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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08/01/2018-25/01/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

impurity 1
Chemical structure
Reference substance name:
Palmitic acid
EC Number:
200-312-9
EC Name:
Palmitic acid
Cas Number:
57-10-3
Molecular formula:
C16H32O2
IUPAC Name:
palmitic acid
impurity 2
Chemical structure
Reference substance name:
Ethane-1,2-diol
EC Number:
203-473-3
EC Name:
Ethane-1,2-diol
Cas Number:
107-21-1
Molecular formula:
C2H6O2
IUPAC Name:
ethylene glycol
Constituent 1
Chemical structure
Reference substance name:
Ethane-1,2-diyl palmitate
EC Number:
210-826-5
EC Name:
Ethane-1,2-diyl palmitate
Cas Number:
624-03-3
Molecular formula:
C34H66O4
IUPAC Name:
ethane-1,2-diyl dihexadecanoate
Constituent 2
Chemical structure
Reference substance name:
2-hydroxyethyl palmitate
EC Number:
224-160-8
EC Name:
2-hydroxyethyl palmitate
Cas Number:
4219-49-2
Molecular formula:
C18H36O3 & C34H66O4
IUPAC Name:
Fatty acids, C16, esters with ethylene glycol
Specific details on test material used for the study:
TEST ITEM : 2-hydroxyethyl palmitate
OTHER NAMES / CODES : 2-HYDROXYETHYL PALMITATE LANOL P ECAILLES 35928C
IPL REGISTRATION NUMBER : 171214
BATCH NUMBER : 170301011115
APPEARANCE : white to off-white solid (flakes by naked eyes)
WATER CONTENT : <1%
PURITY / COMPOSITION : > 99% (dry matter) (Sponsor’s information)
SALT / BASE RATIO : unknown
MOLECULAR WEIGHT : unknown
DENSITY : 1.064 ± 0.013 (Sponsor’s information)
CORRECTION FACTOR : none, at the Sponsor’s request
STORAGE CONDITIONS* : room temperature (+20±5°C)
QUANTITY SUPPLIED : unknown
MANUFACTURING DATE : 27/02/2017
ANALYSIS DATE : 08/03/2017
STABILITY UNDER
STORAGE CONDITIONS : 2 years, up to 27/02/2019 for batch 170301011115
EXPIRY DATE : 27/02/2019

Method

Target gene:
TA1535 and TA100 : his G 46
TA1537 : his C 3076
TA102 : His G 428
TA98 : his D 3052
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
hepatic microsomes from rat livers induced by Aroclor 1254 – Incubation period: ca. 44 h
Test concentrations with justification for top dose:
- First toxicity test : 0, 50, 150, 500, 1500 and 5000 µg/plate. Maximum dose according to OECD Guideline, i.e. 5000 μg/plate. (1 plate / dose).
An important precipitate hindering scoring was observed in all strains both without and with metabolic activation at the 2 highest tested doses of 1500 and 5000 μg/plate. Moderate and slight precipitate was also observed at 500 and 150 μg/plate, respectively. Therefore, due to the important precipitate, the maximum dose retained for the first mutagenicity assay was lowered down to 500 μg/plate in all strains both with and without metabolic activation.
- Mutagenicity test : 0, 5, 15, 50, 150, 500 µg/plate (3 plates / dose)
Vehicle / solvent:
The solvent used is Tetrahydrofuran (THF).
Prior to the implementation of the toxicity assay, trials for solubility were performed in sterile water, dimethylsulfoxide, ethanol and tetrahydrofuran (THF), at initial concentrations of 50 mg/mL, ranging from 100 to 6.25 mg/mL, ranging from 200 to 12.5 mg/mL, and at 250 mg/mL, respectively, at room temperature, demonstrating that the test is not soluble whatever the solvent. After heating the organic preparations at a temperature below the fusion point of 60-65°C (in accordance with the information provided by the Sponsor Representative), only the 250 mg/mL preparation in THF was clear. Noteworthy, when settled at room temperature, the solution in THF solidified.
For the preliminary toxicity assay, the test item 2-hydroxyethyl palmitate was dissolved in THF (Sigma, batch STBF9606V) at an initial concentration of 250 mg/mL, without following the qs (quantum satis) method (see § 11.1), in order to obtain the top dose of 5000 μg/plate when added at 20 μL/plate. Preparations for treatment were kept at ca. 37°C until treatments in order to anticipate the behavior of the test item in solution when temperature decreases.
For both mutagenicity assays, it was also prepared in THF but at an initial concentration of 25 mg/mL, without following the qs (quantum satis) method (see § 11.1), in order to obtain the top dose of 500 μg/plate when added at 20 μL/plate. As at this concentration the solution in THF did not solidified at room temperature, preparations for treatment were kept at room temperature until treatments.
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
6 plates
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
Without S9 mix
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
6 plates
Positive control substance:
benzo(a)pyrene
other: 2-anthramine, 2 μg/plate without pre-incubation, 1 μg/plate with pre-incubation (TA1535, TA1537, TA98, TA100)
Remarks:
With S9 Mix
Details on test system and experimental conditions:
Two test of mutagenicity were realised independently : first with metabolic activation, and the second without (with a preincubation period)

MEDIA : agar, supplemented with 10 % of 0.5 mM biotin histidine solution

TOXICITY TEST :
The toxicity assay was carried out in all the strains to be tested under the same conditions as the first mutagenicity test with and without metabolic activation (See below).
NUMBER OF REPLICATIONS : 1 plates per dose (no positive controls included)
DETERMINATION OF THE CYTOTOXICITY : Microscopic examination

MUTAGENICITY TEST :
WIth metabolic activation:
For each strain : 0.1 mL of a bacterial suspension from a culture agitated overnight at ca. 37°C and 20 μL of the test item at the relevant initial concentrations were successively added to 2 mL of top agar, supplemented with 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at ca. 45°C. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. The plates were then incubated at ca. 37°C for approximately 44 h. At the end of the expression time, colonies of revertants were counted for each plate.
Without metabolic activation : The method was the same as the one described above except that immediately before spreading in the plates, 0.5 mL of the S9-mix metabolic activation system was added in soft agar.
NUMBER OF REPLICATIONS: 3 plates per dose (6 for the positive controls)

Evaluation criteria:
Acceptance criteria :
- Determination of the media sterility (The sterility of the test item was berified in a test where it was incubated for 44 hours at 37°C, no bacterial growth was observed, the test item was considered sterile) : ok
- Determination of the efficiency of the metabolic activation : ok
- Validation of the solvent control (The frequencies of spontaneous revertants (solvent controls) were within the limits generally observed under our experimental conditions) : ok
- Determination of the sensitivity of the strains (Statistically and biologically significant increases in the numbers of revertants were observed in the presence of positive reference test substances) : ok
- For the test, 5 doses available, as required by OECD guideline : ok (5 here)
- For the test, At least 2 plates per dose were available for the assessment of mutagenicity : ok (3 here)
Statistics:
Data were analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control. Statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response and biological relevance of the results should be considered firs

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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 specified
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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, but tested up to precipitating concentrations
Remarks:
Toxicity test : statistically significant decreases were noted in the first assay in strain TA102 at 500 and 150 μg/plate without and with metabolic activation, respectively. Nevertheless, these effects had no meaning in terms of mutagenicity hazard
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
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 specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
a statistically but not biologically significant increase in the number of revertants was observed at the intermediate dose of 15 μg/plate.
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Mutagenicity test :
In two independent assays performed both with and without metabolic activation (the second assay with S9-mix was performed according to the pre-incubation protocol), no biologically significant increases in the mean number of revertants were noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested, in the presence of 2-hydroxyethyl palmitate.
The test item 2-hydroxyethyl palmitate is thus not mutagenic in these conditions.
In the first assay in strain TA100 in presence of metabolic activation without pre-incubation, a statistically but not biologically significant increase in the number of revertants was observed at the intermediate dose of 15 μg/plate. The mean value for revertants was very slightly higher than the upper bound of the intervals of historical data for negative controls, i.e. 162 vs. 161. However, the threshold ratio for a biologically significant effect (set at 2 in this strain) was not reached, with an induction ratio of 1.3, and no dose-effect relationship was observed. Furthermore, the second assay performed under the more sensitive pre-incubation method was clearly negative. The test item 2-hydroxyethyl palmitate was thus considered as not mutagenic in this experimental condition.

Applicant's summary and conclusion

Conclusions:
The mutagenic activity of the test item 2-hydroxyethyl palmitate (Batch 170301011115) sponsored by SEPPIC was assessed by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested both in presence and in absence of metabolic activation, in two independent assays according to OECD guideline (OECD 471, 1997), using the maximum dose compatible with the solubility of the test item in the test system, i.e. 500 μg/plate. The validity criteria for the assay were fulfilled. The study is thus valid. Under these experimental conditions, no mutagenic activity was revealed.

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