Glycerides, C18-36

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 98, TA 100 and TA 102, and in E. coli WP2 uvrA pKM.

Chromosome aberration (OECD 473): negative in Chinese hamster lung cells (CHL/IU) and human lymphocytes with and without metabolic activation.

Gene mutation in mammalian cells (OECD 476): negative in mouse lymphoma L5178Y cells and Chinese hamster ovary (CHO-K1) cells with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium supplemented with 10% calf serum

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9 mix: 891, 1783, 3565 µg/mL
24h treatment without S9 mix: 55.7, 111, 223, 446 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of vehicle: due to limited solubility of the test substance in water, DMSO was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: mitomycin C (MMC), 0.1 µg/mL (6h exposure), 0.05 µg/mL (24h exposure); +S9: cyclophosphamide (CM), 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h (short treatment) and 24 h (continuous treatment)
- Expression time (cells in growth medium): 18 h (after 6 h treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR: 0.2 μg/mL of colcemid (added 2 h prior to harvest)
STAIN: 1.2% Giemsa stain (in sodium phosphoric acid buffer 1/100 mol/L pH 6.8) for 12 min

NUMBER OF REPLICATIONS: 2 replications in one experiment

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative cell growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

100 cells/plate were observed and classified into gap, ctd (chromatid break), cte (chromatid exchange), csb (chromosome break), cse (chromosome exchange) and oth (others). The total number of cells with aberrations excluding gaps was calculated. The number of polyploid cells was also counted.
The test results were considered positive, if a statistically significant increase in the frequency of chromosome aberrations was observed, the increase showed dose-dependency and it was reproducible.

Statistics:

Differences in chromosome aberration frequencies between treated and control cells were analysed with the Fisher's exact test (p < 0.025). Dose-dependency was assessed using the Cochran Armitage trend test.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 446 µg/mL (24 h treatment, without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was not soluble in water.
- Precipitation: visible precipitation was noted at 223 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
To find the appropriate concentration range, a cytotoxicity test was performed at test concentrations of 3565, 1783, 891, 446, 223, 111, 55.7, 27.9, 13.9, 6.96 µg/mL. Cytotoxicity was evaluated both after a continuous 24 h exposure (-S9 mix) and a short-term 6 h exposure (± S9 mix) followed by an 18 h recovery period prior to harvest.
The test substance was not cytotoxic after short-term exposure at any concentration in the presence of S9 mix. Without metabolic activation, relative growth was reduced to about 71% of the control value.
After 24 h continuous treatment without S9 mix, the test substance was not cytotoxic up to 1783 µg/mL. At 3565 µg/mL, relative growth was decreased to ca. 13 % of the control value. The concentration leading to 50% cytotoxicity was calculated to be 2738 µg/mL.
Based on these results, the main test was conducted using the following test substance concentrations:
6 h treatment (± S9 mix): 891, 1783 and 3565 µg/mL
24 h treatment (-S9 mix): 55.7, 111, 223 and 446 µg/mL

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main test, no cytotoxic effects were observed after short-term exposure. The test substance was cytotoxic at 446 µg/mL after the 24 h treatment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Results of main study (short-term treatment) and additional study (continuous treatment)

Test item	Concentration	Relative cell growth	Aberrant cells in %
	in μg/mL	in %	with gaps	without gaps
Exposure period 6h, fixation time 18h, without S9 mix
DMSO		100	1	1
Glycerol monooleate	891 a)	121.8	0	1
	1783 a)	114.8	1	0
	3565 a)	101.1	1.5	0.5
MMC	0.1	77.5	4.5	36*
Exposure period 6h, fixation time 18h, with S9 mix
DMSO		100	0.5	0
Glycerol monooleate	891 a)	135.9	2	2
	1783 a)	123.9	1	0.5
	3565 a)	111.4	1	0.5
CP	12.5	83.4	3.5	19.5*
Exposure period 24h, without S9 mix
DMSO		100	1	1
Glycerol monooleate	55.7	99	0.5	0
	111	105.9	1	1
	223 a)	87.0	0.5	0
	446 a)	91.6	Toxic
MMC	0.05	104.4	6.5	25*

*: Significant difference from control (Fisher's exact test, p < 0.025)

a): Visible precipitation was observed at the end of exposure period.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: 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 (500 mg/kg bw)

Test concentrations with justification for top dose:

First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL

Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)

- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period

When the above mentioned criteria were not met, the test item was considered as non-mutagenic.

Statistics:

Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: First Experiment - 4 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.36	1
625	91	116	57	2.04	0.86
1250	108	94	65	2.44	1.03
2500	106	83	36	2.66	1.12
3600	89	76	11	0.30*	0.12
5000	74	83	8	0.04*	0.017
ENU (50)	53	62	45	#
Table 2: First Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.39	1
625	75	81	65	2.59	1.08
1250	92	44	46	3.04	1.27
2500	93	30	9	0.9	0.38
3600	90	5	1	0.2	0.08
5000	84	4	1	0.47	0.2
DMBA (3.3)	24	50	29	#
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	not reported	not reported
313	82	9	2	not reported	not reported
625	73	4	1	not reported	not reported
1250	68	4	1	not reported	not reported
2500	38	4	1	not reported	not reported
3600	31	1	0	not reported	not reported
ENU (50)	53	35	9	not reported	not reported
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2	1
156	83	95	62	1.95	0.98
313	84	101	70	1.84	0.92
625	82	105	65	1.79	0.9
1250	46	98.5	65	1.84	0.92
2500	38	88	37	1.93	0.97
3600	36	4	0	0	0
DMBA (3.3)	28	52	27	37.3	18.65
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.17	1
`2.5	102	100	62	2.16	1
5	100	114	151	2.02	0.93
10	92	95	145	1.97	0.91
20	105	100	60	2.11	0.97
40	82	95	103	2.17	1
80	69	111	97	1.82	0.84
160	25	44	16	2.32	1.07
320	11	31	6	1.94	0.89
ENU (50)	16	31	9	51.8	23.87

 

ENU: N-ethyl-N-nitrosourea

DMBA: 7,12-dimethyl-1,2-benzanthracene

*: statistically significant with p < 0.01

#: could not be calculated as all wells contained mutant colonies

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Oct - 29 Oct 2001

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

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, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media:
Liquid growth medium (broth): Oxoid Nutrient Broth No. 2 (2.5% (w/v) in distilled water
Selective agar plates: Vogel-Bonner medium composed of bacto agar (1.5%), D-glucose (2%), MgSO4 x 7H20 (0.02%), citric acid (0.2%), K2HPO4 (1%), NaNH4HPO4 x 4 H2O (0.35%) in distilled water (percentages correspond to w/v)
The components were autoclaved separately and mixed afterwards.
Top-agar: bacto agar (0.6%), NaCl (0.5%) in distilled water (percentages correspond to w/v), supplemented with 0.05 mM histidine and 0.05 mM biotin before use

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 (500 mg/kg bw)

Test concentrations with justification for top dose:

50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: sodium azide (SA, 1 µg/plate) for TA 100 and TA 1535; 2-nitrofluorene (2NF, 1 µg/plate) for TA 98 and TA 1537; cumene hydroperoxide (CHP, 100 µg/plate for plate incorporation and 25 µg/plate for preincubation) for TA 102;

Positive control substance:

other: + S9: 2-aminoanthracene (2-AA, 4 µg/plate for TA 102 or 2 µg/plate for remaining strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1. in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

2. preincubation

DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments including 1 plate incorporation and 1 preincubation test


DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of the background lawn of non-revertant bacteria, appearance of micro-colonies and/or reduction in the number of revertant colonies on the test plates comparised to the solvent control plates

Evaluation criteria:

The test was considered to be valid when the following criteria were met:
1. negative and positive control data were consistent and within the range of historical control data (see table 4)
2. positive controls revealed a marked increase over the concurrent solvent controls
3. the evaluation was not restricted by loss of plates (e.g. through contamination)

The test material may be considered mutagenic in this test system if all of the following criteria were met:
1. dose-related increases in the number of revertants at one or more test points
2. reproducible increases in revertants between replicate plates
3. statistically significance in the increases of revertants
4. increases count more than twice the corresponding solvent control values

The test material may be considered non-mutagenic in this test system when no increases in the number of revertants is observed which exceed 1.5 times the solvent control values at any test point. Sporadically ocurring statistically significanct increases in the number of revertants which were not dose-related will usually be considered incidental and not relevant for the evaluation.

Increases between 1.5 and 2 fold compared to the respective solvent controls meeting the other criteria for a positive result were considered to demonstrate weak mutagenicity.

Statistics:

Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA).
In detail, the number of revertant colonies at each treatment test point were compared to the corresponding solvent control values using the Analysis of Variance test. Statistically significant differences were further evaluated via Dunnett´s test to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicates.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no but tested up to limit concentrations (a small reduction in the number of revertants was induced in TA 100 (5000 µg: -15% (preincubation assay, +S9) and TA 1535 (160 µg: -28%; 1600 µg: -46%; 5000 µg: -30% (plate incorporation assay, + S9))

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item did not induce toxicity within the conducted preliminary toxicity test as evaluated by large reductions in the number of revertants or poor growth of the background lawn of non-revertant bacteria either in the absence or presence of S9 mix (see table 1).

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent and positive control values were acceptable and compatible with the historical control values (slight increases have been determined in number of revertants for TA 102 in the plate incorporation (-S9: 470 ± 6 vs 409) and preincubation test (+S9: 460 ± 4 vs 432) and for TA 1535 in the plate incorporation assay after treatment with sodium azide (-S9: 1063 ± 83 vs 908), see table 4).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of the preliminary toxicity test (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard Deviation)
		Frameshift type
		TA98
–	0	36.0 ± 1.7
–	50	30.3 ± 3.1
–	160	28.7 ± 5.1
–	500	33.0 ± 6.0
–	1600	34.3 ± 2.9
–	5000	34.3 ± 7.0
Positive controls, –S9	Name	2NF
	Concentrations (μg/plate)	1
	Mean No. of colonies/plate (average of 3 ± SD)	499.7 ± 88.3
+	0	48 ± 3
+	50	48.3 ± 5.5
+	160	43.3 ± 2.9
+	500	40.0 ± 6.6
+	1600	40.0 ± 1.0
+	5000	39.7 ± 6.1
Positive controls, +S9	Name	2AA
	Concentrations (μg/plate)	2
	Mean No. of colonies/plate (average of 3 ± SD)	682 ± 26.5
2NF = 2 Nitrofluorene
2AA = 2-Aminoanthracene
No statistical analysis was performed.

 

 

Table 2. Test results of main test 1 (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	210.3 ± 7.5	14.7 ± 1.5	469.7 ± 6.1	30.7 ± 7.6	14.0 ± 2.0
–	50	210.7 ± 7.8	15.7 ± 2.5	474.0 ± 3.0	33.3 ± 7.2	17.7 ± 2.5
–	160	206.0 ± 5.3	15.0 ± 1.0	480.0 ± 6.6	27.7 ± 0.6	18.0 ± 2.0
–	500	205.7 ± 3.8	16.3 ± 2.1	478.7 ± 5.7	27.0 ± 2.6	15.0 ± 2.0
–	1600	208.7 ± 9.3	15.0 ± 3.6	479.0 ± 6.2	26.0 ± 5.0	16.3 ± 2.3
–	5000	208.3 ± 2.9	12.3 ± 1.5	483.7 ± 5.5	32.7 ± 3.1	15.7 ± 2.5
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	100	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1136.7 ± 60.8	1063 ± 82.5	968.3 ± 78.4	749.7 ± 100.5	202.0 ± 7.9
+	0	213.7 ± 3.8	20.3 ± 2.9	405.3 ± 1.5	43.3 ± 2.1	16.7 ± 1.5
+	50	213.3 ± 3.1	19.0 ± 1.0	399.7 ± 6.8	41.0 ± 2.0	20.3 ± 0.6
+	160	215.7 ± 3.8	14.7* ± 1.5	401.3 ± 10.2	44.0 ± 2.0	19.7 ± 0.6
+	500	202.0 ± 5.3	17.0 ± 2.6	405.0 ± 11.5	40.3 ± 2.5	18.7 ± 0.6
+	1600	208.0 ± 11.1	11.0 ± 1.7	406.7 ± 9.7	42.0 ± 3.6	18.7 ± 2.1
+	5000	216.7 ± 4.2	14.3* ± 3.1	405.7 ± 4.7	43.0 ± 1.7	19.0 ± 1.7
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	838.0 ± 71.4	276.0 ± 80.0	1030.3 ± 102.6	597.0 ± 25.6	366.7 ± 34.0
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

Table 3. Test results of main test 2 (preincubation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	214.7 ± 4.0	18.0 ± 3.0	403.7 ± 3.1	36.0 ± 1.7	15.7 ± 1.2
–	50	213.3 ± 6.4	17.3± 0.6	405.0 ± 5.6	30.3 ± 3.1	18.3 ± 0.6
–	160	213.7 ± 6.0	18.3 ± 3.5	407.7 ± 2.3	28.7 ± 5.1	18.3 ± 1.5
–	500	213.0 ± 1.7	21.3 ± 4.2	403.7 ± 1.5	33.0 ± 6.0	19.0 ± 1.0
–	1600	211.0 ± 11.5	21.3 ± 3.2	406.7 ± 4.9	34.3 ± 2.9	18.3 ± 2.1
–	5000	212.3 ± 6.5	17.0 ± 1.7	411.3 ± 13.7	34.3 ± 7.0	17.0 ± 1.0
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	25	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1103.0 ± 25.5	596.3 ± 25.5	1158.0 ± 33.2	499.7 ± 88.3	338.7± 22.7
+	0	203.7 ± 7.5	11.7 ± 1.2	459.7 ± 4.2	48.0 ± 3.0	18.7 ± 1.2
+	50	206.0 ± 12.2	17.0 ± 1.0	460.0 ± 2.6	48.3 ± 5.5	20.0 ± 1.0
+	160	213.7 ± 4.7	16.3 ± 4.2	456.7 ± 4.5	43.3 ± 2.9	19.0 ± 1.0
+	500	207.7 ± 6.8	15.7 ± 2.1	455.3 ± 3.8	40.0 ± 6.6	18.0 ± 1.7
+	1600	188.7 ± 3.1	15.0 ± 4.0	458.3 ± 2.5	40.0 ± 1.0	19.0 ± 1.0
+	5000	173.3** ± 9.5	17.7 ± 2.3	459.7 ± 4.2	39.7 ± 6.1	19.0 ± 1.0
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	574.7 ± 30.2	231.7 ± 18.9	1140.7 ± 98.8	682.0 ± 26.5	210.3 ± 16.0
SA = sodium azide
CH = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

 

Table 4. Historical control values
Strain	Treatment (µg/mL)	S9 mix		Number of revertant colonies/plate				Number of plates
				Mean	Standard Deviation	Minimum	Maximum
TA 102	solvent	-		305	40	249	409	114
	solvent	+		321	52	224	432	117
	CHP (25)	-		1262	346	836	2487	54
	CHP (100)	-		1252	258	903	2000	48
	2AA (4)	+		1076	197	500	1522	105
TA 100	solvent	-		144	33	91	221	162
	solvent	+		143	35	90	234	168
	SA (1)	-		1006	196	578	1460	150
	2AA (2)	+		1007	362	123	2163	156
TA 98	solvent	-		51	16	19	82	159
	solvent	+		61	17	21	97	162
	2NF (1)	-		658	247	216	1189	147
	2AA (2)	+		851	284	411	1622	150
TA 1537	solvent	-		15	5	5	28	111
	solvent	+		19	7	7	43	117
	2NF (1)	-		357	154	121	919	99
	2AA (2)	+		301	202	67	1121	105
TA 1535	solvent	-		20	8	7	38	117
	solvent	+		18	7	7	42	123
	SA (1)	-		541	169	206	908	105
	2AA (2)	+		272	129	110	648	111
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Oct - 18 Apr 2002

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: human lymphocytes

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 (500 mg/kg bw)

Test concentrations with justification for top dose:

Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases

Evaluation criteria:

EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:

When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.

Key result

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)	Mitotic index (MI)
	Without S9			With S9
	Individual values		Relative mean MI (%)	Individual values		Relative mean MI (%)
0	4.8	5.3	100	3.8	2.9	100
313	4.8	4.3	90	3.1	3.5	99
625	4.3	4.1	83	2.9	3.5	96
1250	3.4	3.7	70	3.4	2.8	93
2500	3.7	3.3	69	2.1	1.8	58
5000	3.6	2.9	64	0.4	0.3	10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	1 / 0	2 / 1	1 / 0		1 / 0
1250	-	110	1 / 1	0 / 2	2 / 1
2500	-	75	1 / 4	1 / 3	1 / 4
5000	-	70	2 / 3	2 / 1	2 / 4
Daunomycin (0.015 µg/mL)	-	91	17 / 14 **	9 / 5	20 / 14	2 / 1
0	2%	100	0 / 0	1 / 2	0 / 0
625	2%	67	0 / 1	0 / 0	0 / 1
1250	2%	51	0 / 0	2 / 1	0 / 0
2500	2%	42	0 / 1	1 / 1	0 / 1
Cyclophosphamide (6 µg/mL)	2%	20	33a /19b **	16 / 3	36 / 20	15 / 9

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	0 / 3	1 / 5	0 / 3
40	-	84	1 / 4	3 / 6	1 / 4		1 / 0
80	-	59	1 / 2	0 / 2	1 / 2
160	-	49	0 / 2	3 / 1	0 / 2		0 / 1
Daunomycin (0.015 µg/mL)	-	102	13 / 14 **	6 / 9	13 / 13	1 / 1
0	4%	100	2 / 1	3 / 2	1 / 1	1 / 0
625	4%	66	2 / 4	3 / 1	5 / 1		0 / 1
1250	4%	61	1 / 1	3 / 4	1 / 1
2500	4%	33	5 / 1	3 / 0	3 / 4
Cyclophosphamide (6 µg/mL)	4%	77	36 / 33 **	8 / 8	37 / 33	11 / 11

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)	S9 mix	Frequency of metaphases with aberrant chromosomes excluding gaps (%)				Number of cultures
		Mean	SD	Mimimum	Maximum
Negative control	-	0.8	0.8	0	3	32
Daunomycin (0.015 µg/mL)	-	15.1	7.4	7	34	32
Negative control	+	0.7	0.9	0	3	32
Cyclophosphamide (6 µg/mL)	+	43.0	13.6	23	70	32

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 Apr - 24 Apr 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions. Due to growth inhibition in Salmonella, but not in E.coli strains, only low test concentrations used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further specified

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)

Test concentrations with justification for top dose:

0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metablic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.

Statistics:

The results at each concentration were demonstrated with the mean and the standard deviation.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

1st experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	14 ±2	399 ±25	12±2.5 [39]
TA 100	103±5.0	825 ± 34.6	113 ± 9.7 [4.9]
TA 1537	11±1.5	1989± 52.0	13±0.6 [10]
TA 98	19 ±1.7	561 ± 19	18 ±4.4 [1.2]
WP2uvrA	25 ±4.0	243 ±24.7	30 ±2.6 [5000]
with S9-mix
TA 1535	11± 0.6	399 ±25	12 ±2.5 [20]
TA 100	111 ± 6.1	825 ± 34.6	126 ±7.5 [39]
TA 1537	17 ±4.7	1989± 52.0	20±3.8 [20]
TA 98	29±1.5	561 ± 19	28 ±5.0 [39]
WP2uvrA	28 ±0.6	243 ±24.7	33±7.0 [5000]
2nd experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	10±2.3	388± 16.4	8 ±0.6 [10]
TA 100	121 ± 6.0	780 ±7.1	114 ±7.8 [20]
TA 1537	11 ±1.0	2057±200.9	15 ±0 [4.9]
TA 98	17 ±1.2	577 ±32.8	20±1.5  [1.2]
WP2uvrA	20 ±2.0	219 ±28:1	27 ±4.6 [1250]
with S9-mix
TA 1535	9 ±2.1	388 ±16.4	11 ±3.2 [78]
TA 100	114 ±12.7	780 ±7.1	117±9.3 [10]
TA 1537	23 ±4.7	2057 ±200:9	21±2.3 [10]
TA 98	25±3.8	577 ±32.8	29 ±2.6 [39]
WP2uvrA	25± 3.2	219 ±28:1	35 ±2.1 [1250]

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 May 2010 - 12 Jun 2010

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix Migrated to IUCLID6: 0.1 µg/mL ( 6 h treatment), 0.05 µg/mL (24 and 48 h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix Migrated to IUCLID6: 7 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy

Evaluation criteria:

The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more

Key result

Species / strain:

other: CHU/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher

RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Table 1. Effects of the test substance on viability and chromosome aberrations.

Test item	Concentration	Cell viability	Aberrant cells in %
	in µg/mL	in %	Numerical	Structural including gaps
Exposure period 6h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.0	1.5
MMC	0.010	105.5	0.0	39.5
Test substance	0.020	101.0	0.5	0.0
	0.039	98.0	1.0	1.5
	0.078	88.0	0.0	0.0
	0.156	82.5	0.5	1.0
Exposure period 6h with S9 mix
Untreated	--	--	0.0	2.0
Solvent	--	100	0.0	0.5
B(a)P	7.000	96.5	0.0	28.5
Test substance	0.020	96.0	0.0	2.0
	0.039	92.0	0.0	0.0
	0.078	86.0	1.0	0.5
	0.156	84.5	0.5	1.0
Exposure period 24h without S9 mix
Untreated	--	--	1.0	0.5
Solvent	--	100	0.5	0.0
MMC	0.050	102.0	0.0	41.5
Test substance	0.078	90.5	0.0	0.5
	0.156	82.5	0.0	1.0
	0.313	76.0	1.0	1.0
	0.625	66.0	0.0	0.5
	1.250	66.0	0.5	2.0
	2.500	84.5	0.5	0.0
	5.000	92.0	0.0	1.0
Exposure period 48h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.5	0.0
MMC	0.050	94.0	0.0	58.5
Test substance	0.078	99.0	0.0	1.0
	0.156	86.5	0.0	0.5
	0.313	74.5	1.0	0.0
	0.625	70.0	0.0	0.0
	1.250	79.5	0.5	0.0
	2.500	87.5	0.5	0.0
	5.000	92.5	0.5	1.0

MMC: Mitomycin

B(a)P: Benz(a)pyrene

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 Jun - 29 Jun 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions. No TA102 or E. coli strain included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA102 or E. coli strain included

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 Aroclor 1254.

Test concentrations with justification for top dose:

First and second experiment: 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80/bidist. water
- Justification for choice of solvent/vehicle: The suspension medium was chosen according to the solubility properties tested preliminary before start of the study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide (2 µg/plate) for TA100 and TA1535, 9-aminoacridine (80 µg/plate) for TA1537, 4-nitro-o-phenylendiamine (40 µg/plate) for TA98 and TA1538; with S9: 2-aminoanthracene (2.5 or 5 µg/plate) for all strains

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 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn and/or reduced number of revertants versus negative control

Evaluation criteria:

The results were considered positive if the following criteria were met:
- the plate background of not-reverted bacteria did not show any no growth reduction in comparison to the respective negative controls,
- the spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range,
- the positive controls showed mutation rates exceeding the control values of TA100 at least by the factor 2 and those of the other tester strains at least by the factor 3,
-at more than one dose tested, the test substance caused at least a 2-fold increase in comparison with the negative controls in the tester strain TA100. For the other tester strains, an increase in the mutation rate of more than 3 above the corresponding negative controls was considered positive.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate: TA 1535

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Ames Test Results - experiment 1

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 1535	TA 1538	TA 100	TA 98	TA 1537
-	Negative control (water)	8.3	11.0	72.7	28.0	7.7
-	0 (Solvent control)	8.0	11.0	80.3	27.7	6.3
-	8	11.3	11.7	69.7	31.3	8.7
-	40	9.0	11.0	71.7	28.0	6.7
-	200	11.3	8.3	60.0	34.0	5.0
-	1000	8.3	6.7	55.0	24.0	4.7
-	5000	3.7*	6.3	56.7	25.0	4.7
Positive controls - S9	Name	SA	4NP	SA	4NP	9AA
	Concentrations (μg/plate)	2	40	2	40	80
	Number of colonies/plate	251.0	1262.7	207.0	762.3	581.7
+	Negative control (water)	9.7	14.7	69.3	26.7	6.7
+	0 (Solvent control)	9.0	14.7	79.7	29.0	9.0
+	8	9.7	18.7	75.0	32.3	9.0
+	40	9.7	11.7	79.0	30.7	8.0
+	200	11.7	13.7	68.0	30.0	11.0
+	1000	10.3	16.0	73.3	28.3	7.7
+	5000	3.7*	12.7	53.7	24.3	6.3
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	5	5	5	2.5
	Number of colonies/plate	135.7	1403.3	1509.0	1799.7	1654.3

* Partial inhibition of bacterial background lawn

SA = Sodium azide

4NP = 4-Nitro-o-phenylendiamine

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

 

Table 2: Ames Test Results - experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 1535	TA 1538	TA 100	TA98	TA1537
-	Negative control (water)	10.7	12.3	67.7	28.7	6.3
-	0 (Solvent control)	11.7	13.0	72.3	29.3	6.3
-	8	10.3	14.0	68.7	28.0	5.7
-	40	13.7	8.3	75.7	26.0	7.0
-	200	7.3	8.0	54.7	24.7	4.3
-	1000	7.3	8.0	53.7	26.3	4.0
-	5000	2.7*	8.3	46.3	28.7	1.7
Positive controls - S9	Name	SA	4NP	SA	4NP	9AA
	Concentrations (μg/plate)	2	40	2	40	80
	Number of colonies/plate	274.0	1410.0	192.0	738.0	646.7
+	Negative control (water)	13.0	19.7	78.7	37.7	10.3
+	0 (Solvent control)	14.3	20.7	82.7	40.0	9.0
+	8	13.7	20.7	76.0	32.7	9.7
+	40	14.7	16.3	85.0	35.7	10.0
+	200	11.7	16.0	75.3	38.7	7.7
+	1000	13.7	18.7	72.0	36.3	6.7
+	5000	7.7*	14.3	61.3	34.7	3.3
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	5	5	5	2.5
	Number of colonies/plate	218.3	1413.3	1280.3	1637.0	1993.3

* Partial inhibition of bacterial background lawn

SA = Sodium azide

4NP = 4-Nitro-o-phenylendiamine

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

 

 

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication meeting basic scientific principles. Test compound was a mixture of short- and long-chain acyl triglyceride.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

exposure duration not reported;

Principles of method if other than guideline:

The SALATRIM family of triacylglycerols differs from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative
in the three assays.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) CHO-K1 cells obtained from the American Type Culture Collection
- Type and identity of media: in Ham’s F12 medium with 31 µg/mL penicillin, 50 µg/mL streptomycin sulfate, and 5 % heatinactivated
fetal bovine serum (F12/5).

Metabolic activation:

with and without

Metabolic activation system:

final S-9 concentration of 1 %

Test concentrations with justification for top dose:

0, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL
The high dose was limited by the low solubility of the fats in the assay medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
Stock solutions of SALATRIM 23CA lot A014 and corn oil were made in acetone such that the acetone never exceeded 1 % of the culture.

Untreated negative controls:

yes

Remarks:

Corn oil doses were 327.7-1000 μg/mL.

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Positive controls were EMS (200 μg/mL of culture) without metabolic activation and 3-MC (5 μg/mL of culture) with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: not reported
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14-17 days

Phenotypic expression of induced mutants was conducted by incubating the cells for 7 days. Cells were subcultured every 2-3 days by trypsinizing the flask, counting the cells, and replating 2 x 10(6) cells. After the expression period, approximately 3 x 10(6) cells from each culture were seeded in 100 mL of cloning medium supplemented with TG for selection of resistant cells. Approximately 600 cells were seeded in 100 mL of TG-free medium to determine the percentage of viable cells. The cells were incubated for 14-17 days and cell colonies counted with an ARTEK colony counter. Mutant frequency (ratio of mutant cells to nonmutant cells) was calculated by dividing the number of resistant colonies by the number of unselected viable colonies.

SELECTION AGENT (mutation assays): The selective cloning medium contained 30 µM 6-thioguanine (TG).

NUMBER OF REPLICATIONS: The definitive study was done in duplicate using duplicate cultures for each replicate.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by detaching the cells from the culture flask with 0.05% trypsin-0.02% EDTA. A Model 2F Coulter counter was used to determine cell numbers, and an aliquot containing at least 1-2 x 10(6) cells was added to 20 mL of F12/5 to determine phenotypic expression. The remaining cell suspension was diluted in approximately 35 mL of medium so that 500 cells were plated into two Petri dishes. After incubation for 14-17 days, cell colonies were determined. Survival was expressed as the cloning efficiency (CE) relative to the solvent control.

Evaluation criteria:

The test results were considered positive if a dose-related increase in the number of mutant colonies occurred and the mutant frequencies of duplicate cultures treated with one or more concentrations of the test article were at least 3 times the average of those from the solvent control cultures.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Although the test material was soluble in the acetone, turbidity was noted in the cultures, indicating solubility had been exceeded.

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were done to determine cytotoxicity and aid in selection of the dose range.

Table 3: Results of the HPRT Mammalian Cell Gene Mutation Assay of SALATRIM 23CA lot A014

with and without Metabolic Activation*

SALATRIM Dose (μg/mL)	without metabolic activation		with metabolic activation
	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)
0	100	15	100	10
31.3	96	7	94	7
62.5	100	18	84	6
125	89	11	93	10
250	90	17	97	15
500	78	12	69	9
1000	86	17	75	10
corn oil 1000**	91	6	79	15
EMS200	56	170	-	-
3MC 5	-		79	170

 

 

*Data represent the mean of two cultures with the exception of the 0 dose (solvent control), which represents the mean of triplicate cultures.

**Data from a independent experiment with corn oil as test substance instead of SALATRIM

EMS= Ethyl methanesulfonate; positive control for the assay without metabolic activation.

3MC = 3-Methylcholanthrene; positive control for the assay with metabolic activation.

Conclusions:

Interpretation of results (migrated information):
negative

SALATRIM 23CA lot A014 presented no evidence of mutagenic potential under the conditions of the assay.
Positive controls gave the expected results, validating the assay.
Corn oil did not alter either the relative cloning efficiency or the mutation frequency of the cells.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium supplemented with 10% calf serum

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9 mix: 891, 1783, 3565 µg/mL
24h treatment without S9 mix: 55.7, 111, 223, 446 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of vehicle: due to limited solubility of the test substance in water, DMSO was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: mitomycin C (MMC), 0.1 µg/mL (6h exposure), 0.05 µg/mL (24h exposure); +S9: cyclophosphamide (CM), 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h (short treatment) and 24 h (continuous treatment)
- Expression time (cells in growth medium): 18 h (after 6 h treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR: 0.2 μg/mL of colcemid (added 2 h prior to harvest)
STAIN: 1.2% Giemsa stain (in sodium phosphoric acid buffer 1/100 mol/L pH 6.8) for 12 min

NUMBER OF REPLICATIONS: 2 replications in one experiment

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative cell growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

100 cells/plate were observed and classified into gap, ctd (chromatid break), cte (chromatid exchange), csb (chromosome break), cse (chromosome exchange) and oth (others). The total number of cells with aberrations excluding gaps was calculated. The number of polyploid cells was also counted.
The test results were considered positive, if a statistically significant increase in the frequency of chromosome aberrations was observed, the increase showed dose-dependency and it was reproducible.

Statistics:

Differences in chromosome aberration frequencies between treated and control cells were analysed with the Fisher's exact test (p < 0.025). Dose-dependency was assessed using the Cochran Armitage trend test.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 446 µg/mL (24 h treatment, without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was not soluble in water.
- Precipitation: visible precipitation was noted at 223 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
To find the appropriate concentration range, a cytotoxicity test was performed at test concentrations of 3565, 1783, 891, 446, 223, 111, 55.7, 27.9, 13.9, 6.96 µg/mL. Cytotoxicity was evaluated both after a continuous 24 h exposure (-S9 mix) and a short-term 6 h exposure (± S9 mix) followed by an 18 h recovery period prior to harvest.
The test substance was not cytotoxic after short-term exposure at any concentration in the presence of S9 mix. Without metabolic activation, relative growth was reduced to about 71% of the control value.
After 24 h continuous treatment without S9 mix, the test substance was not cytotoxic up to 1783 µg/mL. At 3565 µg/mL, relative growth was decreased to ca. 13 % of the control value. The concentration leading to 50% cytotoxicity was calculated to be 2738 µg/mL.
Based on these results, the main test was conducted using the following test substance concentrations:
6 h treatment (± S9 mix): 891, 1783 and 3565 µg/mL
24 h treatment (-S9 mix): 55.7, 111, 223 and 446 µg/mL

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main test, no cytotoxic effects were observed after short-term exposure. The test substance was cytotoxic at 446 µg/mL after the 24 h treatment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Results of main study (short-term treatment) and additional study (continuous treatment)

Test item	Concentration	Relative cell growth	Aberrant cells in %
	in μg/mL	in %	with gaps	without gaps
Exposure period 6h, fixation time 18h, without S9 mix
DMSO		100	1	1
Glycerol monooleate	891 a)	121.8	0	1
	1783 a)	114.8	1	0
	3565 a)	101.1	1.5	0.5
MMC	0.1	77.5	4.5	36*
Exposure period 6h, fixation time 18h, with S9 mix
DMSO		100	0.5	0
Glycerol monooleate	891 a)	135.9	2	2
	1783 a)	123.9	1	0.5
	3565 a)	111.4	1	0.5
CP	12.5	83.4	3.5	19.5*
Exposure period 24h, without S9 mix
DMSO		100	1	1
Glycerol monooleate	55.7	99	0.5	0
	111	105.9	1	1
	223 a)	87.0	0.5	0
	446 a)	91.6	Toxic
MMC	0.05	104.4	6.5	25*

*: Significant difference from control (Fisher's exact test, p < 0.025)

a): Visible precipitation was observed at the end of exposure period.

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: 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 (500 mg/kg bw)

Test concentrations with justification for top dose:

First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL

Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)

- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period

When the above mentioned criteria were not met, the test item was considered as non-mutagenic.

Statistics:

Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: First Experiment - 4 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.36	1
625	91	116	57	2.04	0.86
1250	108	94	65	2.44	1.03
2500	106	83	36	2.66	1.12
3600	89	76	11	0.30*	0.12
5000	74	83	8	0.04*	0.017
ENU (50)	53	62	45	#
Table 2: First Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.39	1
625	75	81	65	2.59	1.08
1250	92	44	46	3.04	1.27
2500	93	30	9	0.9	0.38
3600	90	5	1	0.2	0.08
5000	84	4	1	0.47	0.2
DMBA (3.3)	24	50	29	#
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	not reported	not reported
313	82	9	2	not reported	not reported
625	73	4	1	not reported	not reported
1250	68	4	1	not reported	not reported
2500	38	4	1	not reported	not reported
3600	31	1	0	not reported	not reported
ENU (50)	53	35	9	not reported	not reported
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2	1
156	83	95	62	1.95	0.98
313	84	101	70	1.84	0.92
625	82	105	65	1.79	0.9
1250	46	98.5	65	1.84	0.92
2500	38	88	37	1.93	0.97
3600	36	4	0	0	0
DMBA (3.3)	28	52	27	37.3	18.65
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.17	1
`2.5	102	100	62	2.16	1
5	100	114	151	2.02	0.93
10	92	95	145	1.97	0.91
20	105	100	60	2.11	0.97
40	82	95	103	2.17	1
80	69	111	97	1.82	0.84
160	25	44	16	2.32	1.07
320	11	31	6	1.94	0.89
ENU (50)	16	31	9	51.8	23.87

 

ENU: N-ethyl-N-nitrosourea

DMBA: 7,12-dimethyl-1,2-benzanthracene

*: statistically significant with p < 0.01

#: could not be calculated as all wells contained mutant colonies

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

18 Oct - 29 Oct 2001

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
Liquid growth medium (broth): Oxoid Nutrient Broth No. 2 (2.5% (w/v) in distilled water
Selective agar plates: Vogel-Bonner medium composed of bacto agar (1.5%), D-glucose (2%), MgSO4 x 7H20 (0.02%), citric acid (0.2%), K2HPO4 (1%), NaNH4HPO4 x 4 H2O (0.35%) in distilled water (percentages correspond to w/v)
The components were autoclaved separately and mixed afterwards.
Top-agar: bacto agar (0.6%), NaCl (0.5%) in distilled water (percentages correspond to w/v), supplemented with 0.05 mM histidine and 0.05 mM biotin before use

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 (500 mg/kg bw)

Test concentrations with justification for top dose:

50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: sodium azide (SA, 1 µg/plate) for TA 100 and TA 1535; 2-nitrofluorene (2NF, 1 µg/plate) for TA 98 and TA 1537; cumene hydroperoxide (CHP, 100 µg/plate for plate incorporation and 25 µg/plate for preincubation) for TA 102;

Positive control substance:

other: + S9: 2-aminoanthracene (2-AA, 4 µg/plate for TA 102 or 2 µg/plate for remaining strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1. in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

2. preincubation

DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments including 1 plate incorporation and 1 preincubation test


DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of the background lawn of non-revertant bacteria, appearance of micro-colonies and/or reduction in the number of revertant colonies on the test plates comparised to the solvent control plates

Evaluation criteria:

The test was considered to be valid when the following criteria were met:
1. negative and positive control data were consistent and within the range of historical control data (see table 4)
2. positive controls revealed a marked increase over the concurrent solvent controls
3. the evaluation was not restricted by loss of plates (e.g. through contamination)

The test material may be considered mutagenic in this test system if all of the following criteria were met:
1. dose-related increases in the number of revertants at one or more test points
2. reproducible increases in revertants between replicate plates
3. statistically significance in the increases of revertants
4. increases count more than twice the corresponding solvent control values

The test material may be considered non-mutagenic in this test system when no increases in the number of revertants is observed which exceed 1.5 times the solvent control values at any test point. Sporadically ocurring statistically significanct increases in the number of revertants which were not dose-related will usually be considered incidental and not relevant for the evaluation.

Increases between 1.5 and 2 fold compared to the respective solvent controls meeting the other criteria for a positive result were considered to demonstrate weak mutagenicity.

Statistics:

Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA).
In detail, the number of revertant colonies at each treatment test point were compared to the corresponding solvent control values using the Analysis of Variance test. Statistically significant differences were further evaluated via Dunnett´s test to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicates.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no but tested up to limit concentrations (a small reduction in the number of revertants was induced in TA 100 (5000 µg: -15% (preincubation assay, +S9) and TA 1535 (160 µg: -28%; 1600 µg: -46%; 5000 µg: -30% (plate incorporation assay, + S9))

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item did not induce toxicity within the conducted preliminary toxicity test as evaluated by large reductions in the number of revertants or poor growth of the background lawn of non-revertant bacteria either in the absence or presence of S9 mix (see table 1).

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent and positive control values were acceptable and compatible with the historical control values (slight increases have been determined in number of revertants for TA 102 in the plate incorporation (-S9: 470 ± 6 vs 409) and preincubation test (+S9: 460 ± 4 vs 432) and for TA 1535 in the plate incorporation assay after treatment with sodium azide (-S9: 1063 ± 83 vs 908), see table 4).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of the preliminary toxicity test (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard Deviation)
		Frameshift type
		TA98
–	0	36.0 ± 1.7
–	50	30.3 ± 3.1
–	160	28.7 ± 5.1
–	500	33.0 ± 6.0
–	1600	34.3 ± 2.9
–	5000	34.3 ± 7.0
Positive controls, –S9	Name	2NF
	Concentrations (μg/plate)	1
	Mean No. of colonies/plate (average of 3 ± SD)	499.7 ± 88.3
+	0	48 ± 3
+	50	48.3 ± 5.5
+	160	43.3 ± 2.9
+	500	40.0 ± 6.6
+	1600	40.0 ± 1.0
+	5000	39.7 ± 6.1
Positive controls, +S9	Name	2AA
	Concentrations (μg/plate)	2
	Mean No. of colonies/plate (average of 3 ± SD)	682 ± 26.5
2NF = 2 Nitrofluorene
2AA = 2-Aminoanthracene
No statistical analysis was performed.

 

 

Table 2. Test results of main test 1 (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	210.3 ± 7.5	14.7 ± 1.5	469.7 ± 6.1	30.7 ± 7.6	14.0 ± 2.0
–	50	210.7 ± 7.8	15.7 ± 2.5	474.0 ± 3.0	33.3 ± 7.2	17.7 ± 2.5
–	160	206.0 ± 5.3	15.0 ± 1.0	480.0 ± 6.6	27.7 ± 0.6	18.0 ± 2.0
–	500	205.7 ± 3.8	16.3 ± 2.1	478.7 ± 5.7	27.0 ± 2.6	15.0 ± 2.0
–	1600	208.7 ± 9.3	15.0 ± 3.6	479.0 ± 6.2	26.0 ± 5.0	16.3 ± 2.3
–	5000	208.3 ± 2.9	12.3 ± 1.5	483.7 ± 5.5	32.7 ± 3.1	15.7 ± 2.5
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	100	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1136.7 ± 60.8	1063 ± 82.5	968.3 ± 78.4	749.7 ± 100.5	202.0 ± 7.9
+	0	213.7 ± 3.8	20.3 ± 2.9	405.3 ± 1.5	43.3 ± 2.1	16.7 ± 1.5
+	50	213.3 ± 3.1	19.0 ± 1.0	399.7 ± 6.8	41.0 ± 2.0	20.3 ± 0.6
+	160	215.7 ± 3.8	14.7* ± 1.5	401.3 ± 10.2	44.0 ± 2.0	19.7 ± 0.6
+	500	202.0 ± 5.3	17.0 ± 2.6	405.0 ± 11.5	40.3 ± 2.5	18.7 ± 0.6
+	1600	208.0 ± 11.1	11.0 ± 1.7	406.7 ± 9.7	42.0 ± 3.6	18.7 ± 2.1
+	5000	216.7 ± 4.2	14.3* ± 3.1	405.7 ± 4.7	43.0 ± 1.7	19.0 ± 1.7
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	838.0 ± 71.4	276.0 ± 80.0	1030.3 ± 102.6	597.0 ± 25.6	366.7 ± 34.0
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

Table 3. Test results of main test 2 (preincubation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	214.7 ± 4.0	18.0 ± 3.0	403.7 ± 3.1	36.0 ± 1.7	15.7 ± 1.2
–	50	213.3 ± 6.4	17.3± 0.6	405.0 ± 5.6	30.3 ± 3.1	18.3 ± 0.6
–	160	213.7 ± 6.0	18.3 ± 3.5	407.7 ± 2.3	28.7 ± 5.1	18.3 ± 1.5
–	500	213.0 ± 1.7	21.3 ± 4.2	403.7 ± 1.5	33.0 ± 6.0	19.0 ± 1.0
–	1600	211.0 ± 11.5	21.3 ± 3.2	406.7 ± 4.9	34.3 ± 2.9	18.3 ± 2.1
–	5000	212.3 ± 6.5	17.0 ± 1.7	411.3 ± 13.7	34.3 ± 7.0	17.0 ± 1.0
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	25	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1103.0 ± 25.5	596.3 ± 25.5	1158.0 ± 33.2	499.7 ± 88.3	338.7± 22.7
+	0	203.7 ± 7.5	11.7 ± 1.2	459.7 ± 4.2	48.0 ± 3.0	18.7 ± 1.2
+	50	206.0 ± 12.2	17.0 ± 1.0	460.0 ± 2.6	48.3 ± 5.5	20.0 ± 1.0
+	160	213.7 ± 4.7	16.3 ± 4.2	456.7 ± 4.5	43.3 ± 2.9	19.0 ± 1.0
+	500	207.7 ± 6.8	15.7 ± 2.1	455.3 ± 3.8	40.0 ± 6.6	18.0 ± 1.7
+	1600	188.7 ± 3.1	15.0 ± 4.0	458.3 ± 2.5	40.0 ± 1.0	19.0 ± 1.0
+	5000	173.3** ± 9.5	17.7 ± 2.3	459.7 ± 4.2	39.7 ± 6.1	19.0 ± 1.0
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	574.7 ± 30.2	231.7 ± 18.9	1140.7 ± 98.8	682.0 ± 26.5	210.3 ± 16.0
SA = sodium azide
CH = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

 

Table 4. Historical control values
Strain	Treatment (µg/mL)	S9 mix		Number of revertant colonies/plate				Number of plates
				Mean	Standard Deviation	Minimum	Maximum
TA 102	solvent	-		305	40	249	409	114
	solvent	+		321	52	224	432	117
	CHP (25)	-		1262	346	836	2487	54
	CHP (100)	-		1252	258	903	2000	48
	2AA (4)	+		1076	197	500	1522	105
TA 100	solvent	-		144	33	91	221	162
	solvent	+		143	35	90	234	168
	SA (1)	-		1006	196	578	1460	150
	2AA (2)	+		1007	362	123	2163	156
TA 98	solvent	-		51	16	19	82	159
	solvent	+		61	17	21	97	162
	2NF (1)	-		658	247	216	1189	147
	2AA (2)	+		851	284	411	1622	150
TA 1537	solvent	-		15	5	5	28	111
	solvent	+		19	7	7	43	117
	2NF (1)	-		357	154	121	919	99
	2AA (2)	+		301	202	67	1121	105
TA 1535	solvent	-		20	8	7	38	117
	solvent	+		18	7	7	42	123
	SA (1)	-		541	169	206	908	105
	2AA (2)	+		272	129	110	648	111
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene

Reference 12

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

16 Oct - 18 Apr 2002

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: human lymphocytes

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 (500 mg/kg bw)

Test concentrations with justification for top dose:

Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases

Evaluation criteria:

EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:

When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.

Key result

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)	Mitotic index (MI)
	Without S9			With S9
	Individual values		Relative mean MI (%)	Individual values		Relative mean MI (%)
0	4.8	5.3	100	3.8	2.9	100
313	4.8	4.3	90	3.1	3.5	99
625	4.3	4.1	83	2.9	3.5	96
1250	3.4	3.7	70	3.4	2.8	93
2500	3.7	3.3	69	2.1	1.8	58
5000	3.6	2.9	64	0.4	0.3	10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	1 / 0	2 / 1	1 / 0		1 / 0
1250	-	110	1 / 1	0 / 2	2 / 1
2500	-	75	1 / 4	1 / 3	1 / 4
5000	-	70	2 / 3	2 / 1	2 / 4
Daunomycin (0.015 µg/mL)	-	91	17 / 14 **	9 / 5	20 / 14	2 / 1
0	2%	100	0 / 0	1 / 2	0 / 0
625	2%	67	0 / 1	0 / 0	0 / 1
1250	2%	51	0 / 0	2 / 1	0 / 0
2500	2%	42	0 / 1	1 / 1	0 / 1
Cyclophosphamide (6 µg/mL)	2%	20	33a /19b **	16 / 3	36 / 20	15 / 9

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	0 / 3	1 / 5	0 / 3
40	-	84	1 / 4	3 / 6	1 / 4		1 / 0
80	-	59	1 / 2	0 / 2	1 / 2
160	-	49	0 / 2	3 / 1	0 / 2		0 / 1
Daunomycin (0.015 µg/mL)	-	102	13 / 14 **	6 / 9	13 / 13	1 / 1
0	4%	100	2 / 1	3 / 2	1 / 1	1 / 0
625	4%	66	2 / 4	3 / 1	5 / 1		0 / 1
1250	4%	61	1 / 1	3 / 4	1 / 1
2500	4%	33	5 / 1	3 / 0	3 / 4
Cyclophosphamide (6 µg/mL)	4%	77	36 / 33 **	8 / 8	37 / 33	11 / 11

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)	S9 mix	Frequency of metaphases with aberrant chromosomes excluding gaps (%)				Number of cultures
		Mean	SD	Mimimum	Maximum
Negative control	-	0.8	0.8	0	3	32
Daunomycin (0.015 µg/mL)	-	15.1	7.4	7	34	32
Negative control	+	0.7	0.9	0	3	32
Cyclophosphamide (6 µg/mL)	+	43.0	13.6	23	70	32

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

09 Apr - 24 Apr 2010

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further specified

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)

Test concentrations with justification for top dose:

0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metablic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.

Statistics:

The results at each concentration were demonstrated with the mean and the standard deviation.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

1st experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	14 ±2	399 ±25	12±2.5 [39]
TA 100	103±5.0	825 ± 34.6	113 ± 9.7 [4.9]
TA 1537	11±1.5	1989± 52.0	13±0.6 [10]
TA 98	19 ±1.7	561 ± 19	18 ±4.4 [1.2]
WP2uvrA	25 ±4.0	243 ±24.7	30 ±2.6 [5000]
with S9-mix
TA 1535	11± 0.6	399 ±25	12 ±2.5 [20]
TA 100	111 ± 6.1	825 ± 34.6	126 ±7.5 [39]
TA 1537	17 ±4.7	1989± 52.0	20±3.8 [20]
TA 98	29±1.5	561 ± 19	28 ±5.0 [39]
WP2uvrA	28 ±0.6	243 ±24.7	33±7.0 [5000]
2nd experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	10±2.3	388± 16.4	8 ±0.6 [10]
TA 100	121 ± 6.0	780 ±7.1	114 ±7.8 [20]
TA 1537	11 ±1.0	2057±200.9	15 ±0 [4.9]
TA 98	17 ±1.2	577 ±32.8	20±1.5  [1.2]
WP2uvrA	20 ±2.0	219 ±28:1	27 ±4.6 [1250]
with S9-mix
TA 1535	9 ±2.1	388 ±16.4	11 ±3.2 [78]
TA 100	114 ±12.7	780 ±7.1	117±9.3 [10]
TA 1537	23 ±4.7	2057 ±200:9	21±2.3 [10]
TA 98	25±3.8	577 ±32.8	29 ±2.6 [39]
WP2uvrA	25± 3.2	219 ±28:1	35 ±2.1 [1250]

Reference 14

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

03 May 2010 - 12 Jun 2010

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix Migrated to IUCLID6: 0.1 µg/mL ( 6 h treatment), 0.05 µg/mL (24 and 48 h treatment)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix Migrated to IUCLID6: 7 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy

Evaluation criteria:

The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more

Key result

Species / strain:

other: CHU/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher

RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Table 1. Effects of the test substance on viability and chromosome aberrations.

Test item	Concentration	Cell viability	Aberrant cells in %
	in µg/mL	in %	Numerical	Structural including gaps
Exposure period 6h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.0	1.5
MMC	0.010	105.5	0.0	39.5
Test substance	0.020	101.0	0.5	0.0
	0.039	98.0	1.0	1.5
	0.078	88.0	0.0	0.0
	0.156	82.5	0.5	1.0
Exposure period 6h with S9 mix
Untreated	--	--	0.0	2.0
Solvent	--	100	0.0	0.5
B(a)P	7.000	96.5	0.0	28.5
Test substance	0.020	96.0	0.0	2.0
	0.039	92.0	0.0	0.0
	0.078	86.0	1.0	0.5
	0.156	84.5	0.5	1.0
Exposure period 24h without S9 mix
Untreated	--	--	1.0	0.5
Solvent	--	100	0.5	0.0
MMC	0.050	102.0	0.0	41.5
Test substance	0.078	90.5	0.0	0.5
	0.156	82.5	0.0	1.0
	0.313	76.0	1.0	1.0
	0.625	66.0	0.0	0.5
	1.250	66.0	0.5	2.0
	2.500	84.5	0.5	0.0
	5.000	92.0	0.0	1.0
Exposure period 48h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.5	0.0
MMC	0.050	94.0	0.0	58.5
Test substance	0.078	99.0	0.0	1.0
	0.156	86.5	0.0	0.5
	0.313	74.5	1.0	0.0
	0.625	70.0	0.0	0.0
	1.250	79.5	0.5	0.0
	2.500	87.5	0.5	0.0
	5.000	92.5	0.5	1.0

MMC: Mitomycin

B(a)P: Benz(a)pyrene

Reference 15

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

20 Jun - 29 Jun 1990

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA102 or E. coli strain included

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 Aroclor 1254.

Test concentrations with justification for top dose:

First and second experiment: 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80/bidist. water
- Justification for choice of solvent/vehicle: The suspension medium was chosen according to the solubility properties tested preliminary before start of the study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide (2 µg/plate) for TA100 and TA1535, 9-aminoacridine (80 µg/plate) for TA1537, 4-nitro-o-phenylendiamine (40 µg/plate) for TA98 and TA1538; with S9: 2-aminoanthracene (2.5 or 5 µg/plate) for all strains

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 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn and/or reduced number of revertants versus negative control

Evaluation criteria:

The results were considered positive if the following criteria were met:
- the plate background of not-reverted bacteria did not show any no growth reduction in comparison to the respective negative controls,
- the spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range,
- the positive controls showed mutation rates exceeding the control values of TA100 at least by the factor 2 and those of the other tester strains at least by the factor 3,
-at more than one dose tested, the test substance caused at least a 2-fold increase in comparison with the negative controls in the tester strain TA100. For the other tester strains, an increase in the mutation rate of more than 3 above the corresponding negative controls was considered positive.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate: TA 1535

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Ames Test Results - experiment 1

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 1535	TA 1538	TA 100	TA 98	TA 1537
-	Negative control (water)	8.3	11.0	72.7	28.0	7.7
-	0 (Solvent control)	8.0	11.0	80.3	27.7	6.3
-	8	11.3	11.7	69.7	31.3	8.7
-	40	9.0	11.0	71.7	28.0	6.7
-	200	11.3	8.3	60.0	34.0	5.0
-	1000	8.3	6.7	55.0	24.0	4.7
-	5000	3.7*	6.3	56.7	25.0	4.7
Positive controls - S9	Name	SA	4NP	SA	4NP	9AA
	Concentrations (μg/plate)	2	40	2	40	80
	Number of colonies/plate	251.0	1262.7	207.0	762.3	581.7
+	Negative control (water)	9.7	14.7	69.3	26.7	6.7
+	0 (Solvent control)	9.0	14.7	79.7	29.0	9.0
+	8	9.7	18.7	75.0	32.3	9.0
+	40	9.7	11.7	79.0	30.7	8.0
+	200	11.7	13.7	68.0	30.0	11.0
+	1000	10.3	16.0	73.3	28.3	7.7
+	5000	3.7*	12.7	53.7	24.3	6.3
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	5	5	5	2.5
	Number of colonies/plate	135.7	1403.3	1509.0	1799.7	1654.3

* Partial inhibition of bacterial background lawn

SA = Sodium azide

4NP = 4-Nitro-o-phenylendiamine

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

 

Table 2: Ames Test Results - experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 1535	TA 1538	TA 100	TA98	TA1537
-	Negative control (water)	10.7	12.3	67.7	28.7	6.3
-	0 (Solvent control)	11.7	13.0	72.3	29.3	6.3
-	8	10.3	14.0	68.7	28.0	5.7
-	40	13.7	8.3	75.7	26.0	7.0
-	200	7.3	8.0	54.7	24.7	4.3
-	1000	7.3	8.0	53.7	26.3	4.0
-	5000	2.7*	8.3	46.3	28.7	1.7
Positive controls - S9	Name	SA	4NP	SA	4NP	9AA
	Concentrations (μg/plate)	2	40	2	40	80
	Number of colonies/plate	274.0	1410.0	192.0	738.0	646.7
+	Negative control (water)	13.0	19.7	78.7	37.7	10.3
+	0 (Solvent control)	14.3	20.7	82.7	40.0	9.0
+	8	13.7	20.7	76.0	32.7	9.7
+	40	14.7	16.3	85.0	35.7	10.0
+	200	11.7	16.0	75.3	38.7	7.7
+	1000	13.7	18.7	72.0	36.3	6.7
+	5000	7.7*	14.3	61.3	34.7	3.3
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	5	5	5	2.5
	Number of colonies/plate	218.3	1413.3	1280.3	1637.0	1993.3

* Partial inhibition of bacterial background lawn

SA = Sodium azide

4NP = 4-Nitro-o-phenylendiamine

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

 

 

Reference 16

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

exposure duration not reported;

Principles of method if other than guideline:

The SALATRIM family of triacylglycerols differs from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative
in the three assays.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) CHO-K1 cells obtained from the American Type Culture Collection
- Type and identity of media: in Ham’s F12 medium with 31 µg/mL penicillin, 50 µg/mL streptomycin sulfate, and 5 % heatinactivated
fetal bovine serum (F12/5).

Metabolic activation:

with and without

Metabolic activation system:

final S-9 concentration of 1 %

Test concentrations with justification for top dose:

0, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL
The high dose was limited by the low solubility of the fats in the assay medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
Stock solutions of SALATRIM 23CA lot A014 and corn oil were made in acetone such that the acetone never exceeded 1 % of the culture.

Untreated negative controls:

yes

Remarks:

Corn oil doses were 327.7-1000 μg/mL.

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Positive controls were EMS (200 μg/mL of culture) without metabolic activation and 3-MC (5 μg/mL of culture) with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: not reported
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14-17 days

Phenotypic expression of induced mutants was conducted by incubating the cells for 7 days. Cells were subcultured every 2-3 days by trypsinizing the flask, counting the cells, and replating 2 x 10(6) cells. After the expression period, approximately 3 x 10(6) cells from each culture were seeded in 100 mL of cloning medium supplemented with TG for selection of resistant cells. Approximately 600 cells were seeded in 100 mL of TG-free medium to determine the percentage of viable cells. The cells were incubated for 14-17 days and cell colonies counted with an ARTEK colony counter. Mutant frequency (ratio of mutant cells to nonmutant cells) was calculated by dividing the number of resistant colonies by the number of unselected viable colonies.

SELECTION AGENT (mutation assays): The selective cloning medium contained 30 µM 6-thioguanine (TG).

NUMBER OF REPLICATIONS: The definitive study was done in duplicate using duplicate cultures for each replicate.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by detaching the cells from the culture flask with 0.05% trypsin-0.02% EDTA. A Model 2F Coulter counter was used to determine cell numbers, and an aliquot containing at least 1-2 x 10(6) cells was added to 20 mL of F12/5 to determine phenotypic expression. The remaining cell suspension was diluted in approximately 35 mL of medium so that 500 cells were plated into two Petri dishes. After incubation for 14-17 days, cell colonies were determined. Survival was expressed as the cloning efficiency (CE) relative to the solvent control.

Evaluation criteria:

The test results were considered positive if a dose-related increase in the number of mutant colonies occurred and the mutant frequencies of duplicate cultures treated with one or more concentrations of the test article were at least 3 times the average of those from the solvent control cultures.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Although the test material was soluble in the acetone, turbidity was noted in the cultures, indicating solubility had been exceeded.

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were done to determine cytotoxicity and aid in selection of the dose range.

Table 3: Results of the HPRT Mammalian Cell Gene Mutation Assay of SALATRIM 23CA lot A014

with and without Metabolic Activation*

SALATRIM Dose (μg/mL)	without metabolic activation		with metabolic activation
	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)
0	100	15	100	10
31.3	96	7	94	7
62.5	100	18	84	6
125	89	11	93	10
250	90	17	97	15
500	78	12	69	9
1000	86	17	75	10
corn oil 1000**	91	6	79	15
EMS200	56	170	-	-
3MC 5	-		79	170

 

 

*Data represent the mean of two cultures with the exception of the 0 dose (solvent control), which represents the mean of triplicate cultures.

**Data from a independent experiment with corn oil as test substance instead of SALATRIM

EMS= Ethyl methanesulfonate; positive control for the assay without metabolic activation.

3MC = 3-Methylcholanthrene; positive control for the assay with metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Analogue justification

No data on the in vitro genetic toxicity in bacterial and mammalian cells are available for Dub TGI 24. The genetic toxicity assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 736150-63-3

The potential mutagenicity of Glycerides, castor-oil, mono, hydrogenated, acetates was assessed in a bacterial mutation assay according to OECD guideline 471 and in compliance with GLP (please refer to IUCLID section 7.6.1). S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to concentrations of 50 - 5000 µg/plate with or without S9-mix in two independent experiments. No dose-dependent increase in the mean number of revertants per plate was observed in any tester strain up to the maximum exposure concentration. Slight cytotoxicity, as indicated by a small reduction in the number of revertants compared to controls, was induced in TA 100 at 5000 µg/plate (with metabolic activation) in the preincubation assay (experiment I) and in TA 1535 at 160 µg, 1600 µg and 5000 µg/plate (with metabolic activation) in the plate incorporation assay (experiment II). The solvent and positive control values were within the historical control values. Based on the results of this study, the test substance did not induce mutagenicity in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

CAS 91052-13-0

A bacterial gene mutation assay with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was performed according to OECD guideline 471 and GLP (please refer to IUCLID section 7.6.1). Two independent experiments were performed both in the presence or absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E. coli WP2 uvrA. In the preliminary toxicity screening, growth inhibitory effects were observed at ≥ 20 µg/plate in S. typhimurium TA 98 and TA 1537 (without metabolic activation), at ≥ 78 µg/plate in S. typhimurium TA 100 and TA 1535 (without metabolic activation), and at ≥ 313 µg/plate in all S. typhimurium strains with metabolic activation. Based on these results, concentrations ranging from 0.61 to 78 µg/plate were used for the tester strains TA 100, TA 1535, TA98 and TA 1537 in the absence of metabolic activation, whereas concentrations ranging from 10 to 313 µg/plate were applied for treatment of the tester strains TA 100, TA 1535, TA 98 and TA 1537 in the presence of metabolic activation. Since no cytotoxicity was seen in E. coli WP2 uvrA, the maximum test concentration of 5000 µg/plate and concentrations of 2500, 1250, 625 and313 µg/plate were selected for treatment in the main assay. Precipitation of the test substance was observed on the plates with E. coli WP2 uvrA at test concentrations ≥ 1250 µg/plate without metabolic activation and at ≥ 2500 µg/plate with metabolic activation in both experiments. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance in the presence or absence of metabolic activation. The positive and negative controls revealed the expected results. Under the conditions of this assay, the test substance did not induce gene mutations in the selected strains of S. typhimurium and in E. coli WP2 uvrA in the absence and presence of metabolic activation, respectively.

CAS 91744-13-7

A bacterial gene mutation assay with Glycerides, C14-18 and C16-22-unsatd. mono- and di- was performed according to the protocol similar to OECD guideline 471 and GLP (please refer to IUCLID section 7.6.1). In two independent experiments, S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to concentrations ranging from 8 to 5000 µg/plate with and without metabolic activation. At the maximum concentration, bacterial growth was partly inhibited in Salmonella strain TA 1535 in the presence or absence of metabolic activation. No increase in the mean number of revertants per plate was observed in any tester strain at the indicated concentrations. The vehicle controls were within the spontaneous mutation ranges of the historical controls and the positive controls induced the expected increase in the number of reverse mutants. Under the conditions of this experiment, the test substance did not exhibit mutagenic properties in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 736150-63-3

An in vitro mammalian cell gene mutation assay was performed with glycerides, castor-oil-mono, hydrogenated, acetates according to OECD guideline 476 and under GLP conditions (please refer to IUCLID section 7.6.1). In the first experiment, mutations at the TK locus of mouse lymphoma L5178Y cells were investigated at concentrations of 625, 1250, 2500, 3600 and 5000 µg/mL. The L5178Y cells were exposed to the test material for a period of 3 h in the presence and for 4 h in the absence of metabolic activation (S9-mix), respectively. At 3600 µg/mL, the relative total growth was 1-11% compared with the negative controls. In the second experiment, cells were exposed to a concentration range of 313 to 3600 µg/ for a period of 24 h, without metabolic activation, and to a concentration range of 156-3600 µg/mL for a period of 4 h, with metabolic activation. Since the relative growth without metabolic activation was very low (0-2%) at all test concentrations, the 24-h treatment of cells in the absence of metabolic activation was repeated with concentrations ranging from 2.5-320 µg/mL, which resulted in appropriate levels of cytotoxicity (10-20% relative growth) at 160 µg/mL. In the presence of metabolic activation, the relative total growth was 37 and 0% at 2500 and 3600 µg/mL in the second experiment, respectively. After a 3-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose concentration. The positive controls significantly increased mutant frequency. In conclusion, the test substance did not induce mutations in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

Short-, medium- and long-chain triglycerides (SCT, MCT, LCT)

The SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols was assessed using a gene mutation assay in cultured mammalian cells (Chinese hamster ovary (CHO-K1) cells) similarly to OECD guideline 476 (please refer to IUCLID section 7.6.1). Gene mutations at the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/mL. Two cultures were tested per dose level. The highest concentration was limited by the low solubility of the fats in the assay medium. No significant cytotoxicity was reported. No increase in mutant frequency was observed at any concentration tested whether with or without metabolic activation. The positive controls significantly increased the mutant frequency. Therefore, it was concluded that under the conditions of the study, the test material was not mutagenic at the HPRT locus of Chinese hamster ovary cells in the absence and presence of metabolic activation.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 736150-63-3

An in vitro mammalian chromosome aberration test was performed with Glycerides, castor-oil, mono, hydrogenated, acetates in human lymphocytes according to OECD guideline 473 and in compliance with GLP (please refer to IUCLID section 7.6.1). An initial cytotoxicity test was performed with concentrations of 313 - 5000 µg/mL in the presence or absence of metabolic activation (S9-mix) for 3 h. A dose-related toxicity was observed. At the highest concentration (5000 µg/mL), the mitotic index was reduced to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation, respectively. Based on these results, concentrations of 625, 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250, 2500, 3600 and 5000 µg/mL (with 2% S9-mix) were chosen for treatment of cells in the main assay for an exposure period of 3 h. Metaphase analysis was performed at concentrations of 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250 and 2500 µg/mL (with 2% S9-mix). In the second main assay, cells were exposed to concentrations of 313-5000 µg/mL (without S9 mix) or 625-5000 µg/mL (with 4% S9 mix) for either 3 or 20 h, respectively. As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups, this part of the experiment was repeated with lower concentrations ranging from 2.5 to 320 µg/mL. Based on the cytotoxicity data obtained, concentrations of 40, 80 and 160 µg/mL (without S9) and 625, 1250 and 2500 µg/mL (with 4% S9-mix) were used for metaphase analysis. The chromosome analysis of the experiments showed no treatment-related increase in the number of cells with chromosomal aberrations compared with controls. The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values and the positive controls were shown to be valid. Based on the results of this chromosome aberration test, the test substance was not clastogenic in human lymphocytes in the presence or absence of metabolic activation under the experimental conditions chosen.

 

CAS 91052-13-0

An in vitro mammalian chromosome aberration test with was performed in Chinese hamster lung cells (CHL/IU) with glycerides, C8-18 and C18-unsatd. mono- and di-, acetates, according to OECD guideline 473 and under GLP conditions (please refer to IUCLID section 7.6.1). In a preliminary cell growth inhibition test with concentrations ranging from 9.8 to 5000 µg/plate, no significant inhibition of cell growth was observed after 4 h exposure in the presence and absence of metabolic activation (S9 mix). However, a moderate reduction of cell growth of 30-40% compared with the controls was observed at concentrations ranging from 156-1250 µg/mL after 24 and 48 h continuous exposure. Based on the results of this study, concentrations of 20, 39, 78 and 156 µg/mL (with and without metabolic activation) were used for the analysis of chromosomal aberrations after short-term exposure (6 h). In addition, concentrations of 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL were chosen for analysis chromosomal aberrations after continuous exposure (24 and 48 h), without metabolic activation. No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. No cytotoxic effects were observed in any of the experiments performed. An oily precipitation of the test substance was observed at concentrations ≥ 78 µg/mL, but did not interfere with chromosomal analysis of the cells. The positive controls included during short-term and continuous exposure were shown to be valid. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU), in the presence and absence of metabolic activation.

CAS 111-03-5

2,3-dihydroxypropyl oleate was assessed in an in vitro mammalian chromosome aberration test in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 and under GLP conditions (please refer to IUCLID section 7.6.1). In a preliminary cytotoxicity test, cells were exposed to concentrations ranging from 6.96 to 3565 µg/mL for a continuous 24-h exposure period with metabolic activation (S9-mix) or a short-term 6 h exposure with and without metabolic activation. The test substance was not cytotoxic after short-term exposure at any concentration in the presence of metabolic activation. Without metabolic activation, relative growth was reduced to about 71% of the control value after a period of 6 h. Continuous exposure for 24 h without metabolic activation caused no cytotoxicity up to concentrations of 1783 µg/mL. At 3565 µg/mL, relative growth was decreased to ca. 13 % of the control value. The concentration leading to 50% cytotoxicity was calculated to be 2738 µg/mL. Based on these results, concentrations of 891, 1783 and 3565 µg/mL (with and without metabolic activation) were selected for chromosome analysis after short-term exposure, and concentrations of 55.7, 111, 223 and 446 µg/mL (without metabolic activation) were chosen for chromosome analysis after continuous exposure. No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. No cytotoxic effects were observed after short-term exposure, but the test substance was cytotoxic at 446 µg/mL after 24 hours continuous treatment. Visible precipitation of the test substance was observed at concentrations ≥ 223 µg/mL; however, this did not interfere with chromosomal analysis. The positive controls included during short-term and continuous exposure were shown to be valid. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU) in the presence and absence of metabolic activation.

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of Dub TGI 24 in bacterial and mammalian cells. Analogue read-across from 5 source substances was applied fromin vitro studies in bacterial cells, from in vitro studies on cytogenicity and from gene mutation in mammalian cells. The results of the available in vitro studies were consistently negative. Based on the available data and following the analogue approach, Dub TGI 24 is not expected to be mutagenic and/or clastogenic.

 

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Dub TGI 24, data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.