Barium(2+) 12-hydroxyoctadecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not show mutagenic properties.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-01-23 to 2018-03-26

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

440/2008/EC, 31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1):

Version / remarks:

June 2012

Deviations:

no

Principles of method if other than guideline:

ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

Target gene:

his/trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

5000; 1600; 500; 160; 50; 16 and 5 μg/plate (±S9 Mix)
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).

Vehicle / solvent:

- Vehicle used: Ethanol
- Justification for choice of vehicle: This vehicle is compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol, ultrapure water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

TA98, 4µg/pate, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol, ultrapure water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA1535; 2 µg/plate, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol, ultrapure water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, 50 µg/plate, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol, ultrapure water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2 uvrA, 2 µL/pate, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol, ultrapure water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All S.typhimrium tester strains, 2 µg/plate; E.coli tester strain 50 µg/plate; with S9 mix

Details on test system and experimental conditions:

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

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies and the background lawn of auxotrophic cells

Evaluation criteria:

The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = mena revertants at the test item (or control) treatments / mean revertants of vehicle control
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was noticed on the plates in the concentration range of 5000-500 μg/plate (±S9 Mix) following plate incorporation and pre-incubation procedures in the absence and presence of exogenous metabolic activation (±S9 Mix). The obtained precipitate did not disturb the scoring in any case.

Table 1: Summary Table of the Results of the Initial Mutation Test

Concentrations (μg/plate)	Salmonella typhimuriumtester strains																E. coli Wp2 uvrA
	TA 98				TA 100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	19.3	0.95	21.3	0.83	82.0	1.14	91.3	0.98	9.3	1.40	9.0	0.96	4.0	1.09	5.3	0.84	33.0	1.19	42.7	1.31
DMSO Control	17.7	1.00	24.0	1.00	–	–	80.7	1.00	–	–	8.7	1.00	3.7	1.00	4.7	1.00	–	–	33.0	1.00
Ultrapure Water Control	–	–	–	–	79.7	1.00	–	–	9.7	1.00	–	–	–	–	–	–	26.0	1.00	–	–
Ethanol Control	20.3	1.00	25.7	1.00	72.0	1.00	93.7	1.00	6.7	1.00	9.3	1.00	3.7	1.00	6.3	1.00	27.7	1.00	32.7	1.00
5000	13.3	0.66	16.7	0.65	55.7	0.77	84.3	0.90	4.0	0.60	7.3	0.79	3.7	1.00	5.3	0.84	31.7	1.14	38.3	1.17
1600	19.0	0.93	17.0	0.66	62.3	0.87	74.0	0.79	8.0	1.20	10.0	1.07	4.7	1.27	5.3	0.84	35.7	1.29	46.3	1.42
500	18.3	0.90	23.7	0.92	67.3	0.94	78.0	0.83	9.3	1.40	14.7	1.57	3.7	1.00	5.7	0.89	38.3	1.39	40.0	1.22
160	18.0	0.89	22.0	0.86	75.7	1.05	76.0	0.81	9.0	1.35	12.0	1.29	7.7	2.09	10.0	1.58	29.3	1.06	38.0	1.16
50	16.7	0.82	24.7	0.96	67.7	0.94	89.7	0.96	9.3	1.40	9.7	1.04	5.3	1.45	5.3	0.84	30.0	1.08	35.0	1.07
16	16.7	0.82	25.3	0.99	67.3	0.94	83.0	0.89	12.3	1.85	11.3	1.21	4.0	1.09	5.7	0.89	34.0	1.23	36.7	1.12
5	27.3	1.34	23.3	0.91	76.0	1.06	87.0	0.93	7.3	1.10	11.0	1.18	7.0	1.91	7.0	1.11	36.7	1.33	41.7	1.28
NPD (4 μg/plate)	481.3	27.25	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg/plate)	–	–	–	–	796.7	10.00	–	–	1093.3	113.10	–	–	–	–	–	–	–	–	–	–
9AA (50 μg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	581.3	158.55	–	–	–	–	–	–
MMS (2 μL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	562.7	21.64	–	–
2AA (2 μg/plate)	–	–	1290.7	53.78	–	–	1629.3	20.20	–	–	194.3	22.42	–	–	123.0	26.36	–	–	–	–
2AA (50 μg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	215.3	6.53
MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene Remarks: Ethanol was applied as vehicle for the test item, ultrapure water was applied as vehicle of the positive control substances SAZ and MMS and DMSO was applied as vehicle of NPD, 9AA and 2AA. The mutation rate of the test item and the untreated control is given referring to the ethanol, the mutation rate of SAZ and MMS is given referring to ultrapure water and the mutation rate of NPD, 9AA and 2AA is given referring to the DMSO.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Concentrations (μg/plate)	Salmonella typhimuriumtester strains																E. coli Wp2 uvrA
	TA 98				TA 100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	23.3	0.97	28.7	1.05	87.7	1.12	108.7	1.06	11.3	1.26	12.0	1.16	8.7	1.30	5.7	1.13	32.7	1.15	35.3	0.96
DMSO Control	17.3	1.00	24.0	1.00	–	–	90.3	1.00	–	–	9.7	1.00	7.3	1.00	6.3	1.00	–	–	40.3	1.00
Ultrapure Water Control	–	–	–	–	80.0	1.00	–	–	12.0	1.00	–	–	–	–	–	–	35.7	1.00	–	–
Ethanol Control	24.0	1.00	27.3	1.00	78.0	1.00	102.7	1.00	9.0	1.00	10.3	1.00	6.7	1.00	5.0	1.00	28.3	1.00	36.7	1.00
5000	12.0	0.50	20.0	0.73	71.3	0.91	82.7	0.81	7.3	0.81	8.7	0.84	2.7	0.40	6.3	1.27	33.7	1.19	37.0	1.01
1600	18.3	0.76	20.0	0.73	75.3	0.97	89.7	0.87	8.7	0.96	14.3	1.39	6.0	0.90	8.3	1.67	35.0	1.24	33.7	0.92
500	34.7	1.44	31.0	1.13	77.3	0.99	84.7	0.82	7.7	0.85	10.0	0.97	6.3	0.95	5.7	1.13	39.0	1.38	38.7	1.05
160	22.0	0.92	24.0	0.88	74.0	0.95	91.3	0.89	8.7	0.96	14.3	1.39	6.0	0.90	7.0	1.40	33.3	1.18	37.7	1.03
50	21.7	0.90	26.3	0.96	74.3	0.95	89.3	0.87	7.7	0.85	10.7	1.03	8.0	1.20	8.3	1.67	39.0	1.38	40.0	1.09
16	21.3	0.89	28.7	1.05	86.3	1.11	87.7	0.85	7.7	0.85	12.0	1.16	5.0	0.75	7.3	1.47	34.0	1.20	35.0	0.95
5	25.7	1.07	31.3	1.15	87.7	1.12	98.3	0.96	8.7	0.96	13.0	1.26	6.0	0.90	7.7	1.53	34.7	1.22	41.3	1.13
NPD (4 μg/plate)	566.7	32.69	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg/plate)	–	–	–	–	976.0	12.20	–	–	682.3	56.86	–	–	–	–	–	–	–	–	–	–
9AA (50 μg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	472.7	64.45	–	–	–	–	–	–
MMS (2 μL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1040.0	29.16	–	–
2AA (2 μg/plate)	–	–	1442.7	60.11	–	–	1088.0	12.04	–	–	133.0	13.76	–	–	109.3	17.26	–	–	–	–
2AA (50 μg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	251.3	6.23
MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene Remarks: Ethanol was applied as vehicle for the test item, ultrapure water was applied as vehicle of the positive control substances SAZ and MMS and DMSO was applied as vehicle of NPD, 9AA and 2AA. The mutation rate of the test item and the untreated control is given referring to the ethanol, the mutation rate of SAZ and MMS is given referring to ultrapure water and the mutation rate of NPD, 9AA and 2AA is given referring to the DMSO.

Conclusions:

In the in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not show mutagenic properties.

Executive summary:

The mutagenic potential of the test item was assessed in an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). In the initial and confirmatory mutation tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2uvrA were investigated. The test item was suspended in ethanol. In the initial and confirmatory mutation tests the following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicates. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the Initial and Confirmatory Mutation Tests unequivocal inhibitory effects of the test item were not observed. The colony and background lawn development were not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. Precipitate was noticed on the plates in the concentration range of 5000-500 μg/plate (±S9 Mix) following plate incorporation and pre-incubation procedures in the absence and presence of exogenous metabolic activation (±S9 Mix). The obtained precipitate did not disturb the scoring in any case. In the present study, apart from three precipitated concentration levels- because of the very low water solubility of the test item - it was decided to test up to the maximum recommended dose level of 5000 μg/plate in order to confirm the absence of mutagenic potential.

The data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro (Ames test)

The mutagenic potential of the test item was assessed in an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). In the initial and confirmatory mutation tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2uvrA were investigated. The test item was suspended in ethanol. In the initial and confirmatory mutation tests the following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicates. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the Initial and Confirmatory Mutation Tests unequivocal inhibitory effects of the test item were not observed. The colony and background lawn development were not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. Precipitate was noticed on the plates in the concentration range of 5000-500 μg/plate (±S9 Mix) following plate incorporation and pre-incubation procedures in the absence and presence of exogenous metabolic activation (±S9 Mix). The obtained precipitate did not disturb the scoring in any case. In the present study, apart from three precipitated concentration levels- because of the very low water solubility of the test item - it was decided to test up to the maximum recommended dose level of 5000 μg/plate in order to confirm the absence of mutagenic potential.

The data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. In vitro results with the test item were negative. As a result the test substance is considered not to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.