n.d.: Not determined

n.s.: Not scorable due to poor metaphase quality / strong cytotoxicity

n.t.: Not tested

Conclusions:

Interpretation of results:
negative

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Str. 7, 55116 Mainz

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
- 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 Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (-/+ S9): 3.13, 6.25, 12.50, 25, 50, 100 µg/mL
Experiment 2 (- S9): 9.38, 18.75, 37.50, 75, 150, 200 µg/mL
Experiment 2 (+ S9): 4.69, 9.38, 18.75, 37.50, 75, 150 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Insolubility in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 300 μg/mL ethyl methanesulfonate (EMS); +S9: 1.25 μg/mL 7.12-Dimethylbenz[a]anthracene (DMBA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): on day 13 to 16, depending on selection period

SELECTION AGENT (mutation assays): 10 µg/mL 6-thioguanine
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate cultures in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (pre-experiment); survival; viability

Evaluation criteria:

The HPRT assay was considered valid if the following criteria were met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range of 0.00 – 19.54 mutants per 106 clonable cells.
• The positive controls both with and without S9 mix had to induce distinctly increased mutant frequencies.
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances were not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding was assessed as positive if the following criteria were met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance was considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups was not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend was judged as statistically significant whenever the p-value (probability value) was below 0.10 and the slope was greater than 0. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1st Experiment, 4-h exposure: -S9 at 100 µg/mL and +S9 at 50 µg/mL onward; 2nd Experiment, 4-h exposure -S9 at 150 µg/mL onward and +S9 at 75 µg/mL onward

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Changes in pH were recorded by a change in the indicator color of the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the vehicle controls with and without S9 mix. The pH values were not influenced by test substance treatment.
- Effects of osmolality: Osmolarity was measured in at least the top dose and the vehicle controls with and without S9 mix. Osmolarity was not influenced by test substance treatment.
- Water solubility: The test substance was found to be insoluble in water.
- Precipitation: Precipitation was observed in culture medium 3 – 4 hours after start of treatment from about 25.0 μg/mL onward in the 1st Experiment and from about 75.0 μg/mL onward in the 2nd Experiment.

RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity, concentrations of 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL were used both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. The 24 hours exposure time was performed additionally to obtain data for an the vitro chromosome
aberration assay in V79 cells (32M0401/12M225). The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses.
In the pretest the parameters pH value and osmolarity were not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in the vehicle dimethyl sulfoxide (DMSO) was not observed in the stock solution (Test group: 5 000 μg/mL) and at the first dilution in the vehicle (2 500 μg/mL). At intermediate concentrations from 1250.0 μg/mL to 39.1 μg/mL
homogeneous, milky suspensions were obtained. At 19.5 μg/mL no precipitation of the test substance in the vehicle DMSO was observed. In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 39.1 μg/mL and above in the absence and presence of S9 mix after 4 hours treatment and at 78.1 μg/mL and above without S9 mix after continuous treatment.
After 4 and 24 hours treatment with the test substance, cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival from 78.1 μg/mL to 2 500.0 μg/mL in the absence of S9 mix and from 78.1 μg/mL to 1 250.0 μg/mL in the presence of S9 mix. Higher concentrations were not cytotoxic, due to strong precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutation frequencies of the vehicle and control groups were within the historical control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

Table 1: Summary of results

						Cytotoxicity***
Experiment	Exposure period [h]	Test groups [µg/mL]	S9 mix	Precipitation*	Genotoxicity** MFcorr. [per 10^6cells]	CE1 [%]	CE2 [%]
1	4	Vehicle Control³	-	-	2.35	100.0	100.0
		3.13	-	-	n.c.^1	104.4	n.c.^1
		6.25	-	-	0.35	89.1	98.4
		12.50	-	-	0.34	85.8	102.6
		25.00	-	+	0.65	74.0	105.1
		50.00	-	+	5.12	73.7	99.3
		100.00	-	+	1.35	27.5	111.3
		Positive Control²	-	-	89.65	103.5	98.4
2	4	Vehicle Control^1	-	-	0.32	100.0	100.0
		9.38	-	-	0.00	105.8	102.0
		18.75	-	-	1.52	95.6	105.9
		37.50	-	-	4.39	84.8	101.1
		75.00	-	+	2.24	80.4	97.5
		150.00	-	+	0.58	10.3	101.7
		200.00	-	+	n.c.^²	1.2	n.c.^²
		Positive Control²	-	-	92.06	105.5	99.2
1	4	Vehicle Control³	+	-	0.32	100.0	100.0
		3.13	+	-	n.c.^1	90.8	n.c.^1
		6.25	+	-	0.33	90.6	94.3
		12.50	+	-	0.32	86.4	102.4
		25.00	+	+	0.78	83.2	94.2
		50.00	+	+	0.70	36.2	91.6
		100.00	+	+	n.c.^²	0.0	n.c.^²
		Positive Control²	+	-	366.23	83.0	72.9
2	4	Vehicle Control³	+	-	0.38	100.0	100.0
		4.69	+	-	0.70	106.4	109.9
		9.38	+	-	0.69	106.1	105.9
		18.75	+	-	0.00	116.8	92.1
		37.50	+	-	2.26	67.0	103.5
		75.00	+	+	n.c.^²	0.0	n.c.^²
		150.00	+	+	n.c.^²	0.0	n.c.^²
		Positive Control²	+	-	518.56	54.0	76.6

*: Precipitation in culture medium at the end of exposure period

**: Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

***: Cloning efficiency related to the respective vehicle control

n.c.¹ : Culture was not continued since a minimum of only four analysable concentrations are required

n.c.²: Culture was not continued due to strong cytotoxicity

²: EMS 300 μg/mL (+S9), DMBA 1.25 μg/mL (-S9)

³: DMSO 1% (v/v)

Conclusions:

Interpretation of results:
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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

RA from source substance (CAS 130905-60-1)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 - 26 Sept 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

no version/adoption date specified

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Version / remarks:

19 Sept 1984

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

other: BOR:NMRI

Details on species / strain selection:

substrain SPF (Han.)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frima Winkelmann, Versuchtierzucht, Gartenstr. 30, Borchen, Germany
- Age at study initiation: adult, about 3 months
- Weight at study initiation: males 22.3 - 25.0 g / females 20.7 - 24.3 g
- Assigned to test groups randomly: yes
- Housing: collective housing
- Cage type: Macrolon type II / max. 5 animals/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 30 ml/kg bw

Details on exposure:

In a preliminare dose-range finding experiment, doses of 15,000, 10,000, 5,000 and 2,000 mg/kg bw were administered orally in a single application to 2 male and 2 female mice. A dose of 15.000 mg/kg was considered to be near the maximal tolerated dose. Based on the results of the dose-range fmding test, the test article was administered in a dose of 15,000 mg/kg bw in the main test. Negative control animals received only the vehicle (corn oil), whilst positive control animals were treated with Cyclophosphamide.

In each case a single oral administration in a volume of 30 ml/kg body weight (10 ml;kg for the positive control) was made. 5 male and 5 female animals were used in each group.prepared in corn oil.

PREPARATION OF DOSING SOLUTIONS: The test solution was prepared by suspending an appropriate amount of the prewarmed test article (70°C) at a volume of 50% in corn oil.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

24 h, 48 h, and 72 h

Dose / conc.:

15 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Neg. control (24 h): 5m, 5f
Neg. control (48 h): 5m, 5f
Neg. control (72 h): 5m, 5f
Pos. control (24 h): 5m, 5f
Treatment (24 h): 5m, 5f
Treatment (48 h): 5m, 5f
Treatment (72 h): 5m, 5f

a total of 35m and 35f

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (trade name: Endoxan)
- Route of administration: oal
- Doses / concentrations: 40 mg/kg bw
- Batch no.: 078487
- Vehicle for positive control: aqua bidest.

Tissues and cell types examined:

Monochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: On the basis of the dose-range finding results, a dose of 15,000 mg/kg body weight was considered to be near the MTD (maximal tolerated dose) and was therefore chosen for the main study.

TREATMENT AND SAMPLING TIMES: 24 h, 48 h, and 72 h

DETAILS OF SLIDE PREPARATION:
After scrifice, femora were removed and the bone marrow was suspended in feta! calf serum. Samples were centrifuged at 1600 x g and subsequently decanted. One drop of each suspension was then smeared on a slide by means of a second slide. Two preparations were made from each animal, dried, fixed in absolute methanol (99%) for 5 min and then allowed to dry in air. Slides were stained with a May-Grunwald and Giemsa solution.

METHOD OF ANALYSIS:
The cells were examined under a microscope at thousandfold magnification. A total of 1000 polychromatic erythrocytes were examined on each slide and the number of micronucleated cells in each sample was recorded. The ratio of polychromatic erythrocytes to normochromatic (mature) erythrocytes was calculated for a sample of 1000 cells.

Evaluation criteria:

Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point. Percentage of polychromatic erythrocytes. Cell counts are based on a total of 1000 cells per animal. Historical control data of the laboratory indicate that an incidence of up to 8 micronucleated cells per 1000 polychromatic erythrocytes may be considered to be within normal limits.

Statistics:

In each test group and the corresponding negative control group, the number of polychromatic erythrocytes with micronuclei, the number of polychromatic erythrocytes, the number of normochromatic erythrocytes and the ratio of poly- to normochromatic erythrocytes were analysed statistically with the t-test for independent samples. Mean values of the negative and positive control groups were compared with the Mann-Whimey U-Test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Dose tested was near MTD (max. tolerable dose), causing reduced activity and piloerection

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

CLINICAL OBSERVATIONS
Clinical findings in all animals treated with a single dose of 15,000 mg/kg bw of the test article were piloerection and reduced activity. No animals died in the main study.

BONE MARROW INVESTIGATION
The number of polychromatic erythrocytes with micronuclei was significantly increased 24 h post-injection in the positive control animals. This result confirms the high sensitivity of this particular strain of mice.

In relation to the untreated controls, the number of polychromatic erythrocytes with micronuclei was not increased in any test group (24, 48, 72 h p.a.).

In none of the parameters investigated, a significant difference was found between female animals treated with the test article and negative control animals. The number of polychromatic erythrocytes without micronuclei in group males (48 h p.a.) as well as the ratio of polychromatic to normochromatic erythrocytes was significantly increased. The same parameters were decreased in group males (72 h p.a.). These effects might be explained by a stimulation of the formation of polychromatic erythrocytes in male mice after 48h by the test article resulting in a subsequent increase of normochromatic erythrocytes, which arise from polychromatic erythrocytes, after 72h.

In conclusion, the test article is considered to be non-mutagenic under the experimental conditions of this study.

Please refer to pdf document attached for detailed tables.

Conclusions:

Interpretation of results:
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read-across

While an investigation on gene mutation in bacteria is available for the target substance, no data on other genotoxitcy endpoints are available. Hence, read across from the relevant source substances 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS 63705-03-3) and Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS 130905 -60-1) was applied to evaluate genetic toxicity.

Read-across from appropriate reference substances is conducted in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. in order to fulfil the standard information requirements defined in Regulation (EC) No. 1907/2006, Annex VIII, 8.6. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

In vitro gene mutation in bacteria

CAS 92044 -91 -2

Data on gene mutation in bacteria are available for both the registered substance as well as adequate structural analogue source substances. The mutagenic potential of Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters (CAS 92044-91-2) was tested in a bacterial reverse mutation study according to OECD TG 471 and under GLP conditions (Emery, 1999). Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were tested. Two independent experiments were carried out. In both experiments, the tester strains were exposed to solutions of the test substance in DMSO. Concentrations used in the first experiment were 8, 40, 200, 1000 and 5000 µg/plate and those in the second experiment were 20, 60, 200, 600 and 1800 µg/plate. Metabolic activation was simulated by a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers treated with Phenobarbital or Naphthoflavone. Appropriate vehicle, negative and positive controls were also included in the study design. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls indicating the satisfactory performance of the test and the activity of the metabolising system. There was no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Slightly toxic effects were noted at the concentration of 1000 µg/plate or higher. The test substance did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

CAS 49553 -76 -6

The result of the key study is further supported by reliable in vitro studies using various analogue source substances. A bacterial gene mutation assay was performed with Oleic acid, monoester with oxybis(propanediol), (CAS 49553-76-6) according to OECD TG 471 and under GLP conditions (DuPont, 2012). The plate incorporation procedure was performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2uver A strains in the absence and presence of metabolic activation (Aroclor 1254-induced rat liver S9-mix). A preliminary cytotoxicity test and a main assay were conducted each in triplicates at concentrations from 33.3 to 5000 µg/plate and 333 to 5000 µg/plate respectively (vehicle: DMSO). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results. Under the study conditions, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

CAS 63705 -03 -3

1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS 63705-03-3) was tested for its mutagenic potential in a bacterial gene mutation assay performed similar to OECD TG 471 and observing GLP conditions (BASF, 1988). Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were tested in two independent experiments using the plate incorporation method and concentrations of 8, 40, 200, 1000 and 5000 µg/plate. Tween 80/bidistilled water was used as vehicle. Experiments were performed in the presence and the absence of a metabolic activation system (Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254). No cytotoxicity was observed and no increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Vehicle and positive controls showed the expected results, demonstrating the validity of the experiments and the activity of the metabolising system. Under the experimental conditions applied, the test substance was found not to induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

CAS 130905 -60 -1

In a bacterial gene mutation assay similar to OECD TG 471 and complying to GLP provisions, the mutagenic potential of Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS 130905-60-1) was investigated. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the indiluted test substance with and without a metabolising system ( cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor or Luminal). As none of the solvents specified for the Ames test could be used, the substance was tested directly and undiluted. Thus, it was not possible to indicate concentrations as µg/plate. The test substance caused no cytotoxicity. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, either with or without metabolic activation. All positive and negative controls yielded the expected results, thus indicating the validity of the test and the activity of the metabolising system. The test substance was considered to be not mutagenic under the experimental conditions applied in this Ames test.

In vitro chromosome aberration in mammalian cells

CAS 49553 -76 -6

An in vitro mammalian chromosome aberration test was performed with Oleic acid, monoester with oxybis(propanediol) (CAS 49553-76-6) in cultured peripheral human lymphocytes according to OECD Guideline 473 and under GLP conditions (DuPont, 2012). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). Cells were exposed for 4 hours to the test substance dissolved in DMSO at concentrations of 25, 50, 100, 150, 200 µg/mL without metabolic activation and for 4 hours at concentration of 50, 100, 200, 300 and 400 µg/mL with metabolic activation and for 22 hours without metabolic activation at concentration of 10, 25, 50, 75 and 100 µg/mL. The test substance induced cytotoxicity at 150 μg/mL in the 4-hour non activated test condition and at 100 μg/mL in the 22-hour non-activated test and in the 4-hour S9-activated test at 200 µg/mL and above. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Mitomycin C and Cyclophosphamide were used as positive control substances inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolising 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 human lymphocytes in vitro.

CAS 63705 -03 -3

A chromosome aberration test was conducted with the test substance according to OECD TG 473 and under GLP conditions with 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS 63705-03-3) in chinese hamster lung fibroblasts (V79). Cultures of V79 cells, treated with the test item, were evaluated for chromosome aberrations in three experiments at dose levels of 1, 3.13, 6.25, 12.5, 25, 50, 100, 200 µg/mL in the first experiment and of 12.5, 25, 50, 100, 200, 400, 800 µg/mL in the second and third experiments. Experiments were performed in the presence and the absence of a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes and all the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and the activity of the metabolising system. Cytotoxicity was observed in the 2nd experiment at 800 µg/mL and in the 3rd experiment from 200 µg/mL onward at a sampling time of 18 hours and from 400 µg/mL onward at 28 hours sampling time. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of S9. In conclusions the test item was considered to be non-clastogenic in V79 cells in vitro.

CAS 130905 -60 -1

In another in vitro mammalian chromosome aberration test, the clastogenic potential of Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS 130905-60-1) was investigated in chinese hamster lung fibroblasts (V79). The study was performed according to OECD TG 473 and GLP conditions were observed (Sasol, 1996). Duplicate cultures of V79 cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In both experiments, cells were exposed for 3 h to the test substance dissolved in ethanol at concentrations of 40, 200, 400 µg/mL with metabolic activation and for 16 and 26 h without metabolic activation. The substance was tested up to the limit concentration and did not induce cytotoxicity. Mitomycin C and Cyclophosphamide were used as positive control substances inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolising 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 V79 cells in vitro.

In vitro gene mutation in mammalian cells

CAS 49553 -76 -6

The in vitro mammalian cell gene mutation of with Oleic acid, monoester with oxybis(propanediol), (CAS 49553-76-6) was investigated according to OECD Guideline 476 under GLP conditions (DuPont, 2012). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Aroclor 1254 rat liver S9). In a preliminary toxicity assay, the maximum concentration of test substance in medium was 5000 µg/mL. In the first experiment, cells were exposed for 4 h to test substance at concentrations of 10 - 150 µg/mL (in DMSO) with and without metabolic activation. The concentrations chosen for cloning were 10, 20, 30, 40 and 50 µg/mL in the presence and absence of metabolic activation. No cloned cultures exhibited induced mutant frequencies ≥ 90 mutants per 10E6 clonable cells. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 5 - 75 µg/mL. The concentrations chosen for cloning were 5, 10, 20, 30 and 40 µg/mL. No cloned cultures exhibited induced mutant frequencies ≥ 90 mutants per 10E6 clonable cells. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. Cytotoxicity was observed at concentrations ≥ 30 µg/mL without metabolic activation and ≥ 50 µg/mL with metabolic activation. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, the test substance did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

CAS 63705 -03 -3

An in vitro mammalian cell gene mutation assay according to OECD Guideline 476 and GLP was performed with 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS 63705-03-3) in Chinese hamster ovary (CHO) cells (BASF, 2013). The test substance was dissolved in DMSO as it proved to be insoluble in water. Experiments were performed with and without a metabolic activation system (co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254). Concentrations used were 3.13, 6.25, 12.50, 25, 50, 100 µg/mL (experiment 1, with and without S9), 9.38, 18.75, 37.50, 75, 150, 200 µg/mL (experiment 2, without S9) and 4.69, 9.38, 18.75, 37.50, 75, 150 µg/mL (experiment 2, with S9). Ethyl methanesulfonate (EMS) and Dimethylbenz[a]anthracene (DMBA) were used as positive control substances. Cytotoxicity of the test substance was observed in experiment 1 in the absence of S9 mix at 100 µg/mL and in the presence of S9 mix at 50 µg/mL onward as well as in the second experiment in the absence of S9 mix at 150 µg/mL onward and in the presence of S9 mix at 75 µg/mL onward. Positive and solvent controls were valid and in range of historical control data. No significant increase in the mutation frequency at the HPRT locus was observed after treatment with the test substance either in the absence or in the presence of S9 mix. It was concluded that the test substance is not mutagenic in the CHO HPRT test system under the experimental conditions described.

Genetic toxicity in vivo

CAS 130905-60-1

Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS 130905-60-1) was found to be not genotoxic in the micronucleus assay in vivo (Sasol, 1993). A single dose of 15000 mg/kg bw/day administered by oral gavage to groups of 5 male and 5 female mice did not induce micro-nuclei formation. The dose level was based on a dose-range finding experiment. A dose of 15,000 mg/kg body weight was considered to be near the MTD (maximal tolerated dose) and was therefore chosen for the main study. Bone marrow samples were taken 24, 48 and 72 h after dosing. In none of the parameters investigated, a significant difference was found between female animals treated with the test article and negative control animals. The number of polychromatic erythrocytes without micronuclei in group males (48 h p.a.) as well as the ratio of polychromatic to normochromatic erythrocytes was significantly increased. The same parameters were decreased in group males (72 h p.a.). These effects might be explained by a stimulation of the formation of polychromatic erythrocytes in male mice after 48h by the test article resulting in a subsequent increase of normochromatic erythrocytes, which arise from polychromatic erythrocytes, after 72h. 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. In conclusion, the test substance was considered to be non-mutagenic under the experimental conditions of the study.

Conclusion on genetic toxicity

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

Justification for classification or non-classification

Based on the analogue read-across approach, the available in vitro and in vivo 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.