Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test (OECD Guideline 471), under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential. The results of an Ames Test,  three in vitro chromosome aberration tests and an in vitro mammalian gene mutation test with closely related structural analogues showed no evidence of mutagenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2014 to 02 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

S. typhimurium, other: TA97a, TA1535, TA98 and TA100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µI/plate) of the test article, two plates per dose.
Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5 µI/plate) of the test article were tested in each of five bacterial tester strains (E. coliWP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).

Vehicle / solvent:

Identity: Acetone, Lot#124017
Supplied by: Fisher Scientific
Date Received: 15 Feb 2013
Expiration Date: Jul 2017
Storage: Room temperature.
Description: Clear colorless liquid
Sample Preparation: Used as received.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene (2AA), Acridine, 6-chloro-9-(3-((2-chloroethyl)amino)propyl)amino-2-methoxy, dihydrochloride (ICR-191), Daunomycin (DM),

Details on test system and experimental conditions:

Sample Preparation
Screen: 50 µI of the test article were brought to a volume of 1 ml with Acetone to yield a 50 µI/ml solution (stock for a dose of 5 µl/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).
Main Assay: 500 µI of the test article were brought to a volume of 10 ml with Acetone to yield a 50 µI/ml solution (stock for a dose of 5 µl/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).
Independent Repeat Assay: 500 µI of the test article were brought to a volume of 10 ml with Acetone to yield a 50 µl/ml solution (stock for a dose of 5 µI/plate) (clear colorless liquid). Additional dilutions were made with Acetone (clear colorless liquids).

Preparation of the Tester Strains: Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. #26-555). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37°C ± 2°C with agitation. The cultures were used after they reached the late exponential growth phase as determined by absorbance readings at 600 nm.

Exogenous Metabolic Activation: An activation buffer containing 10% S9 obtained from the livers of Aroclor 1254-treated adult Sprague Dawley rats was prepared according to the manufacturer's (Moltox) instructions. Each vial of Regensys B (Moltox cat# 60-201) was reconstituted with approximately 1 ml of Regensys A (Moltox cat# 60-200). The solution was transferred back into the Regensys A bottle S9 (Moltox cat# 11-101) was then mixed with Regensys A+B to yield a 10% S9 buffer stock solution. The stock was separated into aliquots in sterile 15 ml conical tubes and refrigerated at 2-8°C until used.
The exogenous metabolic activation mixture was added to one set of all doses - each test article concentration, vehicle control and positive control for each of the bacterial tester strains.

Treatment of the Test System: Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50°C in sterile culture tubes. Dulbecco's Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or -S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, vehicle control or positive control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overiaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37°C ± 2°C for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8°C until scoring.
An independent repeat (confirmatory) test was dosed using a preincubation method. One-hundred microliters of the test article concentration or Control was preincubated with 0.1 ml of the bacteria tester strain, along with 0.5 ml of Phosphate Buffered Saline or the metabolic activation (S9) system. These mixtures were incubated for 20 minutes or more at approximately 3rC prior to being mixed with the overlay agar and poured onto the surface of minimal agar plates. At the request of the Sponsor, the independent repeat (confirmatory) test was aborted after the plates were dosed.

Screen: Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O), Dimethyl sulfoxide (DMSO), and Acetone. The test article was freely soluble at a concentration of 50 µI/ml only in Acetone and the Study Director chose Acetone as the vehicle for the study A cytotoxicity screen was conducted in the TA-100 tester strain using eight concentrations (0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 µI/plate) of the test article, two plates per concentration, with and without S9. A vehicle control (Acetone) was run concurrently, with and without S9. The plates were incubated at 37°C ± 2°C for 48-72 Hours

Main Assay: Five concentrations (0.05, 0.1, 0.5, 1 and 5 µI/plate) of the test article were tested in each of five bacterial tester strains (Escherichia coli WP2 uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and -S9). A vehicle control and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ± 2°C for 48-72 hours.

Revertant Colony Count: Counting of the revertants per plate was performed using an Alphalmager™ 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article.
The plates were also examined visually for test article precipitate.

Independent Repeat Assay: The guidelines recommend that equivocal results be clarified by further testing, preferably using a modification of experimental conditions (e.g. concentrations tested), and that negative results need to be confirmed on a case-by-case basis. Justification should be provided when confirmation of negative results is not considered necessary. There is no recommendation of an independent repeat assay when the assay results are clearly positive.
At the request of the Sponsor, an independent repeat assay (confirmatory test) was aborted after all tester strains had been dosed with test material and plated but before the plates were scored.

Quality Check of the Assay:
Sterility Test: Sterile technique is required throughout the course of the study. To ascertain each component's sterility, a small amount (1 ml) was placed on agar plates and incubated to encourage growth of any contaminating microorganisms. The plates were incubated for 48-72 hours at 37°C ± 2°C and visually observed for microbial growth.
Vehicle Control: The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was calculated and compared to in-house historical ranges.
Positive Control: Positive control treatment for each tester strain of bacteria must result in at least a 2-fold increase of revertants over the mean vehicle control value. The effectiveness of the exogenous metabolic activation mixture was demonstrated by the positive response of the control.

Rationale for test conditions:

In accordance with test guidelines

Evaluation criteria:

Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.
In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose-related increases approaching a 2-fold increase are deemed equivocal.
A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.
Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.

Statistics:

Not specified in the study report.

Key result

Species / strain:

S. typhimurium, other: TA97a, TA1535, TA98 and TA100

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:

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

Additional information on results:

Screen: There was no diminution or clearing of the background lawn observed at any dose level, indicating that the test article was not cytotoxic to tester strain TA-100 at 0.001 to 5 µI/plate. The Study Director chose 5 µI/plate as the top test article concentration for the main test.

Main Assay: The assay was run in all five strains on triplicate plates, Positive and vehicle controls were run concurrently for all five strains, on six plates per strain. All plating was with and without exogenous metabolic activation, S9. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article in the presence or absence of S9, All Positive and Negative Control values were within acceptable ranges, and all criteria for a valid study were met. However, TA-98 (with and without S9) and WP2 uvrA (with S9) initially did not pass QC parameters and had to be repeated. TA-98 with S9 did not pass the negative control QC parameters (average revertant colonies higher than historical range) but passed positive control parameter (greater than 2-fold increase over negative control), TA-98 without S9 did not pass either QC parameter and WP2 uvrA with S9 did not pass the positive control QC parameter, Upon repeating dosing for these two strains (TA-98 with and without S9, and WP2 uvrA with S9) all QC parameters were within acceptable ranges.

Independent Repeat Assay: An independent repeat assay was dosed in all five tester strains using test article concentrations of 0.05, 0.1, 0.5, 1, and 5 µI/plate and the pre-incubation dosing method. However, at the request of the Sponsor the assay was terminated before the plates were scored.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: E.coli EP2 uvrA

Treatment	S9	CFU						Mean	Std. Dev.	Std. Error of Mean	Old Increase over Vehicle
Acetone (Vehicle Control)	+	63	50	51	48	43	52	51.3	6.5	2.7
5µl/plate	+	49	61	61				57.0	6.9	4.0	1.1
1µl/plate	+	47	65	54				55.32	9.1	5.2	1.1
0.5µl/plate	+	57	55	60				57.3	2.5	1.5	1.1
0.1µl/plate	+	47	54	61				54.0	7.0	4.0	1.1
0.05µl/plate	+	47	53	53				51.0	3.5	2.0	1.0
10µg 2AA (Positive Control)	+	132	131	166	152	121	134	141.0	14.7	6.0	2.7*
Acetone (Vehicle control)	-	73	41	98	98	119	83	85.3	26.7	10.9
5µl/plate	-	49	107	38				64.7	37.1	21.4	0.8
1µl/plate	-	95	94	75				88.0	11.3	6.5	1.0
0.5µl/plate	-	62	76	94				77.3	16.0	9.3	0.9
0.1µl/plate	-	45	59	68				57.3	11.6	6.7	0.7
0.05µl/plate	-	69	50	37				52.0	16.1	9.3	0.6
2.5µl MMS (Positive Control)	-	238	440	421	394	368	338	366.5	72.7	29.7	4.3*

*= 2-fold or more increase over Vehicle Control

 

Table 2

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: S. typh. TA-97a

Treatment	S9	CFU						Mean	Std. Dev.	Std. Error of Mean	Old Increase over Vehicle
Acetone (Vehicle Control)	+	63	54	68	61	81	88	69.2	12.9	5.3
5µl/plate	+	57	78	73				69.3	11.0	6.3	1.0
1µl/plate	+	75	80	80				78.3	2.9	1.7	1.1
0.5µl/plate	+	72	71	77				73.3	3.2	1.9	1.1
0.1µl/plate	+	77	75	59				70.3	9.9	5.7	1.0
0.05µl/plate	+	75	65	53				64.3	11.0	6.4	0.9
10µg 2AA (Positive Control)	+	611	564	541	497	496	468	529.5	52.8	21.5	7.7*
Acetone (Vehicle control)	-	40	52	46	58	63	56	52.5	8.4	3.4
5µl/plate	-	54	69	59				60.7	7.6	4.4	1.2
1µl/plate	-	57	59	65				60.3	4.2	2.4	1.1
0.5µl/plate	-	56	57	82				68.3	13.1	7.5	1.3
0.1µl/plate	-	69	58	65				64.0	5.6	3.2	1.2
0.05µl/plate	-	66	69	47				60.7	11.9	6.9	1.2
1µg ICR191 (Positive control)	-	1044	984	936	930	1091	1023	986.3	77.3	31.5	18.8*

*= 2-fold or more increase over Vehicle Control

 

Table 3

Test Article: H2925 (CAS No. 68130-53-0), Lot# 2013090401

Tester Strain: S. typh. TA-1535

Treatment	S9	CFU						Mean	Std. Dev.	Std. Error of Mean	Old Increase over Vehicle
Acetone (Vehicle Control)	+	10	8	10	13	12	9	10.3	1.9	0.8
5µl/plate	+	9	16	11				12.0	3.6	2.1	1.2
1µl/plate	+	5	10	9				8.0	2.6	1.5	0.8
0.5µl/plate	+	8	10	11				9.7	1.5	0.9	0.9
0.1µl/plate	+	10	11	14				11.7	2.1	1.2	1.1
0.05µl/plate	+	12	8	9				9.7	2.1	1.2	0.9
10µg 2AA (Positive Control)	+	47	44	36	44	48	52	45.2	5.4	2.2	4.4*
Acetone (Vehicle control)	-	11	12	7	7	16	8	10.2	3.5	1.4
5µl/plate	-	17	14	13				14.7	2.1	1.2	1.4
1µl/plate	-	8	13	6				9.7	2.9	1.7	1.0
0.5µl/plate	-	9	14	12				11.7	2.5	1.5	1.1
0.1µl/plate	-	11	5	12				9.3	3.6	2.2	0.9
0.05µl/plate	-	11	5	10				8.7	3.2	1.9	0.9
1.5µg NaN3(Positive Control)	-	545	548	534	552	515	482	529.3	26.7	10.9	51.9*

*= 2-fold or more increase over Vehicle Control

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.

Executive summary:

Objective: The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 - Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 - Bacterial Reverse Mutation Test.

 

Method Synopsis: Prior to the cytotoxicity screen, solubility of the test article was checked in tissue culture water (TCH2O), Dimethyl sulfoxide (DMSO), and Acetone. The test article was freely soluble at a concentration of 50µI/mlonly in Acetone and the Study Director chose Acetone as the vehicle for the study. A cytotoxicity screen was conducted in the Salmonella typhimurium TA-100 tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5µI/plate)of the test article, two plates per dose. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlaid onto minimal glucose agar plates. An Acetone vehicle control was run concurrently, with and without S9.

Based on the cytotoxicity results, five concentrations (0.05, 0.1, 0.5, 1 and 5µI/plate)of the test article were tested in each of five bacterial tester strains(E.coliWP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Vehicle controls and positive controls specific to each bacterial strain were treated in a similar manner as the test article concentrations. The plates were incubated at 37°C ± 2°C for 48-72 hours. Revertant colony growth was determined by counting the colonies per plate using an Alphalmager™ imaging system. The number of revertants of the test article treatment plates and positive control plates was divided by the number of revertants of the vehicle plates. In general, a positive result is determined by a 2-fold increase above the vehicle control.

Tester strains WP2 uvrA (with S9) and TA-98 (with and without S9) failed the quality checks. The main test was repeated for these strains and passed the quality checks. The results of the repeat main test are reported; the original data is maintained in the study file.

At the request of the Sponsor, an independent repeat assay (confirmatory test) was aborted after all tester strains had been dosed with test material and plated but before the plates were scored.

 

Summary: Test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, in the vehicle, Acetone, was tested in a Bacterial Reverse Mutation Assay. In the screen, the test article did not show obvious cytotoxicity to tester strain TA-100 at any concentration, with or without S9. In the main test, the test article at 0.05 to 5µI/plate,with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

 

Conclusion: Under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

06. Oct. - 16. Feb. 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD). (Purity of test substance not given, evalation criteria not given.) For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagles essential medium with HEPES buffer (MEM), supplemented with:
L-glutamine, penicillin/streptomycin, amphotericin B, 15% foetel calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Experiment I:
4 hour (with and without): 240, 320, 400 µg/mL

Experiment II
4 hour (with): 240, 320, 400 µg/mL
24 hour (without): 240, 320, 400 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

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

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

no data

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's exact test.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: cloudy precipitates were observed at and above 40 and 80 µg/mL in the 24-hour continuous and 4-hour pulse treatment groups, respectively

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

COMPARISON WITH HISTORICAL CONTROL DATA: The results are in range with historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 3 + 4: Test results of experiment I.

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

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

Table5 +6: Mean Frequency of Polyploid Cells (%)

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

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

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

26 Mar - 08 June 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study. For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from rats pretreated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

First experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (12%, v/v))

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

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

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: No
- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively.
- Expression time (cells in growth medium): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

In naccordance with test guidelines.

Evaluation criteria:

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at and above 333 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 333 µg/mL

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h respectively with a number of increasing test substance concentrations. The highest concentration tested was 750 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h or 24 h incubation.

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Results of experiment 1

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

 

Table 2: Results of experiment 2

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

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Conclusions:

Interpretation of results (migrated information):
negative

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May - 29 Aug 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

September 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 1995

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (Spraque Dawley rats, male, Aroclor 1254 induced)

Test concentrations with justification for top dose:

0, 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide (SA; 1 μg/plate, TA1535 and TA100); 9-Aminoacridine (9AA; 75 µg/plate, TA 1537); 2-Nitrofluorene (2NF; 1 µg/plate, TA98 and TA 1538); Methylmethanesulfonate (MMS; 1000 µg/plate, WP2 uvrA) with S9: 2-Amino-anthracene (2-AA; 1 μg/pl

Details on test system and experimental conditions:

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

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

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial backgroung lawn wit a dissecting microscope

Evaluation criteria:

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

Statistics:

Mean and standard deviation were calculated

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

other: An unacceptable vehicle control value with the tester strain TA1537, contamination of tester strain TA98, experiments were repeated

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: >100 µg/plate

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Experiment 1:

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 1535	TA1537	TA98	TA100	TA 1538	WP2uvrA
-	Vehicle	12	4	17	111	5	21
	10	3	4	15	115	6	16
	33	5	5	19	113	6	11
-	100	9	4	19	98	7	12
-	333	7	5	11	116	4	10
-	1000	8	6	21	120	7	13
Positive controls - S9	Name	SA	9AA	2NF	SA	2NF	MMS
	Concentrations (μg/plate)	1.0	75	1.0	1.0	1.0	1000
	Number of colonies/plate	241	40	105	377	179	143
+	Vehicle	14	5	18	133	7	11
	10	9	5	27	114	12	16
+	33	9	3	21	113	8	13
+	100	8	7	26	108	9	13
+	333	9	4	27	115	6	8
+	1000	10	5	17	111	9	13
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1.0	1.0	1.0	1.0	1.0	10
	Number of colonies/plate	72	127	888	904	783	57

SA: Sodium azide

9AA : 9-Aminoacridine

MMS: Methylmethanesulfonate

2-AA: 2-Aminoanthracene

2NF: 2-Nitrofluorene

 

 

Experiment 2/3:

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 1535	TA1537	TA98	TA100	TA 1538	WP2uvrA
-	Vehicle	14	10	23	126	11	27
	10	7	13	16	117	7	27
	33	9	15	23	124	8	19
-	100	5	13	22	120	6	18
-	333	12	9	17	110	11	16
-	1000	8	14	17	125	5	21
Positive controls - S9	Name	SA	9AA	2NF	SA	2NF	MMS
	Concentrations (μg/plate)	1.0	75	1.0	1.0	1.0	1000
	Number of colonies/plate	429	757	125	601	221	195
+	Vehicle	8	5	19	147	14	24
	10	9	7	17	142	16	30
+	33	10	5	19	136	18	25
+	100	10	5	20	132	13	31
+	333	10	4	17	138	12	19
+	1000	10	7	19	125	12	19
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1.0	1.0	1.0	1.0	1.0	10
	Number of colonies/plate	85	97	530	647	1041	88

SA: Sodium azide

9AA : 9-Aminoacridine

MMS: Methylmethanesulfonate

2-AA: 2-Aminoanthracene

2NF: 2-Nitrofluorene

Conclusions:

Interpretation of results (migrated information):
negative Under the tested experimental conditions the test substance did not induce gene mutations in S. typhimurium strains and in an E. coli strain up to the maximum of solubility. Therefore it is not considered to be mutagenic in this bacterial mutagenicity test.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May - 28 Oct 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. For read-across, maximum reliability score is 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adpoted in 1995

Deviations:

yes

Remarks:

Only basic data on test substance given

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy´s 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/ml and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix (Spraque-Dawley)

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
Chromosomal abberation assay: (157, 313), 625, 1250, 2500, 5000 µg/mL. Concentrations 157 and 313 µg/mL were not evaluated for CA

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, 0.08 and 0.15 µg/mL, -S9; mitomycin C 10 µg/mL, +S9

Remarks:

0.08 and 0.15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h without S9 activation and 4 h with activation.

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) at each concentration used.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

Criteria for cytotoxicity of the test substance: (1) cell growth inhibition relative to the solvent control
Criteria for chromosomal damage: (1) Number and types of aberrations found, the percentage of structurally and numerically damaged cells in the total population were counted.

Statistics:

The frequency of structural aberrations per cell was calculated. The statistical analysis of the percent aberrant cells was performed with Fisher´s Exact Test. It was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. In the event of positive control, the Cochran-Armitage test was used to measure dose- responsiveness.
The dose response was estimated by linear regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

48% in the highest dose for treatment of 4 h without metabolic activation, 33% with metabolic activation and 3% for 20 h exposure without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 8 in the highest dose
- Effects of osmolality: 285 in the highest dose

RANGE-FINDING/SCREENING STUDIES: Yes, cell growth inhibition of 83% without and 3% with metabolic activation at the highest dose tested (5000 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Chromosomal aberration – Summary

Treatment [µg/ml]	S9 activation	Treatment/ harvest time [h]	Mitotic index	Cells scored	Aberrations/cell (mean±SD)	Cells with aberrations [%]
						numerical	structural
Vehicle (Ethanol)	-	4/20	6.9	200	0.005±0.071	2.5	0.5
625	-	4/20	5.9	200	0.000±0.000	2.0	0.0
1250	-	4/20	5.9	200	0.025±0.186	4.0	2.0
2500	-	4/20	6.5	200	0.025±0.186	2.5	2.0
5000	-	4/20	4.4	200	0.000±0.000	0.5	0.0
MMC (0.08)	-	4/20	6.1	200	0.150±0.788	3.5	9.0*
Vehicle (Ethanol)	+	4/20	7.8	200	0.025±0.157	2.0	2.5
625	+	4/20	7.0	200	0.020±0.140	1.0	2.0
1250	+	4/20	6.9	200	0.035±0.184	3.0	3.5
2500	+	4/20	7.4	200	0.030±0.222	2.0	2.0
5000	+	4/20	7.7	200	0.010±0.100	2.5	1.0
CP (10)	+	4/20	2.3	200	0.950±1.591	2.5	45.5*
Vehicle (Ethanol)	-	20/20	7.6	200	0.020±0.140	1.0	2.0
625	-	20/20	6.4	200	0.015±0.158	1.5	1.0
1250	-	20/20	5.5	200	0.025±0.186	2.0	2.0
2500	-	20/20	6.0	200	0.025±0.157	2.5	2.5
5000	-	20/20	6.2	200	0.020±0.172	2.0	1.5
MMC (0.08)	-	20/20	5.8	200	0.220±0.513	1.0	18.0*

MMC = Mitomycine C

CP = Cyclophosphamide

* = p≤0.01; Fisher´s exact test

Conclusions:

Interpretation of results (migrated information):
negative

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

23 Feb - 26 Jul 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (no details on analytical purity of the test substance given).

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no details on analytical purity of the test substance given

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

no details on analytical purity of the test substance given

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

yes

Remarks:

no details on analytical purity of the test substance given

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: in the absence of S9 mix: McCoy's 5A Medium containing 10% foetal bovine serum and 2 mM L-glutamine; in the presence of S9 mix: serum-free McCoy's 5A Medium containing 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Range-finder toxicity test: 0, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL (3 h treatment), with and without S9
Initial and repeat experiment (main assay): 25, 75, 250, 750 and 2500 µg/mL (3 h treatment), with and without S9
Chromosome aberration analysis: 75, 750, and 2500 µg/mL (without S9); 25, 250 and 2500 µg/mL (with S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: a vehicle solubility test was previously performed to assess the solubility of the test substance in DMSO, water and acetone. The results of this test showed that the test substance was soluble in acetone at the concentrations required for this study. The non-cytotoxic dose volume for acetone is 50 µL/flask, therefore based on solubility, 2500 µg/mL was the highest dose that could be achieved in this assay.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

- S): 9,10-dimethyl- 1,2-benzanthracene (DMBA, 10 µg/mL in acetone); + S9: 1-methyl-3-nitro-1-nitrosoguanidine (MNNG, 0.6 µg/mL in acetone

Positive control substance:

9,10-dimethylbenzanthracene

other: 1-methyl-3-nitro-1-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation or harvest of cells):
A) Range-finder toxicity test: 19 h
B) Initial experiment (main assay): 19 h
C) Repeat experiment (main assay): 19 and 43 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL Colcemid®
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell confluency (range-finder toxicity test and main assay); other: cell count (range-finder toxicity test)

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

For a test substance to be considered as positive, one of the following conditions must be met:
- a statistically significant dose-related increase in the mean percentage of aberrant cells and, in at least one of the treatment groups, the mean percentage of aberrant cells exceeds 5%.
OR
- a reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that the test substance induces chromosomal aberrations in cultured mammalian somatic cells. If neither of the above conditions are met, the test substance is considered as non-mutagenic in this system.

Statistics:

The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were used for statistical analysis. The number of aberrant individual chromosomes per cell was not statistically analysed.

The results of the positive control group were compared to the appropriate vehicle control group by the Fisher Exact Test to assure that the assay was performed in an appropriate manner.

To test for homogeneity of the replicates, each pair of replicates was compared by Fisher Exact Test. Two times the sum of the log of the individual two-sided significance levels was compared to chi-square distribution with 2k degrees of freedom (k is the number of replicate pairs). If the test failed, further investigation would be pursued and the remaining analyses would not be performed.

To test for differences among the control and the treated groups, a 2x2-Fisher Exact Test was performed. If differences were shown to exist at the 0.05 level or less, individual 2x2-Fisher Tests were performed to determine which of the treated groups differed from the control group. A permutation test (Hoeffding, 1952) was performed to test for dose-related trends. Statistical analysis included the calculation of means for percent confluency, percent mitotic cells, percent aberrant cells, percent frequency of aberrations, and cell counts. Significance was reported when p < 0.05.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: in the range-finder toxicity test, visual solubility observations were made 3 h after treatment. Cloudiness was evident at all concentrations with S9. Complete solubility was observed at concentrations ranging from 10 to 39 µg/mL without S9. Cloudiness was evident at concentrations of 78 µg/mL through 625 µg/mL without S9. Oil droplets were noted without metabolic activation at concentrations ≥ 1250 µg/mL. A notable decrease in confluency (≥ 50% reduction compared with vehicle controls) were observed at concentrations of 625 µg/mL and 1250 µg/mL with S9 and 2500 µg/mL without S9. Decreases in cell survival and mitotic index (MI) of ≥ 50% from the vehicle control were not observed in either the activated or non-activated series. Cell morphology was normal at all concentrations tested, however debris of undetermined origin was noted in activated flasks at concentrations ≥ 156 µg/mL and non-activated flasks ≥ 313 µg/mL. Based on these results, 25, 75, 250, 750 and 2500 µg/mL were selected as concentrations for the main chromosomal aberration assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in both experiments of the main study, there was no notable decrease in the percent confluency (≥ 50% reduction compared to the vehicle control) at any concentration with or without S9. Cell morphology was normal at all concentrations tested. Similarly, no notable decrease (≥ 50%) in MI values was noted for the test substance concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

CHROMOSOME ANALYSIS

A) INITIAL ASSAY

There were no statistically significant differences or dose-related trends in the percentage of cells with chromosomal aberrations between the treated and the control groups either with or without metabolic activation. The percentage of aberrant cells in the vehicle control groups, with and without metabolic activation, was 2.5 and 1.0%, respectively. Both values fell within the 0 to 5% range of acceptance for the vehicle control. The percentage of aberrant cells in the treatment groups ranged from 2.0 to 3.0% for the metabolically activated series and from 0.5 to 3.0% in the non-activated series.

Table 1. Test results of the initial experiment

Test item	Concentration in µg/mL	Mitotix index in % of vehicle control	Aberrant cells in % per 200 cells scored	Frequency of abberations in % per 200 cells scored
				with gaps	without gaps
Exposure period 3 h, fixation time 19 h, without S9 mix
Acetone	a	100	1.0	1.5	1
MNNG	0.6	26	12.0**	5.0	12.5
Test substance	75	77	0.5	2.5	0.5
	750	80	3.0	1.0	3
	2500	54	2.0	0.0	2
Exposure period 3h, fixation time 19 h, with S9 mix
Acetone	a	100	2.5	2.5	3.0
DMBA	10	59	19.0**	5.0	26.0
Test substance	25	112	3.0	3.0	3.0
	250	100	2.5	1.5	2.5
	2500	104	2.0	3.0	2.0

MNNG = 1-methyl-3-nitro-1-nitrosoguanidine; DMBA = 9,10-dimethyl-1,2-benzanthracene

a = concentration: 50 µL/flask; b = positive controls were not required for the 43 h harvest

*p < 0.05; **p < 0.01

B) REPEAT ASSAY

There was a statistically significant difference (p < 0.05) in the percentage of cells with chromosomal aberrations between the vehicle and the highest concentration (2500 µg/mL) in the 19 h harvest with metabolic activation. The permutation test for the 19 h harvest with metabolic activation also appears to be significant (p<0.01) indicating a dose-related trend in the percentage of cells with chromosomal aberrations. This increase in the percentage of aberrant cells does not appear to be biologically significant due to the fact that the percentage of aberrant cells fell within the acceptable range for the vehicle control (0-5%).

The percentage of aberrant cells in the vehicle control groups at both harvest intervals (with and without metabolic activation) was 2.5% or less. These values fell within the 0 to 5% range of acceptance for the vehicle control. The percentage of aberrant cells observed in the 19 h treated groups ranged from 1 to 3.0%. The percentage of aberrant cells observed in the 43 h treated groups ranged from 0.5% to 2.5%.

Table 2. Test results of the repeat experiment

Test item	Concentration in µg/mL	Mitotix index in % of vehicle control	Aberrant cells in % per 200 cells scored	Frequency of abberations in % per 200 cells scored
				with gaps	without gaps
Exposure period 3 h, fixation time 19 h, without S9 mix
Acetone	a	100	1.0	1.0	1.0
MNNG	0.6	42	8.0**	2.0	13.5
Test substance	75	89	2.0	2.5	2.5
	750	65	1.5	1.0	1.5
	2500	82	1.5	1.0	2.0
Exposure period 3 h, fixation time 43 h, without S9 mix
Acetone	a	100	2.5	2.5	2.5
MNNG	0.6	b	b	b	b
Test substance	75	105	0.5	1.0	0.5
	750	95	0.5	1.5	0.5
	2500	68	1.5	2.0	1.5
Exposure period 3 h, fixation time 19 h, with S9 mix
Acetone	a	100	0.0	1.5	0.0
DMBA	10	66	10.5**	7.0	11.5
Test substance	25	107	1.0	1.0	1.0
	250	105	1.0	1.0	1.0
	2500	60	3.0* #	1.0	3.5
Exposure period 3 h, fixation time 43 h, with S9 mix
Acetone	a	100	1.0	1.0	1.0
DMBA	0.6	b	b	b	b
Test substance	25	89	2.5	2.5	2.5
	250	70	1.5	1.0	1.5
	2500	68	2.0	0.0	2.0

MNNG = 1-methyl-3-nitro-1-nitrosoguanidine; DMBA = 9,10-dimethyl-1,2-benzanthracene

a = concentration: 50 µL/flask; b = positive controls were not required for the 43 h harvest

*p < 0.05; **p < 0.01

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

In an Ames test (OECD Guideline 471), under test conditions, test article H2925 (CAS No. 68130-53-0), Lot# 2013090401, did not have mutagenicity potential.

In an Ames Test with read-across substance, decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS 11138-60-6), was not mutagenic with or without metabolic activation. In an in vitro chromosome aberration test, the same read-across substance was not clastogenic in the CHO cell culture test system, with or without metabolic activation. Regardless of dose level (from 625 g/ml to as high as 5000 g/ml) and dosing regimen, the test substance was concluded to be negative for structural and numerical chromosome aberrations, with or without metabolic activation.

Two further in vitro chromosome aberration tests were conducted with read-across substances, CAS 189120-64-7 and CAS 403507-18-6; both substances provided negative results for structural and numerical chromosome aberrations, with or without metabolic activation.

An in vitro Mammalian Cell Gene Mutation Test was conducted with the structural analogue substance with CAS No. 85186-89-6 (Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane) according to OECD Guideline 476. Mouse lymphoma L5178Y cells were dosed with 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/ml with and without metabolic activation. No signs of toxicity were observed, DMSO was used as vehicle, precipitation was observed at and above 100 µg/ml. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, indicated by the total number of colonies per plate.

Therefore, in conclusion, the test substance is considered to be non‑mutagenic.

Justification for selection of genetic toxicity endpoint

Klimisch 1 study.

Justification for classification or non-classification

Based on the results of above studies with closely related structural analogues, the substance does not need to be classified according to the CLP Regulation (EC) 1272/2008.

A report justifying the read-across approach is included in IUCLID Chapter 13.