Decanoic acid, mixed esters with heptanoic acid, isovaleric acid, octanoic acid and pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Test

Not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Chromosome Abberation Test and Mammalian Cell Gene Mutation Test

The substance, CAS 68130-51-8; EC  268-594-6, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. The substance is not considered to be mutagenic on the basis of read across.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26. Sep. 2017 to 06. Oct. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment: 5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate
The following nominal concentrations were prepared for the second experiment: 5 μL/plate, 2.5 μL/plate, 1.25 μL/plate, 0.63 μL/plate, 0.31 μL/plate, 0.16 μL/plate and 0.08 μL/plate
Justifcation not specified in the study report.

Vehicle / solvent:

Ethanol was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO), Demineralised water and Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine; 2-Amino-Anthracene

Details on test system and experimental conditions:

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix or phosphate buffer (for test without metabolic activation).
-100 μL bacteria suspension
-2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix or phosphate buffer (for test without metabolic activation).
-100 μL bacteria suspension. After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.

References and Validity
Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.

Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.

UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 seconds, the plates for the strain TA 98 were irradiated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W).
Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535.
Keeping a distance of 66 cm for the following strains: TA98, TA100.
Incubation for 24 hours at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough/rfa)
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs ( 9 mm), each soaked with 10 μL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.

Spontaneous Revertants
Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.

Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.

Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ±1°C, four replicates.

Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual inspection.

Positive Controls
Using diagnostic mutagens, three replicates were prepared.
The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.

Rationale for test conditions:

In accordance with the test guidelines.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

Not specified.

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.To verify this result, a further experiment was performed.

Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.

Mutagenicity of Test Item
The test item Hatcol 1570 showed no increase in the number of revertants in all bacteria strains in both experiments.
Nearly all negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Hatcol 1570 is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Acceptability of Study, Discussion
In all experiments, no precipitation of the test item Hatcol 1570 was observed at any of the tested concentrations up to 5 μL/plate.
In both experiments, the test item caused no cytotoxicity towards all bacteria strains
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.
Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.

Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	74	73	35	38	94	108	291	288	19	20
	sd	12.4	7.5	2.0	8.6	22.5	12.2	39.5	10.6	2.0	3.5
DMSO	Mean	83	81	35	37	83	86	248	225	20	15
	sd	22.7	18.5	3.2	4.0	1.2	7.5	24.0	97.9	2.5	3.6
Ethanol	Mean	79	83	35	43	98	93	211	225	15	16
	sd	9.2	22.3	4.0	9.1	12.4	2.5	62.0	66.3	4.6	4.6
Positive Controls*	Mean	587	1001	493	299	327	557	668	1488	219	144
	sd	86.3	0.0	69.0	18.5	39.3	12.2	34.9	34.9	68.0	17.8
	f(l)	7.07	12.36	14.09	8.08	3.48	6.48	2.69	6.61	11.53	9.60
5 μL/plate	Mean	96	76	38	39	79	113	271	297	24	23
	sd	12.9	4.6	7.0	7.0	13.1	15.5	28.1	24.1	4.4	2.6
	f(l)	1.22	0.92	1.09	0.91	0.81	1.22	1.28	1.32	1.60	1.44
1.5 μL/plate	Mean	88	73	39	39	107	103	261	315	21	20
	sd	32.1	8.0	7.2	2.1	6.4	15.1	50.0	37.0	3.1	4.5
	f(l)	1.11	0.88	1.11	0.91	1.09	1.11	1.24	1.40	1.40	1.25
0.5 μL/plate	Mean	96	85	60	68	99	81	244	319	19	23
	sd	12.2	10.5	2.3	8.9	7.8	6.1	54.1	68.0	2.1	7.2
	f(l)	1.22	1.02	1.71	0.88	1.01	0.87	1.16	1.42	1.27	1.44
0.15 μL/plate	Mean	90	82	38	34	83	92	332	271	17	17
	sd	15.0	16.1	1.2	3.2	16.5	19.1	27.7	44.1	4.2	3.0
	f(l)	1.14	0.99	1.09	0.79	0.85	0.99	1.57	1.20	1.13	1.06
0.05 μL/plate	Mean	74	77	33	36	86	112	181	209	18	16
	sd	1.5	6.5	2.1	3.1	7.2	22.7	8.1	15.1	4.5	5.0
	f(l)	0.94	0.93	0.94	0.84	0.88	1.20	0.86	0.93	1.20	1.00

f(l) = increase factor

* Different positive controls were used

 

Mean Revertants Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	90	79	35	37	114	100	307	356	22	23
	sd	9.0	7.6	6.9	5.3	18.7	16.4	10.1	10.6	3.5	2.9
DMSO	Mean	88	83	40	39	114	109	291	329	20	21
	sd	4.0	10.4	11.8	4.0	3.5	8.1	11.5	39.3	0.6	1.2
Ethanol	Mean	80	81	38	38	109	96	260	363	15	17
	sd	9.8	8.7	9.9	3.0	5.0	15.9	65.5	41.1	5.0	4.0
Positive Controls*	Mean	365	915	685	188	341	816	1235	1235	346	153
	sd	14.7	28.1	92.4	38.0	20.1	83.1	119.8	180.0	50.3	12.9
	f(l)	4.15	11.02	17.13	4.82	2.99	7.49	4.24	3.75	15.73	7.29
5 μL/plate	Mean	80	100	36	33	114	115	385	392	21	23
	sd	16.2	11.0	5.0	3.1	8.1	36.3	34.9	60.5	2.9	2.5
	f(l)	1.00	1.23	0.95	0.87	1.05	1.20	1.48	1.08	1.40	1.35
2.5 μL/plate	Mean	100	72	41	34	101	97	325	287	20	19
	sd	9.5	6.0	2.0	10.1	18.1	21.7	50.3	4.6	1.2	4.0
	f(l)	1.25	0.89	1.08	0.89	0.93	1.01	1.25	0.79	1.33	1.12
1.25 μL/plate	Mean	96	101	36	39	78	96	257	233	18	20
	sd	18.2	11.5	4.7	3.2	3.8	20.6	77.6	37.2	2.9	3.5
	f(l)	1.20	1.25	0.95	1.03	0.72	1.00	0.99	0.64	1.20	1.18
0.63 μL/plate	Mean	100	83	35	30	122	114	268	257	22	14
	sd	10.1	7.2	7.2	9.5	1.2	2.0	46.1	55.6	1.5	2.1
	f(l)	1.25	1.02	0.92	0.79	1.12	1.19	1.03	0.71	1.47	0.82
0.31 μL/plate	Mean	67	83	33	31	107	97	347	276	19	19
	sd	10.4	5.2	6.0	5.5	5.0	21.9	8.3	14.4	5.3	1.2
	f(l)	0.84	1.02	0.87	0.82	0.98	1.01	1.33	0.76	1.27	1.12
0.16 μL/plate	Mean	77	99	36	41	108	114	333	355	20	19
	sd	9.0	27.5	3.5	2.5	8.1	2.0	16.2	31.1	3.2	1.5
	f(l)	0.96	1.22	0.95	1.08	0.99	1.19	1.28	0.98	1.33	1.12
0.08 μL/plate	Mean	84	91	39	35	115	108	290	344	20	18
	sd	7.0	13.2	10.4	5.1	10.1	3.2	93.6	48.7	2.9	3.2
	f(l)	1.05	1.12	1.03	0.92	1.06	1.13	1.12	0.95	1.33	1.06

f(l) = increase factor

* Different positive controls were used

 

 

Historical Data of Spontaneous Revertants

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. H2O	Mean	91	96	17	19	92	97	280	298	17	17
	Min	60	63	6	8	51	64	85	67	6	7
	Max	144	138	52	50	141	141	425	511	31	33
	SD	19	16	9	8	16	15	62	74	6	6
	Exp 1	74	73	35	38	94	108	291	288	19	23
	Exp 2	90	79	35	37	114	100	307	356	22	23
DMSO	Mean	90	100	17	18	90	92	279	294	17	17
	Min	58	67	7	8	44	62	79	80	8	6
	Max	135	144	46	41	136	199	393	459	33	32
	SD	18	17	9	9	16	18	59	66	7	6
	Exp 1	83	81	35	37	83	86	248	225	20	15
	Exp 2	88	83	40	39	114	109	291	329	20	21
Ethanol	Mean	88	99	19	20	86	91	287	276	17	17
	Min	57	65	8	9	61	69	111	141	9	9
	Max	181	205	47	45	116	129	368	339	29	36
	SD	23	28	11	10	13	16	50	44	6	7
	Exp 1	79	83	35	43	98	93	211	225	15	16
	Exp 2	80	81	38	38	109	96	260	363	15	17
Positive Controls*	Mean	552	506	397	95	512	736	1142	1232	257	119
	Min	264	237	100	39	223	273	491	408	55	45
	Max	1152	1181	793	487	984	1912	2331	6083	484	712
	SD	165	148	139	74	151	300	443	636	86	77
	Exp 1	587	1001	493	299	327	557	668	1488	219	144
	Exp 2	365	915	685	188	341	816	1235	1235	346	153

*Different positive controls were used

Conclusions:

Based on the results of the study it is concluded that Hatcol 1570 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the study.

Executive summary:

Determination of the mutagenic potential of Hatcol 1570 with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

 

Findings and Results:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item Hatcol 1570 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

In the first experiment, the test item (dissolved in ethanol) was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Based on the first experiment, the test item was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of the study it is concluded that Hatcol 1570 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the study.

Reference 2

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

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:
negative

Reference 3

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)

Remarks:

2 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

See the full attached justification in section 13 for details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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

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

Remarks:

CAS 85186-89-6

Conclusions:

The substance, CAS 68130-51-8; EC 268-594-6, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of mutagenicity. The substance is not considered to be mutagenic on the basis of read across.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar - 08 June 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Reverse Mutation Test

The test item Hatcol 1570 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

In the first experiment, the test item (dissolved in ethanol) was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Based on the first experiment, the test item was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of the study it is concluded that Hatcol 1570 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the study.

Chromosome abberation test

In an in vitro chromosome aberration 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. 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.

In vitro Mammalian Cell Gene Mutation Test

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.

Justification for classification or non-classification

Based on the available in vitro ames test conducted on the substance and utilising the read across approach with a closely related structural analogue, 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.