Glycerides, C12-18 mono-, di- and tri-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/12/2017 to 26/02/2018

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

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Regulation EC 400/2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: Artile Appendix II to the article D523-8 of the French environment code

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: EC Commission Directive 2004/10/EC of 11th February 2004 (Official Journal No. L050)

Principles of method if other than guideline:

Mutagenic compounds are detected by the reverse mutation induced at the his- locus in the histidine
auxotrophic strains of Salmonella typhimurium.
The increase, over the spontaneous rate, in the number of revertants that do not require histidine
(prototrophic revertants) and that grow in the presence of the test item in a histidine-free medium, provides
an indication of the mutagenic activity of the test item. The use of different strains allows demonstrating the
different mechanisms of gene mutation.
The test item is studied with and without metabolic activation in order to demonstrate the activity of
promutagens and direct mutagens, respectively.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

TEST ITEM : Glycerides, C12-18 mono-, di-and tri-
OTHER NAMES / CODES : 34327M Monoglyceride Copra
MONOGLYCERIDE COPRAH BASE
34327M
NAME ON IDENTIFICATION TAG : 34327M Monoglyceride Copra
IPL REGISTRATION NUMBER : 171213
BATCH NUMBER : 170519013326
APPEARANCE : white to off-white solid (waxy by naked eyes)
WATER CONTENT : unknown
PURITY / COMPOSITION : considered as 100% (Sponsor’s information)
SALT / BASE RATIO : unknown
MOLECULAR WEIGHT : unknown
DENSITY : unknown
CORRECTION FACTOR : none, at the Sponsor’s request
STORAGE CONDITIONS* : room temperature (+20±5°C)
QUANTITY SUPPLIED : unknown
MANUFACTURING DATE : 15/05/2017
ANALYSIS DATE : 29/05/2017
STABILITY UNDER
STORAGE CONDITIONS : 2 years, up to 15/05/2019 for batch 170519013326
EXPIRY DATE : 15/05/2019

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:

using hepatic microsomes from rat livers induced by Aroclor 1254 – Incubation period: ca. 44 h

Test concentrations with justification for top dose:

5000 μg/plate
METHODS
Strains used : TA1535, TA1537, TA98, TA100, TA102
Solvent : ethanol
Stability in the solvent : unknown (preparations for treatments were made extemporaneously)
Purity / composition : considered as 100% (Sponsor’s information)
Correction factor : none, at the Sponsor’s request
Expression of the doses used
in the assays : μg/plate of Glycerides, C12-18 mono-, di-and tri- as supplied
TOXICITY ASSAY
Preliminary test of toxic activity : carried out on 5 strains both with and without metabolic activation
using hepatic microsomes from rat livers induced by Aroclor 1254 –
Incubation period: ca. 44 h
Sterility test : absence of contamination
Limiting factor for maximum dose : maximum dose according to OECD Guideline, i.e. 5000 μg/plate
In all strains and whatever the metabolic activation condition, an important precipitate hindering scoring
was observed at the highest tested dose of 5000 μg/plate. Moderate and slight precipitate was also noted
at 1500 and 500 μg/plate, respectively. Therefore, the highest tested dose of 5000 μg/plate was not
analysable for revertant counting. It is also noteworthy that the assessment of toxicity was difficult, due to
the precipitate.
Regarding toxicity, strong toxicity was noted at 1500 and 5000 μg/plate whatever the metabolic activation
condition in all strains, except in strain TA1535 (in which a slight to moderate toxicity was observed). A
slight toxicity was noted at 500 μg/plate in all strains and whatever the metabolic activation condition.
Therefore, due to the solubility and/or the toxicity, the maximum dose retained for the first mutagenicity
assay was lowered down to:
- 500 μg/plate in strains TA1537, TA98, TA100 and TA102 both without and with metabolic
activation, and 4 lower doses were also tested;
- 1000 μg/plate in strain TA1535 both without and with metabolic activation, and 5 lower doses were
also tested.

Vehicle / solvent:

Solvent : ethanol
Stability in the solvent : unknown (preparations for treatments were made extemporaneously)
Purity / composition : considered as 100% (Sponsor’s information)
Correction factor : none, at the Sponsor’s request
Expression of the doses used
in the assays : μg/plate of Glycerides, C12-18 mono-, di-and tri- as supplied
TOXICITY ASSAY
Preliminary test of toxic activity : carried out on 5 strains both with and without metabolic activation
using hepatic microsomes from rat livers induced by Aroclor 1254 –
Incubation period: ca. 44 h
Sterility test : absence of contamination
Limiting factor for maximum dose : maximum dose according to OECD Guideline, i.e. 5000 μg/plate

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

APPEARANCE : white to off-white solid (waxy by naked eyes)
WATER CONTENT : unknown
PURITY / COMPOSITION : considered as 100% (Sponsor’s information)
SALT / BASE RATIO : unknown
MOLECULAR WEIGHT : unknown
DENSITY : unknown
CORRECTION FACTOR : none, at the Sponsor’s request
STORAGE CONDITIONS* : room temperature (+20±5°C)
This test item, the characteristics of which are given in Appendix No. 4, was tested in accordance with the
Final Study Plan FSP-IPL 171110 (see Appendix No. 6), and Study Plan Adherence
Prior to the implementation of the toxicity assay, trials for solubility were performed in sterile water,
dimethylsulfoxide, ethanol and tetrahydrofuran, at initial concentrations of 50, 100, 200 and 250 mg/mL,
respectively, demonstrating that the better solubility was obtained in ethanol.
For the preliminary toxicity assay, the test item Glycerides, C12-18 mono-, di-and tri- was dissolved in
ethanol (Merck, batch K47900183 622) at an initial concentration of 50 mg/mL in order to obtain the top
dose of 5000 μg/plate when added at 100 μL/plate (without and with S9-mix).
For the first mutagenicity assay, the test item Glycerides, C12-18 mono-, di-and tri- was also dissolved in
ethanol at an initial concentration of 10 mg/mL in order to obtain the top dose of 1000 μg/plate in strain
TA1535 when added at 100 μL/plate (without and with S9-mix).
For the second mutagenicity assay, the test item Glycerides, C12-18 mono-, di-and tri- was also
dissolved in ethanol at an initial concentration of 40 mg/mL in order to obtain the top dose of 1000 μg/plate
in strains TA1535, TA1537 and TA98 when added at 25 μL/plate (with S9-mix, with pre-incubation). This
solution was then quarter diluted to obtain an initial concentration of 10 mg/mL and a corresponding top
dose of 1000 μg/plate in strains TA1535 and TA100 when added at 100 μL/plate (without S9-mix).
Lower concentrations were also prepared with ethanol by serial dilutions and used at either 100 or
25 μL/plate (without and with S9-mix, without pre-incubation or with S9-mix, with pre-incubation,
respectively).
The stability of the test item in the solvent was unknown. Therefore, preparations for treatments were
performed just before use.
In the preliminary toxicity assay and the main assays, the appearance of a precipitate is noted as follows:
- : no precipitate
+ : slight precipitate
++ : moderate precipitate
+++ : heavy precipitate hindering scoring of revertants
In the preliminary toxicity assay, in all strains and whatever the metabolic activation condition, an important
precipitate hindering scoring was observed at the highest tested dose of 5000 μg/plate. Moderate and
slight precipitate was also noted at 1500 and 500 μg/plate, respectively. (see Table 1).
In both mutagenicity assays performed up the maximum dose compatible with the toxic activity (see § 6.1
and 6.2), i.e. 1000 or 500 μg/plate (depending on the strain and/or the metabolic activation condition and/or
the assay), a slight precipitate was observed in all strains and metabolic activation conditions at the doses
of 500 and 1000 μg/plate

Rationale for test conditions:

The Ames' test in Salmonella typhimurium strains has been validated by many international studies and is
well standardized (Maron and Ames, 1983). That test has been the subject of an OECD (1997) and EC
(2008) Guidelines.

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
- For the test, At least 2 plates per dose were available for the assessment of mutagenicity : ok

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 first.
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of
the experimental conditions examined all the following criteria are fulfilled:
- at least one of the test doses exhibits a biologically significant increase (2 or 3 fold increase in the
mean number of revertants depending on the strain) compared with the concurrent negative
control and,
- the increase is dose-related,
- and the mean numbers of revertants of the test doses are outside the distribution of negative
control data
The test item is then considered able to induce mutations in this test system.
If, in all experimental conditions examined, none of the above criteria are fulfilled, a test item is considered
clearly negative and unable to induce mutations in this test system.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The search for any mutagenic activity of Glycerides, C12-18 mono-, di-and tri- (Batch 170519013326)
sponsored by SEPPIC, was studied by means of the Ames’ test (Salmonella his-/microsome system) in
compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471, using the
maximum dose compatible with the toxic activity, i.e. 1000 or 500 μg/plate (depending on the strain and/or
the metabolic activation condition and/or the assay) for the mutagenicity assays. Four or five lower doses
were also tested.
The summaries of the test results are given in Appendix No. 1 (Tables 2 and 3). The individual results for
mutagenicity assays are shown in Appendix No. 2 (Tables 5 to 14).
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 Glycerides, C12-18 mono-, di-and tri-.
The test item Glycerides, C12-18 mono-, di-and tri- was thus not mutagenic in these conditions.
In the second assay in strain TA98 in presence of metabolic activation with pre-incubation (Table 12),
statistically but not biologically significant increases in the number of revertants were observed at the 2
intermediate doses of 50 and 150 μg/plate, with induction ratios of 1.4, i.e. below the threshold ratio for a
biologically significant effect (set at 2 in this strain). Moreover, the mean values for revertants were within
the intervals of historical data for negative controls, i.e. 39.3 and 37.7, respectively vs. 9-57, and no doseeffect
relationship was observed..
The test item Glycerides, C12-18 mono-, di-and tri- was thus considered as not mutagenic in this
experimental condition.

Applicant's summary and conclusion

Conclusions:

The mutagenic activity of the test item Glycerides, C12-18 mono-, di-and tri- (Batch 170519013326)
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 toxic activity, i.e. 1000 or 500 μg/plate
(depending on the strain and/or the metabolic activation condition and/or the assay).
The validity criteria for the assay were fulfilled. The study is thus valid.
Under these experimental conditions, no mutagenic activity was revealed.