Reaction products of n-octanol and acrylic acid, first distillation pitch

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key studies: OECD Guideline 471 (Bacterial Reverse Mutation), 473 (Chromosome Aberration) and 476 (Mammalian Cell Gene Mutation). GLP studies. The test substance appeared to be not mutagenic in the Ames test and in a chromosome aberration study and it was negative in a Mouse Lymphoma assay with and without metabolic activation.

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:

From january 30, 2012 to March 8, 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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine mutation :
TA1537: hisC3076, TA98: hisD3052/R-factor*, TA1535: hisG46, TA100: hisG46/R-factor*
*: R-factor = plasmid pKM101 (increases error-prone DNA repair).

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate
Experiment 2: 33, 100, 333, 1000, 3330 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; ethanol; physiol. saline

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol)

Negative solvent / vehicle controls:

yes

Remarks:

(Saline)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation Migrated to IUCLID6: TA1535

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

With metabolic activation Migrated to IUCLID6: TA1535

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

With and without metabolic activation

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

With and without metabolic activation Migrated to IUCLID6: TA98

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

With and without metabolic activation Migrated to IUCLID6: TA100

Untreated negative controls:

yes

Remarks:

(The vehicle of the test substance, which was ethanol)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

With and without metabolic activation Migrated to IUCLID6: WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10E9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies, histidine independent (His+), for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted.

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
- Method: The plates were examinated for a reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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:

cytotoxicity

Remarks:

(at a dose level of 3330 and 5000µg/plate, only in experiment 2 and in absence of S-9 mix for strain TA100)

Untreated negative controls validity:

valid

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose range of the test material used in the preliminary toxicity study was 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix. The test material exhibited toxicity at 3330 and 5000 μg/plate to the strain of Salmonella used (TA100). In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.


COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Experiment 1: Mutagenic response of Reaction products of n-octanol and acrylic acid, first distillation pitch in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Dose                             Mean number of revertant colonies/3 replicate plates (± S.D.) with (ug/plate)                      different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	807 ± 25	848 ± 22	858 ± 40	812 ± 17	977 ± 9
Solvent control	10 ± 0	6 ± 2	23 ± 3	107 ± 3	21 ± 5
3				90 ± 6	23 ± 5
10				100 ± 9	25 ± 5
33	7 ± 1	4 ± 2	13 ± 1	101 ± 21	27 ± 4
100	10 ± 3	4 ± 2	17 ± 3	99 ± 10	17 ± 3
333	9 ± 2	4 ± 1	14 ± 3	101 ± 10	19 ± 5
1000	8 ± 3	5 ± 2	18 ± 2	87 ± 13	18 ± 1
3330	8 ± 2	3 ± 2	18 ± 3	50 ± 7	21 ± 3
5000 SP	6 ± 2	2 ± 2	11 ± 1	17 ± 5	20 ± 3
With S9-mix^1
Positive control	252 ± 9	394 ± 23	886 ± 20	955 ± 51	381 ± 35
Solvent control	7 ± 2	5 ± 2	22 ± 2	146 ± 13	21 ± 2
3				92 ± 10	22 ± 4
10				86 ± 11	20 ± 3
33	7 ± 2	6 ± 1	21 ± 0	95 ± 4	19 ± 2
100	7 ± 2	6 ± 2	23 ± 1	87 ± 3	19 ± 1
333	7 ± 4	4 ± 2	20 ± 2	99 ± 12	20 ± 2
1000	8 ± 3	6 ± 4	21 ± 2	89 ± 12	19 ± 5
3330	9 ± 5	4 ± 1	21 ± 3	70 ± 7	18 ± 3
5000 SP	7 ± 2	3 ± 2	19 ± 3	41 ± 13	16 ± 3

Solvent control: 0.1 ml ethanol

1    The S9-mix contained 5% (v/v) S9 fraction

SP  Slight Precipitate

 

Table 2. Experiment 2: Mutagenic response of Reaction products of n-octanol and acrylic acid, first distillation pitch in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Dose                             Mean number of revertant colonies/3 replicate plates (± S.D.) with (ug/plate)                      different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	698 ± 45	543 ± 61	989 ± 45	842 ± 63	977 ± 104
Solvent control	5 ± 1	4 ± 1	15 ± 4	90 ± 14	13 ± 4
33	6 ± 2	3 ± 1	14 ± 3	84 ± 7	13 ± 2
100	6± 2	4 ± 1	11± 3	81 ± 9	17 ± 3
333	5 ± 1	3 ± 2	14 ± 3	85± 2	17± 3
1000	8 ± 0	3 ± 1	14 ± 2	71 ± 6	14± 3
3330	4 ± 3	3 ± 1	13 ± 6	56 ± 8	12 ± 4
5000 SP	5± 2	3 ± 1	13± 3	42 ± 7	20± 2
With S9-mix^1
Positive control	171 ± 13	329 ± 31	715 ± 23	1138 ± 183	429 ± 13
Solvent control	4 ± 2	3 ± 1	19 ± 3	115 ± 7	15 ± 2
33	6 ± 2	7 ± 1	23 ± 3	111 ± 26	14 ± 4
100	5 ± 1	5± 3	21 ± 3	99 ± 13	18 ± 2
333	7 ± 1	2± 1	22 ± 5	118 ± 27	18 ± 3
1000	7 ± 1	3 ± 2	21 ± 2	110 ± 14	21 ± 2
3330 SP	6± 1	4 ± 2	18 ± 3	107 ± 7	17 ± 1
5000 SP	5 ± 1	2 ± 2	19 ± 5	109 ± 8	11 ± 4

Solvent control: 0.1 ml ethanol

1    The S9-mix contained 10% (v/v) S9 fraction

SP  Slight Precipitate

 

 

 

 

 

 

Conclusions:

Interpretation of results: negative

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

Executive summary:

Evaluation of the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli reverse mutation assay (with independent repeat) was determinated according to OECD 471, EC B.13/14 Guidelines and with GLP. Four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a tryptophan-requiring strain of Escherichia coli (WP2uvrA) were used for the assay. The test was performed in two independent experiments in the presence and absence of S9-mix (5% (v/v) S9 fraction in expertiment 1 and 10% (v/v) S9 franction in experiment 2). The dose range was determined in a prelimirary toxicity assay and was 3 to 5000µg/plate in the first experiment. The experiment was repeated using a dose range of 33 to 5000 µg/plate.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Reaction products of n-octanol and acrylic acid, first distillation pitch is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 12, 2012 to March 21, 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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Blood was collected from healthy adult, non-smoking, male volunteers. Dose range finding study: First cytogenetic assay: age 31, AGT = 13.4 h; Second cytogenetic assay: age 26, AGT = 15.3 h

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
- Properly maintained: yes (at a temperature of 37.0 ± 1.0°C and 5.0 ± 0.5% CO2 in air).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

-Experiment 1: 10, 33 and 100 µg/mL culture medium (3 h exposure time, 24 h fixation time) with and without S9-mix.

-Experiment 2: Without S9-mix : 10, 100, 150, 200, 250 and 300 μg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time). With S9-mix : 10, 33 and 100 μg/mL culture medium (3 h exposure time, 48 h fixation time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test substance solvent: Dimethyl sulfoxide (DMSO); positive control solvent: Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium.

Untreated negative controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 0.5 μg/mL for a 3 h exposure period, 0.2 μg/mL for a 24 h exposure period and 0.1 μg/mL for a 48 h exposure period.

Untreated negative controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix) Migrated to IUCLID6: 10 μg/mL for a 3 h exposure period (24 h fixation time).

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1 : After 3 h exposure to the test substance in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).

Experiment 2: After 3 h exposure, the cells exposed to the test substance in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44 - 46 h (48 h fixation time). The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

DURATION
- Exposure duration: Experiment 1: 3h; Experiment 2: 3, 24 and 48 h.
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment 1: 24 h; Experiment 2: 24 and 48h.

SPINDLE INHIBITOR (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium).
STAIN (for cytogenetic assays): 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in tap water.

NUMBER OF REPLICATIONS: duplicate culture

NUMBER OF CELLS EVALUATED: One hundred metaphase chromosome spreads per culture; only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, but was not counted as an aberration.
- Determination of endoreplication: no

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: blood samples were obtained from healthy male subjects.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: At the dose range finding study at the 24 h and 48 h continuous exposure time blood cultures were reated with 1, 3, 10, 33, 100, 333 and 1000 μg test substance/mL culture medium without S9-mix. The test substance was tested beyond the limit of solubility to obtain adequate toxicity data. At a concentration of 100 μg/mL test substance precipitated in the culture medium.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Chromosome aberrations in human lymphocyte cultures treated with Reaction products of n-octanol and acrylic acid, first distillation pitch in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time).
Conc	DMSO			10			33			100			MMC-C
	(1.0% v/v)			µg/mL			µg/mL			µg/mL			0.5 µg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic index (%)	100			101			97			85			87
No. of	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
cells scored
No. of cells with aberrations	1	1	2	0	0	0	0	0	0	0	0	0	13	21	***)
(+gaps)^a)															34
No. of cells with aberrations (- gaps)	1	0	1	0	0	0	0	0	0	0	0	0	13	20	***)
															33
g´		1												1
g”
b`	1												11	17
b”													1	4
m`
m”
exch.													1
dic
d´
misc.
Total aberr (+gaps)	1	1		0	0		0	0		0	0		13	22
Total aberr	1	0		0	0		0	0		0	0		13	21
(-gaps)
^a) Abbreviations used for various types of aberrations.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
*^)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
Table 2. Chromosome aberrations in human lymphocyte cultures treated with Reaction products of n-octanol and acrylic acid, first distillation pitch in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time).
Conc	DMSO			10			33			100			CP
	(1.0% v/v)			µg/mL			µg/mL			µg/mL			10 µg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic index (%)	100			94			92			84			49
No. of cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of cells with aberrations	0	0	0	0	0	0	0	0	0	0	2	2	21	24	***)
(+gaps)^a)															45
No. of cells with aberrations (- gaps)	0	0	0	0	0	0	0	0	0	0	2	2	21	24	***)
															45
g´
g”
b`											2		17	19
b”													3	7
m`
m”
exch.													1	1
dic
d´
misc.					poly		poly
Total aberr (+gaps)	0	0		0	0		0	0		0	2		21	27
Total aberr	0	0		0	0		0	0		0	2		21	27
(-gaps)
^a) Abbreviations used for various types of aberrations.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
The numerical variations polyploidy (poly) was not counted as an aberration.
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
Table 3. Chromosome aberrations in human lymphocyte cultures treated with Reaction products of n-octanol and acrylic acid, first distillation pitch in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time).
Conc	DMSO			100			200			250			MMC-C
	(1.0% v/v)			µg/mL			µg/mL			µg/mL			0.2 µg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic index (%)	100			101			66			47			96
No. of cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of cells with aberrations	0	2	2	0	0	0	1	1	2	1	1	2	17	18
(+gaps)^a)
															***)
															35
No. of cells with aberrations (- gaps)	0	2	2	0	0	0	1	1	2	1	1	2	17	18
															***)
															35
g´
g”
b`							1				1		3	4
b”		2						1		1			12	15
m`
m”
exch.													3	1
dic
d´
misc.
Total aberr (+gaps)	0	2		0	0		1	1		1	1		18	20
Total aberr (-gaps)	0	2		0	0		1	1		1	1		18	20
^a) Abbreviations used for various types of aberrations.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
Table 4. Chromosome aberrations in human lymphocyte cultures treated with Reaction products of n-octanol and acrylic acid, first distillation pitch in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time).
Conc	DMSO			10			100			200			MMC-C
	(1.0% v/v)			µg/mL			µg/mL			µg/mL			0.1 µg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic index (%)	100			95			67			44			82
No. of cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of cells with aberrations	3	2	5	0	1	1	0	0	0	0	0	0	20	22
(+gaps)^a)
															***)
															42
No. of cells with aberrations (- gaps)	3	2	5	0	1	1	0	0	0	0	0	0	20	22
															***)
															42
g´
g”
b`	1	1											2	6
b”	2	1											15	12
m`
m”
exch.					1								5	5
dic
d´
misc.
Total aberr (+gaps)	3	2		0	1		0	0		0	0		22	23
Total aberr (-gaps)	3	2		0	1		0	0		0	0		22	23
^a) Abbreviations used for various types of aberrations.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
Table 5. Chromosome aberrations in human lymphocyte cultures treated with Reaction products of n-octanol and acrylic acid, first distillation pitch in the presence of S9-mix in the second cytogenetic assay (3 h exposure time, 48 h fixation time).
Conc	DMSO			10			33			100			CP
	(1.0% v/v)			µg/mL			µg/mL			µg/mL			10 µg/mL
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic index (%)	100			98			95			98			-^b)
No. of cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of cells with aberrations	1	2	3	0	1	1	0	0	0	0	0	0	22	17	***)
(+gaps)^a)															39
No. of cells with aberrations (- gaps)	1	2	3	0	1	1	0	0	0	0	0	0	22	17	***)
															39
g´													1
g”
b`		2											17	6
b”	1				1								10	11
m`
m”
exch.													1	4
dic
d´
misc.
Total aberr (+gaps)	1	2		0	1		0	0		0	0		29	21
Total aberr (-gaps)	1	2		0	1		0	0		0	0		28	21
^a) Abbreviations used for various types of aberrations.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
^b)CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

Conclusions:

Interpretation of results: negative

The test substanceis not clastogenic in human lymphocytes.

Executive summary:

Evaluation of the ability of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes was determinated according to OECD 473, EC B.10 Guidelines and with GLP. The chromosome aberrations were evaluated in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of the test substance was tested in two independent experiments.

In the first cytogenetic assay, the test substance was tested up to 100 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction; and showed precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test substance was tested up to 250 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 200 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix the test substance was tested up to 100 μg/mL for a 3 h exposure time with a 48 h fixation time and showed precipitated in the culture medium at this dose level.

Positive control chemicals, MMC-C and CP, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations and did not presented any effects on the number of polyploid cells and cells with endoreduplicated chromosomes in the absence and presence of S9-mix in either of the two independently repeated experiments.

Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

In conclusion, the test substance is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 10, 2012 to March 5, 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

Principles of method if other than guideline:

“International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
3 to 333 μg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
Experiment 1:
in the absence and presence of 8% (v/v) S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 μg/ml exposure medium.
Experiment 2:
in the absence and presence of 12% (v/v) S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 μg/ml exposure medium.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

The solvent for the test article, i.e. dimethyl sulfoxide.

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 15 and 5 μg/ml for a 3 and 24 hours treatment period, respectively

Untreated negative controls:

yes

Remarks:

The solvent for the test article, i.e. dimethyl sulfoxide.

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix) Migrated to IUCLID6: 7.5 μg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: in the absence and presence of (8%) S9-mix (3 hour treatment period)
Experiment 2: in the absence (24 hour treatment period) and presence of (12%) S9-mix(3 hour treatment period)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 μg/ml trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10E6 survivors, and for CP not below 700 per 10E6 survivors.

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: precipitating dose level was observed at 333 μg/ml in the dose range finding test.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity in the RSG was observed up to and including the highest test substance concentration of 333 μg/ml compared to the suspension growth of the solvent control (in absence and presence of S9-mix at 3 and 24hours).

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay, with the exception of mutation frequency of the positive control in the absence of S9-mix (second experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

Remarks on result:

other: strain/cell type: L5178Y/TK+/--3.7.2C mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

Table 1. Experiment 1: Cytotoxic and mutagenic response of Reaction products of n-octanol and acrylic acid, first distillation pitch in the mouse lymphoma L5178Y test system.

dose	RSG	CEday2	RSday2	RTG	mutation frequency
(µg/ml)	(%)	(%)	(%)	(%)	per 10^6survivors total
					Total	( small	large)
without metabolic activation 3 hours treatment
SC1 SC2	100	80 71	100	100	50 66	( 26 ( 30	22) 34)
0.03	107	65	86	92	70	( 34	34)
0.1	112	90	119	133	73	( 30	41)
0.3	117	71	94	110	61	( 31	28)
1	124	63	83	103	65	( 32	32)
3	113	70	93	105	49	( 24	24)
10	97	110	145	141	67	( 22	43)
33	118	79	104	123	59	( 32	26)
100^(1)	100	63	83	84	78	( 39	37)
MMS	83	27	35	29	899	( 364	482)
With 8% (v/v) metabolic activation 3 hours treatment
SC1 SC2	100	74 115	100	100	96 68	( 36 ( 32	56 ) 33)
0.03	99	108	115	114	65	( 30	33)
0.1	120	85	90	109	74	( 34	38)
0.3	110	88	93	102	71	( 23	46)
1	112	72	77	86	58	( 31	26)
3	96	101	107	103	47	( 17	29)
10	124	105	112	138	54	( 26	26)
33	113	115	122	138	44	( 15	28)
100^(1)	122	95	101	124	76	( 25	49)
CP	61	55	58	35	1351	( 543	563)

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = Ethanol; MMS = Methylmethanesulfonate;

CP = Cyclophosphamide

(1)      = Condensation products of cyclopentanone and pentaldehyde, fractionation pitch precipitated in the exposure medium

Table 2. Experiment 2: Cytotoxic and mutagenic response of Reaction products of n-octanol and acrylic acid, first distillation pitch in the mouse lymphoma L5178Y test system.

 

dose	RSG	CEday2	RSday2	RTG	mutation frequency
(µg/ml)	(%)	(%)	(%)	(%)	per 10^6survivors total
					Total	( small	large)
without metabolic activation 24 hours treatment
SC1	100	105 89	100	100	62  92	( 33 ( 36	27) 52)
SC2
0.03	106	91	94	100	69	( 40	27)
0.1	68	94	97	66	77	( 32	43)
0.3	108	99	103	111	37	( 18	18)
1	123	84	86	106	81	( 32	46)
3	126	80	83	104	44	( 20	24)
10	133	97	100	132	35	( 13	21)
33	98	81	84	83	86	( 27	56)
100^(1)	72	91	94	68	59	( 17	40)
MMS	131	83	85	112	494	( 227	218)
With 12% (v/v) metabolic activation 3 hours treatment
SC1 SC2	100	93 115	100	100	97 92	( 32  ( 32	61) 55)
0.03	124	123	119	148	52	( 24	26)
0.1	126	98	95	119	75	( 30	43)
0.3	115	98	95	109	71	( 23	46)
1	97	95	92	89	83	( 40	40)
3	97	93	89	87	80	( 32	44)
10	108	98	95	102	82	( 24	55)
33	113	121	117	132	49	( 25	23)
100^(1)	100	108	104	105	74	( 29	42)
CP	74	77	74	55	960	( 342	447)

 Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth;

SC = Solvent control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

[1]      = Condensation products of cyclopentanone and pentaldehyde, fractionation pitch precipitated in the exposure medium

Conclusions:

Interpretation of results: negative

The test substance is not mutagenic in the TK mutation test system.

Executive summary:

Evaluation of the mutagenic activity of the test substance in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was deterinated according to the OECD 476, EC B.17 Guidelines and with GLP. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

Although the mutation frequency of the positive control in the absence of S9-mix (second experiment) was not within the acceptability criteria, the mutagenic response was more than 8 times greater than the concurrent solvent control values, therefore the validity of the test was considered to be not affected.

The test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a second experiment with modifications in the duration of treatment time (in the absence of S9-mix) and with modifications in the concentration of the S9 for metabolic activation (in the presence of S9-mix).

It is concluded that Reaction products of n-octanol and acrylic acid, first distillation pitch is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study: Ames Test:

The reverse mutation was carried out according to OECD Guideline 471 (GLP study). The test substance is not mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and, TA 100 and E. Coli WP2 uvr A with and without metabolic activation. Cytotoxicity was only observed in tester strain TA100 in the absence of S9 -mix in the second experiment, at 3330 and 5000 µg/plate.

Key study: Chromosome aberrations:

The chromosome aberration in cultured peripheral human lymphocytes was carried out according to OEDC Guideline 473 (GLP study). No chromosomal aberrations were induced in human lymphocytes tested with and without metabolic activation. No toxicity was observed up to and including the highest precipitating tested dose in the absence and presence of S9 -mix at 3, 24 and 48 hours exposure and 24 and 48 hours fixation time.

Key study: Mammalian cell gene mutation:

The mutagenic activity of test substance was carried out according to the OECD Guideline 476 (GLP study) showing no effects on the thymidine kinase locus in L5178Y mouse lymphoma cells. No toxicity was observed up to and including the highest test substance concentration of 100 µg/mL in the absence and presence of S9 -mix.

Justification for classification or non-classification

Based on all information available, Reaction products of n-octanol and acrylic acid, first distillation pitch does not have to be classified for genotoxicity.