Palmitoyl chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Palmitoyl chloride is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Available date for the source substances docosanoic acid CAS 112-85-6 and decanoic acid CAS 334-48-5.

Gene mutation (OECD 471/472): negative, CAS 112-85-6 (Nakajima, 2002)
Chromosome aberration (OECD 473): negative, CAS 112 -85 -6 (Nakajima, 2002)

Chromosome aberrration (similar to OECD 473 ): negative, CAS 334-48-5 (MHW Japan, 2018)

Gene mutation in mammalian cells (TK locus; OECD 476): negative; CAS 334-48-5 (Trenz, 2010)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle-MEM liquid medium
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
-S9 mix (24 h continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48-hour continuous exposure): 0, 288, 575, 1150, 2300 µg/mL

Vehicle / solvent:

1.0 % carboxymethylcellulose sodium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: continuous exposure: mitomycin C (0.05 µg/mL for 24 hours and 0.025 µg/mL for 48 hours); short-term exposure: cyclophosphamide (12.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: 6 (short-term exposure), 24, 48 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The frequency of polyploid cells or cells with abnormal structure of each test group were determined according to the criteria of Ishidate.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble in water, soluble in alcohol, ether, chloroform and acetone
- Precipitation: observed on the slide of continuous exposure high dose group

RANGE-FINDING/SCREENING STUDIES: see 'Any other information on material and method incl. tables'

COMPARISON WITH HISTORICAL CONTROL DATA: this test was valid, since the frequency of chromosomal aberration in positive control was within background data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test substance did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun - 23 Aug 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

yes

Remarks:

Several deviations based on the changes from OECD TG 476 (1997) to OECD TG 490 (2016).

Principles of method if other than guideline:

Deviations from OECD test guideline 490 (2016):
The cell line used for the MLA was described as L5178Y without further differentiation if the required TK+/- -3.7.2C subline of L5178Y cells was used. The expression period for the short-term exposure experiments was 3 days (instead of 2 days). The mutation frequency was calculated based on number of mutant colonies adjusted by cloning efficiency instead of determination of cloning efficiency for the selection and cloning efficiency plates. Differences in acceptability criteria, i.e. suspension growth was not assessed as acceptability criterium. The Global Evaluation factor (GEF) was not evaluated.

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

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 complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
: prepared at BSL BIOSERVICE GmbH
Male rats were induced with phenobarbital (80 mg/kg bw) and beta-naphtoflavone (100 mg/kg bw). A stock of the supernatant containing the microsomes was frozen and stored at <= 75°C. The protein concentration in the S9 preparation (Lot: 110310) was 31 mg/mL.
- method of preparation of S9 mix: according to Ames et al., 1977
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the culture. Cofactors were added to the S9 mix to reach the concentration below:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP in
in 100 mM sodium-phosphate-buffer pH 7.4.
- quality controls of S9: The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and biological activity in the chromosome aberration assay using benzo[a]pyrene and cyclophosphamide.
b) Sterility test

Test concentrations with justification for top dose:

Pre-Test:
Experiment 1:
- with and without metabolic activation: 0.5, 2, 4, 6, 8, 10 mM
Experiment 2:
- without metabolic activation. 0.005, 0.05, 0.2, 0.7, 1.3, 2.0 mM

Main Test:
Experiment 1:
- with metabolic activation: 0.70, 0.82, 0.94, 1.06, 1.18, 1.30, 1.42, 1.54 mM
- without metabolic activation. 0.22, 0.46, 0.58, 0.70, 0.82, 0.94, 1.06, 1.18 mM
Experiment 2:
- with metabolic activation: 1.0, 1.12, 1.24, 1.36, 1.48, 1.60, 1.72, 1.84 mM
- without metabolic activation. 0.0005, 0.001, 0.002, 0.005, 0.01, 0.06, 0.18, 0.3 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

since medium was used as solvent, no further solvent control was necessary

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: methylmethanesulfonate (10 µg/mL, dissolved in 0.9% NaCl); ethylemethanesulphanate (200 and 500 µg/mL, dissolved in medium); +S9: benzo(a)pyrene (3.5 µg/mL, dissolved in DMSO (1% final concentration in medium))

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h (short-term exposure) with and without metabolic activation
Experiment 2: 4 h (short-term exposure) with metabolic activation and 24 h (long-term exposure) without metabolic activation
- Expression time (cells in growth medium): 3 days (short-term exposure) or 2 days (long-term exposure)
- Selection time (if incubation with a selection agent): 11 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 18 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: single cultures per concentration each in two independent experiments; cells from each experimental group were seeded in 4 96-well-plates for determination of mutation frequencies

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; cloning efficiency

OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations
- Criteria for small (slow growing) and large (fast growing) colonies: Small colonies approx. <= 1/4 of well diameter; large colonies approx. > 1/4 of well diameter
- Method used: microwell plates for the mouse lymphoma assay

Evaluation criteria:

There are several criteria for a positive result:
- clear and dose-related increase in the mutant frequency
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/Small colonies ratio (1.5 times the ratio of clastogenic control MMS and/or B[a]P) is an indication for potential clastogenic effects and/or chromosomal aberrations.

The test substance is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1.54 mM with metabolic activation and at 1.18 mM without metabolic activation in experiment 1, respectively; at 1.84 mM with metabolic activation and at 0.30 mM without metabolic activataion in experiment 2, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: within the physiological range
- Effects of osmolality: within the physiological range
- Precipitation: in the pre-test with metabolic activation from concentrations of 4 mM and higher

RANGE-FINDING/SCREENING STUDIES:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)

COMPARISON WITH HISTORICAL CONTROL DATA:
All mutant values were found to be within the range of the historical negative control data of the test facility (about 51 to 170 mutants per 10^6 cells). For more information on the historical control data (HCD), please refer to Table 5 under "Any other information on results incl. tables".

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Growth inhibition was observed in both experiments with and without metabolic acitivation:
In experiment I with S9 the relative total growth (RTG) was 9.84% for the highest concentration (1.54 mM) evaluated. The highest concentration evaluated without S9 was 1.18 mM with a RTG of 11.04%.
In experiment II with S9 the RTG was 19.98% for the highest concentration (1.84 mM) evaluated. The highest concentration evaluated without S9 was 0.30 mM with a RTG of 19.77%.

- Genotoxicity results:
In experiment I with S9 mix all mutant values found were within the HCD of the test facility. A slight dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase compared to the negative controls. Mutation frequencies of the negative controls were found to be 93.87 and 79.67 mutants/10^6 cells and in the range of 76.43 and 139.92 mutants/10^6 cells with the test item, respectively. The highest mutation factor was 1.61 compared to the negative controls found at a concentration of 1.54 mM with a RTG of 9.84%. Without metabolic activation no dose-response relationship or any biologically rel
evant increase compared to the negative controls were observed. In experiment II with and wihout S9 mix all mutant values were within the HCD of the test facility. No dose-response relationship nor any biologically relevant increase were observed. For more information, please refer to the tables under "Any other information on results incl. tables".

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colony ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5), is an indication for potential clastogenic effects and/or chromosomal aberrations.

Although in experiment 1 with metabolic activation an increased number of small colonies was noted at doses of 1.30 mM and 1.42 mM (26 and 31 small colonies, respectively, compared to 11 and 9 at control) all dose groups were considered as not clastogenic since no mutagenicity was found at these doses.

All other dose groups in the other experiments were also found not to be clastogenic, respectively.

 

 

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	86.77	1	3.66
0.7	96.63	95.51	95.86	1.10	--
0.82	104.12	92.71	88.56	1.02	--
0.94	109.36	95.60	76.43	0.88	--
1.06	110.11	78.73	78.58	0.91	--
1.18	109.36	60.06	86.55	1.00	--
1.30	116.10	40.14	100.88	1.16	1.77
1.42	112.36	31.61	121.24	1.40	1.55
1.54	96.63	9.84	139.92	1.61	2.78
B[a]P, 3.5 µg/mL	99.63	69.03	623.89	7.19	1.24

B[a]P: Benzo[a]pyrene

  

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation 

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	79.39	1	2.75
0.22	100.33	90.09	78.53	0.99	--
0.46	86.38	79.76	124.63	1.57	--
0.58	91.03	79.70	77.88	0.98	--
0.70	98.34	89.53	70.89	0.89	--
0.82	98.34	71.30	69.28	0.87	--
0.94	95.02	64.50	62.74	0.79	1.60
1.06	99.00	47.66	74.59	0.94	3.17
1.18	91.69	11.04	97.49	1.23	1.12
EMS, 500 µg/mL	86.38	62.27	1337.77	16.85	--
MMS, 10 µg/mL	85.71	66.62	841.03	10.59	0.69

EMS: Ethyl methane sulphonate

MMS: Methyl methane sulphonate

 

 Table 3: Experiment II - 4 h Exposure - With Metabolic Activation

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	82.64	1.00	2.07
1	96.97	85.91	82.23	1.00	--
1.12	101.68	89.08	61.54	0.74	--
1.24	98.99	86.20	86.92	1.05	--
1.36	99.66	83.65	75.75	0.92	--
1.48	90.91	64.04	118.05	1.43	--
1.60	96.07	35.51	70.23	0.85	2.38
1.72	91.58	38.25	84.77	1.03	2.13
1.84	98.99	19.98	98.81	1.20	4.73
B[a]P, 3.5 µg/mL	86.20	60.39	829.02	10.03	0.89

B[a]P: Benzo[a]pyrene

 

 

Table 4: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	104.66	1	2.61
0.0005	95.24	94.64	76.34	0.73	--
0.001	101.36	103.58	68.22	0.65	--
0.002	94.56	95.29	66.56	0.64	--
0.005	101.36	105.73	52.63	0.50	--
0.01	102.04	98.98	64.06	0.61	--
0.06	102.72	96.75	61.54	0.59	3.30
0.18	96.60	55.56	91.91	0.88	2.24
0.30	91.16	19.77	99.26	0.95	1.94
EMS, 200 µg/mL	70.75	34.84	2516.55	24.05	--
MMS, 10 µg/mL	59.18	27.33	2625.00	25.08	0.81

EMS: Ethyl methane sulphonate

MMS: Methyl methane sulphonate

 

Table 5: Historical laboratory control data (Jan - Aug 2010)

	Negative Control		Positive Control
	-S9	+S9	EMS (500 µg/mL)	EMS  (200 µg/mL)	MMS  (10 µg/mL)	B[a]P  (3.5 µg/mL)
Mean of mutants per 1E+06 cells	90.81	89.22	1464.40	2245.20	1281.16	691.08
Minimum of mutants per 1E+06 cells	51.46	50.83	244.01	593.04	202.45	332.60
Maximum of mutants per 1E+06 cells	173.95	170.04	2531.86	4745.21	2654.69	1148.82
Standard deviation	28.66	28.13	642.39	973.54	2654.69	216.65
Relative standard deviation [%]	31.56	31.53	43.87	43.36	2859.71	31.35
Number of control values	50	50	14	13	not reported	25

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item decanoic acid is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y. The study was conducted according to OECD TG 476 (1997) and in accodance with GLP. Even though there are some deviations from the current OECD TG 490 (2016), the study is considered valid and suitable for the assessment of the potential of the test item to cause gene mutations in mammalian cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: (his-)
Escherichia coli: (trp-)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

20 - 5000 µg/ plate (Standard plate test, SPT)
4 - 2500 µg/ plate (Preincubation test, PIT)

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see free text

Details on test system and experimental conditions:

Standard plate test
Test tubes containing 2 ml soft agar kept in a water bath at 45°C, and remaining components added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or 0 .5 ml phosphate buffer (in tests without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates.

Preincubation test
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.

In each experiment 3 test plates per dose or per control used; after fter incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his or trp revertants) are counted.

Positive control
with metabolic activation: 2.5 and 60 μg/plate 2-aminoanthracene for each S. typhimurium strain and the E.coli strain, respectively;
without metabolic activation: 5 μg/plate N-methyl-N'-nitro-N-nitrosoguanidine for TA 100 and TA 1535, 10 μg/plate 4-nitro-o-phenylendiamine
for TA 98, 100 μg 9-aminoacridine chloride monohydrate for TA 1537 and 10 µg N-ethyl-N'-nitro-N-nitrosoguanidin for E. coli. All substances were dissolved in DMSO.
The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.

Evaluation criteria:

Positive results
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Statistics:

Mean and standard deviation calculated in result tables. No further data.

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537; E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed from about 500 - 2500 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A test substance precipitation was found from about 500 µg/plate onward.

Standard plate test (20 - 5000 µg/plate)
Strain	Metabolic activation system	Replicates	maximum revertant factor	dose dependency	Assessment
TA 98	no	3	0.9	no	negative
	yes	3	1.0	no	negative
TA 100	no	3	1.0	no	negative
	yes	3	0.8	no	negative
TA 1535	no	3	1.1	no	negative
	yes	3	1.1	no	negative
TA 1537	no	3	0.8	no	negative
	yes	3	0.9	no	negative
WP2 uvr A	no	3	1.0	no	negative
	yes	3	1.0	no	negative
Preincubation test (4 - 2500 µg/plate)
Strain	Metabolic activation system	Replicates	maximum revertant factor	dose dependency	Assessment
TA 98	no	3	1.0	no	negative
	yes	3	1.0	no	negative
TA 100	no	3	1.0	no	negative
	yes	3	1.0	no	negative
TA 1535	no	3	0.9	no	negative
	yes	3	0.9	no	negative
TA 1537	no	3	0.9	no	negative
	yes	3	0.8	no	negative
WP2 uvr A	no	3	0.9	no	negative
	yes	3	0.8	no	negative

Conclusions:

According to the results of the present study, the test substance Palmitoyl chloride is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Executive summary:

The study was conducted according to the OECD Guidelines 471 and 472, respectively and is reliable without restrictions. Palmitoyl chloride was tested in the standard plate test and in the preincubation test with and without metabolic activation in S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA at dose levels of 20 - 5000 and 4 - 2500 µg/plate, respectively. No increase in the number of revertants was detected in any strain with and without MA. Vehicle controls and positive controls were valid. Cytotoxicity was found depending on the strain and metabolic activation. Precipitation was reported.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No further details.

Additional information

For the target substance palmitoyl chloride only a study for gene mutation in bacteria are available. To cover the other endpoints studies from the source substances docosanoic acid (CAS 112-85-6) and decanoic acid (CAS 334-48-5) are taken.

Gene mutation in bacteria

Palmitoyl chloride was tested in the standard plate test and in the preincubation test with and without metabolic activation in S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA at dose levels of 20 - 5000 and 4 - 2500 µg/plate, respectively. The study was conducted according to the OECD Guidelines 471 and 472, respectively and is reliable without restrictions. No increase in the number of revertants was detected in any strain with and without MA. Vehicle controls and positive controls were valid. Cytotoxicity was found depending on the strain and metabolic activation. Precipitation was reported. According to the results of the pesent study, the test substance palmitoyl chloride ist not nutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay under the experimantaa conditions chosen here. (BASF, 1998).

 

Available studies for the source substance decanoic acid (CAS 334-48-5) and docosanoic acid (CAS 112-85-6).

Genetic toxicity in bacteria was evaluated in a study performed according to GLP and OECD guideline 471and 472, and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals. S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 andE. coli WP2 uvr Awere treated with docosanoic acid (CAS 112-85-6) dissolved in DMSO at concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation by S9 mix from livers of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone. No increase in revertants compared to the vehicle control was observed in any of the strains at all test concentrations. No toxicity was noted up to a concentration of 5000 µg/plate. Thus, docosanoic acid can be regarded as non-mutagenic in bacteria (Nakajima, 2002).  

Decanoic acid (CAS 334-48-5) was tested for genetic toxicity in the S. typhimurium strains TA 97, TA 98, TA 100, TA1535 and TA1537 similar to OECD guideline 471. For metabolic activation, cofactor supplemented post-mitochondrial fraction (S9 mix) were used, containing rat or hamster liver enzymes (S9) in a concentration of 10% or 30%. Using the preincubation method 10, 33, 100, 333 and 666 µg decanoic acid/plate with metabolic activation did not reveal any genotoxicity. Also concentrations of 3, 10, 33, 100, 333 and 666 µg/plate without metabolic activation did not reveal an increase in the mean number of revertants compared to the negative control. Slight cytotoxicity was observed at 666 µg/plate (NTP, 1985).  

 

Cytogenicity in mammalian cells

In an vitro mammalian chromosome aberration test was conducted with docosanoic acid (CAS 112-85-6) in accordance with GLP and OECD guideline 473 and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals. Properly maintained Chinese hamster lung (CHL) cells were treated with docosanoic acid dissolved in 1% carboxymethylcellulose sodium at concentrations of 875, 1750 and 3500 µg/mL for 6 hours with and without metabolic activation by S9 mix prepared from phenobarbital- and 5,6-benzoflavone-induced rat livers. The highest test concentration of 3500 µg/mL reflect 0.01 M of the test substance as required in the in OECD guideline 473. In addition, the cells were incubated with 350, 700, 1400, 2800 µg/mL without metabolic activation for 24 hours and with 288, 575, 1150 and 2300 µg/mL without metabolic activation for 48 hours, respectively. The highest concentration of the test item used was set to the maximum one showing no apparent cytotoxic effects during continuous treatment. No increase in chromosomal aberrations nor polyploidy were observed up to the maximum concentration under short-term and continuous treatment with and without metabolic activation.The positive controls included during short-term and continuous exposure showed the expected results and thus verified the sensitivity of the assay (Nakajima, 2002).

 

The chromosomal aberration-inducing capability (equal or similar to OECD 473) of decanoic acid (CAS 334-48-5) was investigated using female Chinese hamster lung-derived CHL/IU cells, by the short-term treatment method with and without metabolic activation, and by the 24-hour continuous treatment method. Chromosomal observation was performed at 4 doses up to 216 µg/mL (27.0, 54.1, 108 and 216 µg/mL) in the short-term treatment method, and at 4 doses up to 90.0 µg/mL (11.3, 22.5, 45.0 and 90.0 µg/mL) in the 24-hour continuous treatment method. On the short-term treatment at a dose of 54.1 µg/mL without metabolic activation, the incidence of cells having structural aberration was statistically significantly higher than in the negative control. Nevertheless, the incidence of cells having structural aberration was 1.7%, which is within the fluctuation range of the negative control calculated from the background data, the incidence of cells having structural aberration in the negative control was low, at 0%, and the increase was not dosedependent, and so this increase was judged to be accidental rather than a biologically meaningful increase. Moreover, no statistically significant increases were observed at other doses on short-term treatment without metabolic activation, short-term treatment with metabolic activation, or 24-hour continuous treatment. It was therefore judged that exposure to the test substance afforded no biologically significant increase in the incidence of cells having structural chromosomal aberration, or of polyploidy. From the above results, it was judged that this test substance has no chromosomal aberration inducing capability under the conditions of this study (MHW Japan, 2018).

 

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed with decanoic acid (CAS 334-48-5) under GLP according to OECD guideline 476. In two independent experiments, mouse lymphoma L5178Y cells were treated with decanoic acid at concentrations up to 1.18 mM without metabolic and up to 1.54 mM with metabolic activation by phenobarbital and beta-naphthoflavone-induced rat liver S9-mix, respectively. The exposure duration was 4 hours and 24 hours in experiments without S9 mix and 4 hours in the experiments with S9 mix. The treatment of cells in all experiments was followed by an expression period of 2 days and a selection period of 11-14 days in the presence of trifluorothymidine. Although cytotoxicity was observed at the highest concentrations tested, all mutant values were found to be within the range of the historical control data of the test facility, so that decanoic acid was regarded not to be mutagenic. In addition, colony sizing was performed for the highest concentrations used to detect potential clastogenic effects and/or chromosomal aberrations. As result, decanoic acid was found not to be clastogenic at all dose groups tested. The positive controls caused a pronounced increase in the mutation frequency demonstrating the sensitivity of the test system (Trenz, 2010).

 

 

Justification for classification or non-classification

No classification suggested for mutagenicity as criteria of regulation 1272/2008/EC are not met.