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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Based on the analogue approach, two studies for genetic toxicity assesment of other substances, CAS#91052 -13 -0 and CAS#736150 -63 -3 respectively, could be used for Glycerides, C12-18 mono-, di- and tri- by read-across way.

The substance of CAS#736150 -63 -3 was not mutagenic in this in vitro mammalian cell gene mutation test performed in mouse lymphoma L5178Y cells with the absence and presence of S-9 mix.

The substance of CAS#91052 -13 -0 was not mutagenic in this in vitro Chromosome Aberration Test performed in cultured Mammalian CHL/IU Cells with the absence and presence of S-9 mix.

So, according to analogue aaproach, Glycerides, C12 -18 mono-, di- and tri- could be considered to be not mutagenic in these two studies.

In conclusion, Glycerides, C12 -18 mono-, di- and tri- has no classification for genetic toxicity.

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:

22/12/2017 to 26/02/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Regulation EC 400/2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

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

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

Target gene:

TA1535 and TA100 : his G 46
TA1537 : his C 3076
TA102 : His G 428
TA98 : his D 3052

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

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.

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

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

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

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

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.

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.

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

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Details on mammalian cell type (if applicable):

The cells of the following passage number( lot N°) were used in the study
Cell growth inhibition test: 9th passages (lot N°20100422(5))
Chromosome aberration test:
-short-term exposure exp.:13th passages (lot N° 20100422(5))
-Continuous exposure exp.:17th passages (lot N° 20100422(5))
The cells were cultured every three to four days.

Additional strain / cell type characteristics:

other: modal chromosome number of 25

Metabolic activation:

with and without

Metabolic activation system:

Positive controls without cofactor supplemented post-mitochondrial fraction (S9 mix), Mitomycin C(MMC) solvent for MMC=Physiological salinewith cofactor supplemented post-mitochondrial fraction (S9 mix),Benzo[a]pyrene

Test concentrations with justification for top dose:

5.0 mg/ml

Vehicle / solvent:

Solvent:acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

other: Physiological saline; DMSO

Evaluation criteria:

In accordance with evaluation criteria defined by Sofuni, et al. 4), the following criteria were set to evaluate the frequency of aberrant cells in each dose group.
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps. No statistical analysis was performed to the results.
Negative (-) less than 5%
Equivocal 5% or more, less than 10%
Positive (+) 10% or more
When more than 50 cells in meta phase per dish were not observed by the cytotoxicity, it was excluded from the judgment, and recorded as “TOX".
The short“term exposure experiment was performed first. When the result of the short-term exposure experiment was positive, the continuous exposure experiment wasn't to be performed. When the results of the short”term exposure experiment with and without S9 mix were negative, the continuous exposure experiment was to be performed. Furthermore, when the result of chromosome aberration test was positive but not definitely dose-dependent (the positive result was found only in a single dose group) or that was equivocal, a confirmatory test was to be performed including the same dose level which was positive or equivocal. In the results of the confirmatory test, it was to be judged equivocal if the equivocal result was observed again and to be judged positive if the positive result was induced without dose-dependence.

Key result

Species / strain:

other: CHI/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Based on the above results, it is concluded that the test substance does not induce chromosome aberrations in CHL/IU cells.

Conclusions:

Under the test conditions, it is concluded that the tested substance does not induce chromosome aberrations in the cell line of CHL/IU cells.

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)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Details on mammalian cell type (if applicable):

The cells of the following passage number( lot N°) were used in the study
Cell growth inhibition test: 9th passages (lot N°20100422(5))
Chromosome aberration test:
-short-term exposure exp.:13th passages (lot N° 20100422(5))
-Continuous exposure exp.:17th passages (lot N° 20100422(5))
The cells were cultured every three to four days.

Additional strain / cell type characteristics:

other: modal chromosome number of 25

Metabolic activation:

with and without

Metabolic activation system:

Positive controls without cofactor supplemented post-mitochondrial fraction (S9 mix), Mitomycin C(MMC) solvent for MMC=Physiological salinewith cofactor supplemented post-mitochondrial fraction (S9 mix),Benzo[a]pyrene

Test concentrations with justification for top dose:

5.0 mg/ml

Vehicle / solvent:

Solvent:acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

other: Physiological saline; DMSO

Evaluation criteria:

In accordance with evaluation criteria defined by Sofuni, et al. 4), the following criteria were set to evaluate the frequency of aberrant cells in each dose group.
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps. No statistical analysis was performed to the results.
Negative (-) less than 5%
Equivocal 5% or more, less than 10%
Positive (+) 10% or more
When more than 50 cells in meta phase per dish were not observed by the cytotoxicity, it was excluded from the judgment, and recorded as “TOX".
The short“term exposure experiment was performed first. When the result of the short-term exposure experiment was positive, the continuous exposure experiment wasn't to be performed. When the results of the short”term exposure experiment with and without S9 mix were negative, the continuous exposure experiment was to be performed. Furthermore, when the result of chromosome aberration test was positive but not definitely dose-dependent (the positive result was found only in a single dose group) or that was equivocal, a confirmatory test was to be performed including the same dose level which was positive or equivocal. In the results of the confirmatory test, it was to be judged equivocal if the equivocal result was observed again and to be judged positive if the positive result was induced without dose-dependence.

Key result

Species / strain:

other: CHI/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Based on the above results, it is concluded that the test substance does not induce chromosome aberrations in CHL/IU cells.

Conclusions:

Under the test conditions, it is concluded the registered substance does not induce chromosome aberrations in the cell line of CHL/IU cells.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Dec. 2001 - 17 Feb. 2002

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase gene (TK+/-)

Species / strain / cell type:

other: Mouse lymphoma L5178Y cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9.

Test concentrations with justification for top dose:

See table 1

Vehicle / solvent:

Vehicle: DMSO
Justification: Solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Migrated to IUCLID6: 3.3 µg/ml(with S-9 mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 50 µg/ml(without S-9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exposure period with metabolic activation was 3 hours and exposure period without metabolic activation was 4 hours
- Expression time (cells in growth medium): The cultures were incubated for an expression period of three days counting from the start of treatment.
- Selection time (if incubation with a selection agent): Incubation with trifluorothymidine for approximately 10 days at 5% CO2 and 37 ºC.

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: triplicate cultures

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cloning efficiency

Evaluation criteria:

If mutagenic effect, the following criteria should be met:

-increases in frequency of metaphases with aberrant chromosomes at one or more concentrations
-reproducible between replicate cultures and tests
-statistically significant effects
-increase exceeds historical negative control range
-increase not associated with changes in pH or osmolarity
-Evidence of dose-response

Statistics:

The analysis of variance test was used to compare the mutation frequencies for the treated groups with the corresponding negative control.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>2500 µg/ml (test 2) and >1250 µg/ml (test 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>160 µg/ml (test 2) and >3600 µg/ml (test 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no information
- Effects of osmolality: no information
- Evaporation from medium: no information
- Water solubility: TS-ED 532 was not soluble in water
- Precipitation: none
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: No range-finding/screening test was performed. Two independent tests and a repeat of the second test without S-9 mix were performed.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutation frequencies of the negative and positive controls were all within the historical control range from the laboratory with exception that one of the positive control values was slightly higher than the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: See Table 2

Remarks on result:

other: Migrated from field 'Test system'.

Table 1 Concentration, exposure period and expression time

Metabolic Activation	Test Substance Concentration (μg/mL)	Exposure Period	Expression Time
Present
Test 1	625, 1250, 2500, 3600, 5000	3	72
Test 2	156, 313, 625, 1250, 2500, 3600	3	72
Absent
Test 1	625, 1250, 2500, 3600, 5000	4	72
Test 2	2.5, 5, 10, 20, 40, 80, 160, 313, 320, 625, 1250, 2500, 3600, 5000	24	72

Table 2 Results

Metabolic Activation	Test Substance Concentration (µg/mL) Resulting in:
	Cytotoxicity in PreliminaryTest	Cytotoxicity in Main Test	Precipitation	Genotoxic Effect
Present
Test 1	Not stated	>1250	none	none
Test 2	Not stated	>2500	none	none
Absent
Test 1	Not stated	>3600	none	none
Test 2	Not stated	>160	none	none

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with mouse lymphoma L5178Y cells in the absence and presence of S-9 mix.

Executive summary:

Acetic acid esters of monoglyceride of fully hydrogenated castor oil was tested in vitro in a mammalian cell gene mutation test using mouse lymphoma L5178Y cells (OECD 476). Cultures of mouse lymphoma L5178Y cells were treated with solutions of Acetic acid esters of monoglyceride of fully hydrogenated castor oil  prepared in cell culture medium at concentrations of 2.5 to 5000 µg/ml. In the absence of S‑9 mix, the cells were treated for 4 hours in the first test and 24 hours in the second test. In the presence of S-9 mix, the cells were treated for 3 hours in both tests. The concentration of S-9 homogenate in the mix was 1.5-fold higher in the second test.
No biologically or statistically significant increases in the frequency of total mutant colonies were observed at any Acetic acid esters of monoglyceride of fully hydrogenated castor oil

concentration in either test, either in the absence and presence of S-9 mix. Large increases in mutation frequency observed in cultures treated with the positive control agents demonstrated the sensitivity of the test system and the efficacy of the S-9 mix.

Based on these results,Acetic acid esters of monoglyceride of fully hydrogenated castor oil

is non clastogenic in this in vitro mammalian cell gene mutation test performed with mouse lymphoma L5178Y cells in the absence and presence of S‑9 mix.

 

Based on these results,TS-ED 532 is non clastogenic in this in vitro mammalian cell gene mutation test performed with mouse lymphoma L5178Y cells in the absence and presence of S‑9 mix.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase gene (TK+/-)

Species / strain / cell type:

other: Mouse lymphoma L5178Y cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9.

Test concentrations with justification for top dose:

See table 1

Vehicle / solvent:

Vehicle: DMSO
Justification: Solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Migrated to IUCLID6: 3.3 µg/ml(with S-9 mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 50 µg/ml(without S-9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exposure period with metabolic activation was 3 hours and exposure period without metabolic activation was 4 hours
- Expression time (cells in growth medium): The cultures were incubated for an expression period of three days counting from the start of treatment.
- Selection time (if incubation with a selection agent): Incubation with trifluorothymidine for approximately 10 days at 5% CO2 and 37 ºC.

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: triplicate cultures

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cloning efficiency

Evaluation criteria:

If mutagenic effect, the following criteria should be met:

-increases in frequency of metaphases with aberrant chromosomes at one or more concentrations
-reproducible between replicate cultures and tests
-statistically significant effects
-increase exceeds historical negative control range
-increase not associated with changes in pH or osmolarity
-Evidence of dose-response

Statistics:

The analysis of variance test was used to compare the mutation frequencies for the treated groups with the corresponding negative control.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>2500 µg/ml (test 2) and >1250 µg/ml (test 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>160 µg/ml (test 2) and >3600 µg/ml (test 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no information
- Effects of osmolality: no information
- Evaporation from medium: no information
- Water solubility: TS-ED 532 was not soluble in water
- Precipitation: none
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: No range-finding/screening test was performed. Two independent tests and a repeat of the second test without S-9 mix were performed.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutation frequencies of the negative and positive controls were all within the historical control range from the laboratory with exception that one of the positive control values was slightly higher than the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: See Table 2

Remarks on result:

other: Migrated from field 'Test system'.

Table 1 Concentration, exposure period and expression time

Metabolic Activation	Test Substance Concentration (μg/mL)	Exposure Period	Expression Time
Present
Test 1	625, 1250, 2500, 3600, 5000	3	72
Test 2	156, 313, 625, 1250, 2500, 3600	3	72
Absent
Test 1	625, 1250, 2500, 3600, 5000	4	72
Test 2	2.5, 5, 10, 20, 40, 80, 160, 313, 320, 625, 1250, 2500, 3600, 5000	24	72

Table 2 Results

Metabolic Activation	Test Substance Concentration (µg/mL) Resulting in:
	Cytotoxicity in PreliminaryTest	Cytotoxicity in Main Test	Precipitation	Genotoxic Effect
Present
Test 1	Not stated	>1250	none	none
Test 2	Not stated	>2500	none	none
Absent
Test 1	Not stated	>3600	none	none
Test 2	Not stated	>160	none	none

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with mouse lymphoma L5178Y cells in the absence and presence of S-9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No study available and not relevant as no effect was observed in in vitro studies

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance is not classified as a genotoxic/mutagen